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Topic:Analytical Methods
Analytical methods in equine research encompass a variety of scientific techniques and tools used to study and evaluate different aspects of horse health, performance, and physiology. These methods help advance our understanding of equine biology, diagnosing conditions, and improving management practices. Common analytical methods include molecular techniques like PCR and ELISA for detecting pathogens and measuring biomarkers, imaging technologies such as ultrasound and MRI for assessing musculoskeletal health, and statistical models for analyzing genetic data and performance metrics. This page compiles peer-reviewed research studies and scholarly articles that explore the development, application, and impact of various analytical methods in equine science.
A sensitive liquid chromatographic procedure for the analysis of camphor in equine urine and plasma. A sensitive method was required to analyze low levels of camphor in equine urine and plasma. Camphorated oil (20% w/w camphor) was administered topically (6 g) and intratracheally (1 g) to standardbred mares. The drug was extracted from urine and plasma by diethyl ether and analyzed as its 2,4-dinitrophenylhydrazone derivative by reverse phase HPLC with UV detection. The UV detector was set at 368.5 nm and the samples were eluted from the C18 column by 82% acetonitrile in water. The detection limit achieved was about 10 ng/mL urine and about 20 ng/mL plasma. After topical administration, only ...
Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse. Microquantitative measurements of ADH-activity were carried out on the livers of male and female rats, guinea-pigs and horses (two geldings and a mare). Lyophilized cryostat sections of liver parenchyma were microdissected the whole way along the sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ADH activity in samples of about 50-150 ng was measured in a microbiochemical assay using the oil-well technique without enzymatic cycling, by direct luminometric determination of NADH. On the basis of the single measurements, mean values of total hepatic ADH activit...
Qualitative detection of corticosteroids in equine biological fluids and the comparison of relative dexamethasone metabolite/dexamethasone concentration in equine urine by micro-liquid chromatography-mass spectrometry. Several important corticosteroids were qualitatively determined in the plasma and urine of horses by micro-liquid chromatography-mass spectrometry (micro-LC-MS). The sensitivity and specificity of micro-LC-MS are demonstrated as is the ability of micro-LC-MS to deal with endogenous interferences. In turn, the relative amount of dexamethasone and its major unconjugated metabolite were determined in equine urine by micro-LC-MS; the conclusions drawn are reported.
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...
Pharmacologic and pharmacokinetic properties of methocarbamol in the horse. The hemodynamic, respiratory, and behavioral effects, as well as the pharmacokinetic properties of methocarbamol, were determined in horses. Heart rate, cardiac output, arterial and venous blood pressures, respiratory rate, and arterial blood gases did not change after IV methocarbamol (4.4, 8.8, 17.6 mg/kg) administration. There were no signs of behavior modification or ataxia observed. Analysis of plasma concentration time data indicated that the disposition of methocarbamol may be dose-dependent. Clearance and steady-state volume of distribution decreased as the dose increased. Plasma conce...
Rapid screening and confirmation for drugs and metabolites in racing animals by tandem mass spectrometry. A screening and confirmation procedure for drugs and metabolites in the blood serum and urine of racing animals was developed. Equine blood serum was spiked with low concentrations of several drugs of interest. Canine blood serum and urine were collected following oral doses of diethylcarbamazine, procaine, and phenylbutazone. Serum, urine, and extracts of each were analyzed, using a triple quadrupole mass spectrometer. Simultaneous screening of up to 50 drugs was possible in a single sample, in less than 2 minutes. Detection limits for most compounds were in the ng/ml to microgram/ml range, u...
Mare lactotransferrin: purification, analysis and N-terminal sequence determination. Mare lactotransferrin has been purified and analyzed. Its molecular mass is 81 kDa. A 28 amino acid long N-terminal sequence was established and a first series of comparisons with other transferrins was performed.
Assessment of a reflectance photometer in a veterinary laboratory. This report is an assessment of clinical chemistry dry reagent methodology for veterinary use. A portable reflectance photometer and dry reagent strips were used to measure canine whole blood hemoglobin, and total bilirubin, glucose, cholesterol, creatinine and urea in canine, bovine, equine and feline sera. Creatine kinase and lactate dehydrogenase were assayed in canine, bovine and equine sera. The following aspects of performance are reported: within run variation determined on canine samples, between run variation using a commercial control, correlations between dry reagent and wet reagent...
Natural protease inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis. An electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors is reported. This assay is particularly suited for investigations of crude biological materials when specific antisera are not available. The supporting medium consists of agarose into which denatured fibrinogen is incorporated as the substrate for proteases. The processing then is divided into two steps: (1) electrophoretic resolution of the inhibitor containing material and (2) detection of the inhibitor bands through their protease inhibiting activity. The inhibitor position is thus made v...
Isolation, partial identification and quantitative determination of four guaiphenesin glucuronides in plasma and urine of the horse by high-performance liquid chromatography. The isolation, partial identification and quantitative determination of four guaiphenesin glucuronides in plasma and urine of the horse is described. The identity of the glucuronides was checked by UV and fluorescence spectrophotometry, by NMR spectrometry and by mass spectrometry after permethylation. The applicability of the procedure to pharmacokinetic studies is demonstrated.
[An analysis of reproducibility in the determination of the activity of selected enzymes in the blood serum of horses]. Single biochemical analyses can be used for the diagnosis of animal diseases only with the knowledge of the effects that may distort the single result. The study of the repeatability of analyses is described in the four basic enzymes (AST, ALP, GMT, LD), which are most frequently used for diagnosis. The experiment was conducted in a group of ten Kladrub mares. Six blood samples were taken from each of the mares within ten days. The measured values were subjected to statistical processing and repeatability coefficients (r op) were calculated. All the r op values were high (ALP--0.96, LD--0.93, ...
Characterisation of glycoproteins in the sweat of the horse (Equus caballus). The two major polypeptides H (Mr 49,000) and L (Mr 33,000) of equine sweat have been purified by gel filtration and characterised by gel electrophoresis and compositional analysis. Both H and L are glycoproteins containing sialic acid, neutral sugars, N-acetylglucosamine and N-acetylgalactosamine, but the two polypeptides differ considerably in the extent of glycosylation. H and L also differ in amino acid composition, but both contain only low levels of sulphur containing amino acids and histidine. These glycoproteins may behave as surfactants.
The identification of C-18 neutral steroids in normal stallion urine. As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yr...
Determination of flunixin in equine plasma by reversed-phase liquid chromatography. Flunixin is determined in equine plasma by liquid chromatography on LiChrosorb RP-18 with 70% methanol in phosphate buffer pH 3.1 as the eluent, with detection at 284 nm. Plasma is deproteinized with methanol and the supernatant is then injected directly into the system. With a short pre-column (5 x 3 mm i.d.), which is replaced after 25-40 injections of sample, 420 plasma samples could be analysed on one analytical column. The detection limit in plasma is 0.30 micromol/l (89 ng/ml) and the method can be used in pharmacokinetic studies.
Analysis of propionylpromazine and its metabolites in horse urine. The metabolism of propionylpromazine in the horse was studied. Although propionylpromazine is not currently approved or recommended for use in horses, it has been used illegally to alter their performance. Propionylpromazine hydrochloride was administered intramuscularly at clinical and subclinical doses. Three metabolites were detected in urine. The major metabolite was identified as 2-(1-hydroxypropyl) promazine sulfoxide. The detection of this metabolite in routine drug testing has been described.
A distinct environment for iron (III) in the complex with horse spleen apoferritin observed by x-ray absorption spectroscopy. Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-spec...
Studies on prolactin: conformational comparison of human, equine, and porcine pituitary prolactins. The conformations of human, equine, and porcine pituitary prolactins, as evidenced by various optical properties, have been compared. The alpha-helix contents of all three proteins are essentially identical to each other (60 +/- 5%), as well as to prolactins isolated from other mammalian species. Direct absorption (zero and second-order), difference absorption, fluorescence emission, and circular dichroism spectra suggest that the majority of tyrosine and tryptophan side chains in these three proteins exist in very similar microenvironments within the folded forms of the hormones. Thus, the ge...
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...
Sodium and potassium ion-dependent change in oligomerization of Na,K-ATPase in C12E8 detected by low-angle laser light scattering technique in combination with high performance porous silica-gel chromatography. Approximate molecular weights and the subunit structures of Na,K-ATPase from horse kidney were estimated by means of the combination of porous silica gel chromatography, laser light scattering (LS) and refractive index (RI) measurements in C12E8. When the enzymes were eluted with NaCl- or KCl-containing solution, 3 or 4 protein peaks, respectively were detected except that of low molecular weight range. These peaks were tentatively named Na-1, Na-2, Na-2', Na-3 (NaCl-containing eluents), K-1, K-2, K-3 (KCl-containing eluents), respectively. Na,K-ATPase and K-p-nitrophenylphosphatase activities...
Radioimmunological measurement of beta-endorphin in equine plasma. Radioimmunoassay procedures were developed and validated for the quantification of beta-endorphin (beta-EP)-like immunoreactivity in equine plasma. beta-EP could be quantitatively extracted from plasma with silicic acid powder and subsequently assayed, however, valid estimates of this hormone could also be obtained on unextracted plasma. Although beta-lipotropin (beta-LPH) cross-reacted in the assay, it was not necessary to correct for beta-LPH activity when assaying unextracted plasma because chromatographic analyses showed that 92% of the immunoreactivity in plasma extracts was similar in mo...
Studies related to the metabolism of anabolic steroids in the horse: the metabolism of 1-dehydrotestosterone and the use of fast atom bombardment mass spectrometry in the identification of steroid conjugates. The metabolism and urinary excretion of 1,2(n)-3H-1-dehydrotestosterone were studied in cross-bred gelded horses. Approximately 40% of the dose was excreted in 24 h. The steroid metabolites were extracted by Amberlite XAD-2 resin and fractionated into glucuronides and sulphoconjugates. Unchanged 1-dehydrotestosterone was the only component identified by gas chromatography mass spectrometry after solvolysis of the sulphoconjugates. Positive and negative ion fast atom bombardment mass spectra were obtained on the purified 1-dehydrotestosterone sulphoconjugate isolated from horse urine and on the...
Selective crystallization of horse isoferritins. Various precipitating agents were examined in order to crystallize horse heart and spleen ferritins. Cadmium sulfate induced the crystallization of the spleen ferritin, while 2-methyl-2,4-pentanediol and poly(ethylene glycol) only induced that of the heart ferritin. Isoelectric focusing analysis showed that the crystals grown from cadmium sulfate contained only the more acidic isoferritins, and those grown from methyl pentanediol only the less acidic isoferritins. Heart ferritin crystallizes in a cubic space group, as previously reported for spleen ferritin crystals grown from cadmium sulfate....
The equine spleen: an electron microscopic analysis. The capacity of the equine spleen to store and rapidly release as much as half the circulating blood volume after adrenergic stimulation depends upon the size of the spleen, its muscular capsule, and the distinctive structure of its red pulp. The unit, or lobule, of red pulp is a cylinder of pulp spaces organized in a reticular meshwork, supplied by a peripheral ring of arterial capillaries, and drained by a central venule. Reticular cells, which make up the meshwork of the pulp, contain an extraordinarily large complement of microfilaments and intermediate filaments and are richly innervated ...
