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Topic:Biochemistry
The study of biochemistry in horses encompasses the chemical processes and substances that occur within equine organisms. This field investigates the molecular interactions and pathways that are fundamental to horse physiology, including metabolism, enzyme activity, and genetic expression. Key areas of interest include the examination of metabolic disorders, nutrient absorption, and the biochemical basis of muscle function and energy production. Researchers utilize biochemical analysis to understand health and disease mechanisms in horses, contributing to the development of diagnostic tools and therapeutic strategies. This page gathers peer-reviewed studies and scholarly articles that explore various biochemical processes and their implications for equine health and performance.
Changes in MMP-2 and -9 activity and MMP-8 reactivity after amphotericin B induced synovitis and treatment with bufexamac. The objective here was to evaluate the acute effects of induced arthritis on synovial fluid (SF) levels of matrix metalloproteinases (MMP) -2, -8 and -9 in horses. To evaluate MMP-2 and -9 activities and the effect of non-steroidal anti-inflammatory drug (NSAID) bufexamac during remission from acute arthritis. Aseptic arthritis was induced in 24 Standardbred horses using 20 mg of amphotericin B as a single intra-articular (IA) injection in the right intercarpal joint. After 1 week and 2 weeks, horses were treated intra-articularly with 10, 20, or 40 mg of bufexamac suspension or with sterile s...
Transcriptional and proteolytic regulation of the insulin-like growth factor-I system of equine articular chondrocytes by recombinant equine interleukin-1beta. Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-...
Urine cartilage oligomeric matrix protein (COMP) measurement is useful in discriminating the osteoarthritic Thoroughbreds. To quantify the urinary concentration of cartilage oligomeric matrix protein (COMP), and to evaluate the relationship between urinary COMP concentration and the catabolic activity of synovial fluid (SF) in diseased horses. Methods: COMP in horse urine was detected by immunoblotting with a monoclonal antibody (mAb; 14G4) raised against equine COMP from articular cartilage. Urine and serum samples were obtained from 83 Thoroughbred horses with aseptic joint diseases (AJD, 79 horses) or septic joint diseases (SJD, four horses) at the time of anesthesia induction, and samples of SF were obtained d...
Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes. Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity. ...
Serum concentrations of calcium, phosphorus, magnesium and calciotropic hormones in donkeys. To provide reference values for serum biochemical variables that are used for evaluation of mineral metabolism in donkeys and compare values with those in horses. Methods: 18 donkeys and 18 horses. Methods: Total calcium (tCa), total magnesium (tMg), and inorganic phosphorus (P) concentrations were measured in serum samples via spectrophotometry. Ionized calcium (iCa) and magnesium (iMg) concentrations were quantified with selective electrodes. By use of a micropartition system, tCa and tMg were fractionated to separate protein-bound (pCa, pMg) and ultrafiltrable fractions. Complexed calcium (...
Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin. Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react ...
Mapping of donor and recipient site properties for osteochondral graft reconstruction of subchondral cystic lesions in the equine stifle joint. To improve osteochondral graft reconstruction of subchondral cystic lesions in the medial and lateral femoral condyles by matching the material properties of donor and recipient sites. Objective: To measure biomechanical and biochemical parameters that influence the function and healing of osteochondral grafts used to reconstruct subchondral cystic lesions. Objective: Suitable donor sites are available within the stifle joint for reconstructing the femoral condyles, despite considerable regional property variation. Methods: Fifty-six osteochondral cores were harvested from 6 distal femurs for ...
Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids. Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, d...
Comparison of 2 endothelin-receptor antagonists on in vitro responses of equine palmar digital arterial and venous rings to endothelin-1. The goals of this study were to determine the concentration-response (C-R) relationship of endothelin-1 (ET-1), compare 2 ET-receptor antagonists and determine the antagonist concentrations that block the vasomotor effects of ET-1, and compare the effectiveness of ET-1 and previously studied vasoconstrictors in equine palmar digital arterial and venous rings in vitro. Vessel rings from 8 nonlaminitic horses were placed in Tyrode's solution, 1 side fixed to the floor of an organ bath and the other side fixed to a force-displacement transducer. Two separate studies were conducted: (I) incubation...
Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia. The aim of the current study was to clone the equine adenosine A(2A) receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) ...
Cloning and pharmacological characterization of the equine adenosine A3 receptor. The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and an...
Cytochemical characterization of glycoconjugates in the apocrine glands of the equine scrotal skin. Cytochemistry of glycoconjugates in the apocrine glands in the scrotal skin of the horse was studied using cytochemical methods for electron microscopy, particularly lectin cytochemistry. The secretory cells possessed a variable number of secretory vesicles, a well-developed Golgi apparatus, and abundant cisternae of the rough endoplasmic reticulum. Additionally, the basolateral plasma membrane formed numerous interdigitating folds. Glycoconjugates with vicinal diol groupings were present predominantly in the secretory vesicles, the Golgi apparatus, the surface coat of the plasma membrane, and...
Temporal relationship between proliferating and apoptotic hormone-producing and endothelial cells in the equine corpus luteum. The temporal relationship between endothelial cell death, vascular regression and the death of hormone-producing cells in the mare has not been established. To determine the dynamics of cell proliferation and death throughout the luteal phase, corpora lutea were studied at the early, mid- and late luteal phase, and after treatment with cloprostenol in the mid-luteal phase to induce premature luteolysis. Changes in cell proliferation and apoptosis were investigated utilising specific markers (phosphorylated histone-3 and activated caspase-3 respectively). Histone-3 positive cells were most abun...
von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen. Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively. Objective: Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen. Results: In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-...
Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique. The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon re...
Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses. The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive C...
Neurologic signs and hyperammonemia in a horse with colic. A 23-year-old Thoroughbred gelding was referred for the evaluation of acute onset of ataxia and depression, and a 2-day history of fever. On physical examination, the gelding was profoundly depressed and 10-12% dehydrated. The horse appeared very unstable, with a wide-based stance in the hind limbs, severe symmetric ataxia in all 4 limbs, and proprioceptive deficits in both hind limbs. Nasogastric intubation produced 4 L of brown, fetid reflux, and rectal examination revealed mild small intestinal and cecal distention. Hematologic abnormalities included neutropenia with toxic change, compatibl...
Oral and intravenous carbohydrate challenges decrease active ghrelin concentrations and alter hormones related to control of energy metabolism in horses. This study tested the hypothesis that grain and intravenous dextrose challenges would alter plasma concentrations of active ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in Standardbred mares. To deliver 0.5 g of glucose (dextrose solution for the intravenous test)/kg of BW, mares received intravenous dextrose (50% solution) or oral grain administration in 2 trials. In response to the oral grain challenge, plasma glucose and insulin concentrations increased (P < 0.001) by 56 and 802%, respectively. Plasma ghrelin concentration initially decreased (P < 0.001) by 40%, then s...
Evidence for the expression and enzymatic activity of haem oxygenase-1 in the lungs of horses. The aim of this study was to characterise the expression of haem oxygenase-1 (HO-1) in healthy lung tissue from horses and to measure its activity. Samples of lung tissue were collected from six horses euthanased for reasons other than respiratory disease. HO-1 expression and activity were detected in type II alveolar epithelial cells, macrophages and neutrophils in all the samples examined. The activity was dependent on the presence of NADPH and inhibited quantitatively by the addition of increasing concentrations of a competitive inhibitor of HO-1, tin mesoporphyrin IX.
A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase. Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate ...
Extraction and detection of mRNA from horsehair. After RNA extraction from horsehair shafts and roots, the mRNAs of beta-actin, muscle-type phosphofructokinase, and transforming growth factor-beta1 were detected by reverse transcription polymerase chain reaction assay. Low amounts of RNA were present in the horsehair. These specific mRNA transcripts were readily detected when more than three hair roots were used. However, detection of the mRNA transcripts was difficult in the hair shaft. These findings indicate that the small amounts of residual RNA in horsehair roots can be utilized as samples for molecular biological analysis.
Thrombospondin-4 and cartilage oligomeric matrix protein form heterooligomers in equine tendon. Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with...
Assessment of the catabolic effects of interleukin-1beta on proteoglycan metabolism in equine cartilage cocultured with synoviocytes. To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. Methods: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. Methods: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectropho...
Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid? In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-3...
New staining methods for sperm evaluation estimated by microscopy and flow cytometry. New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing ac...
Determination of acrosin amidase activity in equine spermatozoa. Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each o...
Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81...
Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility. Semen samples were collected from six fertile stallions and seven stallions with unexplained infertility. Percentages of motile sperm (77.5 +/- 11.3 versus 67.5 +/- 12.2, P = 0.2), and progressively motile sperm (70.8 +/- 13.6 versus 60.7 +/- 14.0, P = 0.2) were similar between fertile and subfertile stallions, respectively. Morphologic characteristics in ejaculates of control and affected stallions (% normal: 60.2 +/- 18.2 versus 52.9 +/- 11.3, P = 0.4; % abnormal heads 7.3 +/- 4.8 versus 12.1 +/- 5.0, P = 0.11; and % abnormal acrosomes 1.6 +/- 2.1 versus 3.0 +/- 3.4, P = 0.4) did not differ....
Detection of 17alpha-hydroxyprogesterone caproate in equine plasma by gas chromatography/tandem mass spectrometry. A method was developed for the analysis of the synthetic progestin 17alpha-hydroxyprogesterone caproate in equine plasma following its administration by intramuscular injection. The method employed a reversed-phase solid-phase extraction followed by enol-trimethylsilylation and analysis by gas chromatography/tandem mass spectrometry. The intact ester was detectable in the plasma for up to 2 weeks after a single therapeutic dose, and was found to be stable in equine whole blood for at least 2 months.
