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Topic:Enzymes
Enzymes are biological catalysts that facilitate biochemical reactions in horses by lowering the activation energy required for these processes. They are involved in various physiological functions, including digestion, metabolism, and cellular repair. Common enzymes in equine biology include amylase, lipase, and lactate dehydrogenase, each playing a specific role in the breakdown of nutrients and energy production. The activity and concentration of these enzymes can vary in response to different physiological and pathological conditions, serving as potential indicators in veterinary diagnostics. This page compiles peer-reviewed research studies and scholarly articles that explore the function, regulation, and clinical implications of enzymes in equine health.
Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro. Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Co...
Role of the equine CYP3A94, CYP3A95 and CYP3A97 in ketamine metabolism in presence of medetomidine, diazepam and methadone studied by enantioselective capillary electrophoresis. The anesthetic ketamine is often combined with analgesics and benzodiazepines in equine medicine. Therefore, drug-drug interactions are possible. Enzyme kinetics for ketamine N-demethylation were determined using equine CYP3A94, CYP3A95 and CYP3A97, and the effect of medetomidine, diazepam and methadone on the ketamine metabolism was studied in vitro. Ketamine was incubated with the CYPs or equine liver microsomes (ELM) alone or in presence of medetomidine, diazepam and/or methadone for different times. Norketamine levels were determined using enantioselective capillary electrophoresis (CE) wi...
Validation of a paraoxon-based method for measurement of paraoxonase (PON-1) activity and establishment of RIs in horses. Paraoxonase-1 (PON-1) is an antioxidant compound that is considered a negative acute phase protein. No information on the analytic performance of the paraoxon method for measuring PON-1 in horse serum is available. Objective: The aim of this study was to validate a paraoxon-based method to measure PON-1 in horses and to establish RIs in healthy horses and foals. Methods: Horses and foals classified as healthy after physical examination and routine biochemistry were used in the study. Serum PON-1 activity was measured with an automated spectrophotometer and an enzymatic method validated in othe...
Effects of stanozolol on normal and IL-1β-stimulated equine chondrocytes in vitro. Intra-articular administration of stanozolol has shown promising results by improving the clinical management of lameness associated with naturally-occurring osteoarthritis (OA) in horses, and by decreasing osteophyte formation and subchondral bone reaction in sheep following surgically induced OA. However, there is limited evidence on the anti-inflammatory and modulatory properties of stanozolol on articular tissues. The objective of the current study was to evaluate the effects of stanozolol on chondrocyte viability and gene expression in normal equine chondrocytes and an inflammatory in vit...
Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding-induced endometritis and the effect of a specific iNOS inhibitor in vitro. Emerging research suggests that the nitric oxide system may play a role in persistent breeding-induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose-dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activ...
Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation. The joint synovium consists of a heterogeneous cell population, chiefly comprised of macrophages, and fibroblast-like synoviocytes (FLS). An inter-species co-culture model was developed to examine interactions between these cells. Equine FLS and the canine macrophage line DH82 were differentially labeled using fluorescent markers and results from direct co-culture compared with those from both indirect co-culture, and conditioned media experiments. The transcript expression of IL-1β, IL-6, ADAMTS4, and ADAMTS5 in each cell type were determined using species-specific qPCR assays. Lipopolysacch...
Expression of indoleamine 2,3-dioxygenase 1 as transcript and protein in the healthy and diseased equine endometrium. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) acts immunomodulatory and restricts bacterial growth. In the uterus of women and mice, it likely contributes to tissue homeostasis and disease pathogenesis. Pregnancy failure in mares is often caused by endometritis and endometrosis. The pathogenesis of nonsuppurative endometritis and endometrosis is still uncertain. To the authors' knowledge, no information on IDO1 expression in the equine endometrium is published. Aim of this study was to examine the presence of IDO1 as transcripts and proteins in the healthy and diseased endometrium of 25 mare...
Submaximal exercise training, more than dietary selenium supplementation, improves antioxidant status and ameliorates exercise-induced oxidative damage to skeletal muscle in young equine athletes. Exercise is associated with increased production of reactive oxygen species (ROS) as metabolism is upregulated to fuel muscle activity. If antioxidant systems become overwhelmed, ROS can negatively affect health and performance. Adaptation to exercise through regular training has been shown to improve defense against oxidative insult. Given selenium's role as an antioxidant, we hypothesized that increased Se intake would further enhance skeletal muscle adaptations to training. Quarter Horse yearlings (18 ± 0.2 mo; 402 ± 10 kg) were randomly assigned to receive either 0.1 or 0.3 mg Se/kg DM a...
Interaction between the sequence of feeding of hay and concentrate, and boiling of barley on feed intake, the activity of hydrolytic enzymes and fermentation in the hindgut of Arabian mares. The interaction between the sequence of feeding of hay and concentrate and the hydrothermal processing of barley in alleviating concentrate effects on intake, and hindgut fermentation in horses was tested. Six Arabian mares (4-10 years of age, 410 ± 35 kg body weight) were used to evaluate the effects of feeding sequence (FS) and type of barley (TB) on intake, and faecal volatile fatty acids (VFA), activities of α-amylase (AA: EC 3.2.1.1), carboxymethyl cellulase (CMCase: EC 3.2.1.4), microcrystalline cellulase (MCCase: EC 3.2.1.91) and general filter paper degrading activity (FPD). Mare...
Inhibition of 5α-reductase alters pregnane metabolism in the late pregnant mare. In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metab...
Intestinal and hepatic contributions to the pharmacokinetic interaction between gamithromycin and rifampicin after single-dose and multiple-dose administration in healthy foals. Standard treatment of foals with severe abscessing lung infection caused by Rhodococcus equi using rifampicin and a macrolide antibiotic can be compromised by extensive inhibition and/or induction of drug metabolising enzymes (e.g. CYP3A4) and transport proteins (e.g. P-glycoprotein), as has been shown for rifampicin and clarithromycin. The combination of rifampicin with the new, poorly metabolised gamithromycin, a long-acting analogue of azithromycin and tulathromycin with lower pharmacokinetic interaction potential, might be a suitable alternative. Objective: To evaluate the pharmacokinetic ...
Characterization of equine GST A3-3 as a steroid isomerase. Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ-androstene-3,17-dione to Δ-androstene-3,17-dione and of Δ-pregnene-3,20-dione to Δ-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the ...
Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State. The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, NVCWAK, which differs from that of papain (CGSCWAFS). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited s...
Effects of body condition score (BCS) on steroid- and eicosanoid-metabolizing enzyme activity in various mare tissues during winter anoestrus. The objective of this study was to determine the activity of steroid- and eicosanoid-metabolizing enzymes in horses with varying BCSs. The BCSs of twenty non-pregnant, anoestrous mares were determined prior to euthanasia, and tissue samples were collected from the liver, kidney, adrenal gland, ovary and endometrium. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activities were determined using luminogenic substrates. The MIXED procedure of SAS was used to test the effect of BCS on enzyme activity and differences between tissues. Activ...
Steroidogenic enzyme activities in the pre- and post-parturient equine placenta. Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP a...
Inhibition of microsomal prostaglandin E-synthase-1 (mPGES-1) selectively suppresses PGE2 in an in vitro equine inflammation model. Inhibition of prostaglandin E2 (PGE2) production effectively limits inflammation in horses, however nonspecific prostaglandin blockade via cyclooxygenase (COX) inhibition elicits deleterious gastrointestinal side effects in equine patients. Thus, more selective PGE2 targeting therapeutics are needed to treat inflammatory disease in horses. One potential target is microsomal prostaglandin E-synthase-1 (mPGES-1), which is the terminal enzyme downstream of COX-2 in the inducible PGE2 synthesis cascade. This enzyme has yet to be studied in equine leukocytes, which play a pivotal role in equine inf...
Equine fetal adrenal, gonadal and placental steroidogenesis. Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher bu...
Effect of conservative mutations (L94V and L94I) on the structure and stability of horse cytochrome c. A sequence alignment of horse cytochrome c (cyt c) with all known cyts c shows that Leu at position 94 is conserved, except in 14 species which have either Val or Ile at this position. It is also known that Leu94 of the mammalian cyt c plays an important role in folding and stability. The important question here is as to what will happen in terms of folding and stability if Leu94 of the mammalian cyt c is substituted by Val or Ile. To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c. The purified L94V and L94I mutants under native condition (pH 6.0...
Spectroscopic Study of the Interaction between Horse Heart Myoglobin and Zirconium(IV)-Substituted Polyoxometalates as Artificial Proteases. A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and H and P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 Zr -...
Rapid Mechanistic Evaluation and Parameter Estimation of Putative Inhibitors in a Single-Step Progress-Curve Analysis: The Case of Horse Butyrylcholinesterase. Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing ...
Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one pos...
Evaluation of an in vitro system to simulate equine foregut digestion and the influence of acidity on protein and fructan degradation in the horse’s stomach. The aim of this study was to improve an in vitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated in vitro (Part 1). The effect of different pH values on in vitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass ...
Function of bacteriophage G7C esterase tailspike in host cell adsorption. Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and funct...
High metabolic activity of tissue-nonspecific alkaline phosphatase not only in young but also in adult bone as demonstrated using a new histochemical detection protocol. Tissue-nonspecific alkaline phosphatase (TNAP) is playing a key role in bone calcification, as has been demonstrated in different mammalian species including human and rodents. However, to investigate age-related changes during life history, histochemical demonstration of TNAP is severely hampered, particularly in the elderly, by technical difficulties associated with sectioning calcified tissue. Sufficient fixation must precede decalcification since poorly fixed bone tissue is exposed to the deleterious effects of decalcification reagents. In order to find a method that would allow cryosectio...
Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa. Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, ...
Comparison and validation of ELISA assays for plasma insulin-like growth factor-1 in the horse. Insulin-like growth factor-1 (IGF-1) plays several important physiological roles, and IGF-related pathways have been implicated in developmental osteochondral disease and endocrinopathic laminitis. This factor is also a downstream marker of growth hormone activity and its peptide mimetics. Unfortunately, previously used assays for measuring equine IGF-1 (radioimmunoassays and ELISAs) are no longer commercially available, and many of the kits on the market give poor results when used on horse samples. The aim of the present study was to compare three different ELISA assays (two human and one ho...
Effect of the α2 -receptor agonists medetomidine, detomidine, xylazine, and romifidine on the ketamine metabolism in equines assessed with enantioselective capillary electrophoresis. The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Necrotizing hepatitis associated with Clostridium novyi in a pony in western Canada. Severe icterus, peritoneal effusion, localized fibrinous peritonitis, and necrotizing hepatitis were found at necropsy of a 20-year-old female pony with a history of acute onset depression, inappetence, fever, and marked elevation in hepatic enzymes. Gross pathology, histopathology, and immunohistochemistry were compatible with a diagnosis of clostridial hepatitis caused by Clostridium novyi-group bacteria. This is believed to be the first reported case of clostridial hepatitis in an equid in Canada, and only the third report of this rare disease in North America. Hépatite nécrosante associÃ...
Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4. Cytochrome P450 enzymes (CYPs) are responsible for the phase I metabolism of drugs, xenobiotics and endogenous substances. Knowledge of single CYPs and their substrates is important for drug metabolism, helps to predict adverse effects and may prevent reduced drug efficacy in polypharmacy. In this study, three equine isoenzymes of the 3A subfamily, the equine flavoprotein NADPH-P450 oxidoreductase (POR), and the cytochrome b5 (CYB5) were cloned, sequenced and heterologously expressed in a baculovirus expression system. Testosterone, the standard compound for characterization of the human CYP3A...
Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks. To investigate various biochemical components of seminal plasma in Marwari stallions and Poitou Jacks and to find out their correlation with that of the seminal characteristics. Methods: In this study, semen was collected from six Marwari stallions and six Poitou jacks aged from 4 to 6 years and with known fertility status. The semen collection from the stallions were collected during the breeding season, i.e., between the months of April and June. From the collected semen ejaculates, we estimated the values of some biochemical components, viz., total protein content, total lipid content, and ...
