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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.
Tenocyte response to cyclical strain and transforming growth factor beta is dependent upon age and site of origin. The effect of strain and transforming growth factor beta on equine tendon fibroblasts (tenocytes) was assessed in vitro. Tenocytes were isolated from flexor and extensor tendons of horses from foetal to 10 years of age. These cells were cultured until confluent on collagen-coated silicone dishes. Cyclic biaxial strain of 9+/-1% was applied at 0.5 Hz for 24 hours with or without added TGFbeta1 or 3 (10 ng/ml). Proliferation and synthetic responses were dependent on the tendon of origin. Neither strain nor TGFbeta caused flexor tenocytes to proliferate significantly, while strain alone did proli...
Characterization of the role of glutathione in repin-induced mitochondrial dysfunction, oxidative stress and dopaminergic neurotoxicity in rat pheochromocytoma (PC12) cells. Repin, a major constituent in extracts of the plant Centaurea repens is thought to be the active principal responsible for the development of equine nigropallidal encephalomalacia (ENE), a fatal Parkinson-like neurodegenerative disorder in horses. Although the exact mechanism by which ingestion of this weed causes ENE is uncertain, a limited body of experimental evidence suggests a critical role for the glutathione redox system. In the present study, the mechanism of repin neurotoxicity was examined in PC12 cells with a focus on determining the role of glutathione (GSH) in repin-induced mitoch...
Feeble weekly rhythmicity in hematological, cardiovascular, and thermal parameters in the horse. The homeostatic control of physiological processes is affected by a variety of temporal programs, such as circadian rhythms, estrous cycles, and circannual rhythms. The existence of circaseptan rhythms (endogenous rhythms with the duration of a week) has been postulated but not properly verified. In this study, we compared plasma concentration of lactic acid, systolic and diastolic blood pressure, and rectal temperature in athletic horses (maintained under a weekly training schedule) with those in sedentary horses (maintained under a constant schedule throughout the week). Although exercise ha...
Molecular characterisation of fructose transport in equine small intestine. Fructose can be a suitable carbohydrate supplement for horses before and/or during endurance exercise. In comparison to glucose, the ingestion of fructose results in a lower insulin peak and less marked fluctuations in blood glucose during exercise, potentially avoiding hypoglycaemia-induced exhaustion. Objective: To assess the capacity of the equine small intestine to absorb fructose and to determine the mechanism, molecular structure and properties of equine intestinal fructose transport. Methods: Using PCR-based strategies, RNA isolated from equine small intestine and primers designed to ho...
Protopine alkaloids in horse urine. Protopine was extracted from Fumaria officinalis and purified by column chromatography. Urine samples were collected from horses and a human volunteer that had been administered either F. officinalis or protopine free base. Plant and urine samples were acetylated and analysed by GCMS after solid-phase extraction (SPE). The urinary metabolites of protopine were identified as 4,6,7,13-tetrahydro-9,10-dihydroxy-5-methyl-benzo[e]-l,3-benzodioxolo [4,5-1][2] benzazecin-12(5H)-one, 4,6,7,13-tetrahydro-10-hydroxy-9-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-1][2] benzazecin-12(5H)-one and 4,6,7,1...
3-Methoxytyramine as an indicator of dopaminergic manipulation in the equine athlete. The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the populatio...
Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry. The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion ...
Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry. The metabolism of clemastine was studied in dogs, horses, and humans after a single dose of Tavegyl. The urine collected was extracted by solid-phase extraction or hydrolyzed with beta-glucuronidase and then extracted by liquid-liquid extraction, prior to analysis for unchanged drug and phase I and II metabolites by liquid chromatography/tandem mass spectrometry. The metabolites were identified by their molecular mass and interpretation of the product ion spectra, since no standard substances were available. Unchanged drug was recovered in urine samples from dogs and humans, but not from horse...
The detection of piroxicam, tenoxicam and their metabolites in equine urine by electrospray ionisation ion trap mass spectrometry. An investigation has been conducted into the metabolism and urinary excretion of orally administered piroxicam and tenoxicam in the horse. The major component detected in urine after the administration of piroxicam was 5'-hydroxypiroxicam, which was detectable up to 24 h post-administration. Unchanged piroxicam was present only as a minor component. In contrast, unchanged tenoxicam was the major component observed after the administration of tenoxicam, being detectable for 72 h post-administration, while 5'-hydroxytenoxicam was a minor component. Phase II beta-glucuronide conjugation in each c...
Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface. The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension...
Structural investigation of pig metmyoglobin by 129Xe NMR spectroscopy. The potentiality of xenon's sensitivity to its local magnetic environment is thoroughly investigated to probe internal structural differences between pig and horse metmyoglobin (MMb). These MMb's differ by 14 amino acids. One of these, Ile142 in horse MMb, is located in the proximal cavity, which is the xenon-binding site in horse MMb, and is replaced by Met142 in pig MMb. Specific and non-specific xenon-protein interactions are investigated here by 129Xe NMR chemical shifts and relaxation rate in aqueous solutions of pig MMb as a function of the xenon and protein concentrations. The results a...
Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry. Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human pl...
Comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation. To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. Methods: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. Methods: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical var...
Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles. In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work...
Insulin sensitivity and skeletal muscle glucose transport in horses with equine polysaccharide storage myopathy. Equine polysaccharide storage myopathy (PSSM) is an inherited disorder characterized by the accumulation of glycogen and abnormal polysaccharide in muscle with normal glyco(geno)lytic enzyme activities. The purpose of this study was to evaluate in vivo insulin sensitivity and glucose excursion in PSSM using a euglycemic hyperinsulinemic clamp. In addition, the content of muscle glucose transporters (GLUT1 and GLUT4) and the insulin receptor was determined in muscle biopsies using Western blot analysis. The glycogen content was 1.8-fold higher, and isolated polysaccharide analyzed by iodine abs...
Influence of fasting and exercise on the daily rhythm of serum leptin in the horse. The hormone leptin is secreted by white adipocytes and regulates food intake and energy expenditure in rodents and humans. The goal of the present study was to investigate the existence of a daily rhythm of serum leptin in horses and its dependence on fasting and physical exercise. A robust daily rhythm of leptin was found in both athletic and sedentary horses, with a daytime trough and a peak in the dark phase. While physical exercise never induced changes in circulating leptin, fasting reliably affected serum leptin levels. Food deprivation did not abolish the daily rhythm of serum leptin, b...
Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season. The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory fol...
Anticorrelated motions as a driving force in enzyme catalysis: the dehydrogenase reaction. Molecular dynamics and cross-correlation analysis of the horse liver alcohol dehydrogenase HLADH.NAD(+).PhCH(2)O(-) complex has established anticorrelated motions between the NAD(+)-binding domain and other portions of the enzyme. Four pairs of anticorrelated interactions are (i and ii) cofactor-binding domain: C(alpha) of V292 and the CG1 of V203 with C7 of PhCH(2)O(-); (iii) cofactor-binding domain: amide carbonyl oxygen of I318 with amide N of H67; and (iv) cofactor domain: C(alpha) of T178 with carbonyl oxygen of L141. The average distances between pairs are 9.2 A for i, 8.2 A for ii, 14.7...
An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone. In articular joints, the forces generated by locomotion are absorbed by the whole of cartilage, subchondral bone and underlying trabecular bone. The objective of this study is to test the hypothesis that regional differences in joint loading are related to clear and interrelated differences in the composition of the extracellular matrix (ECM) of all three weight-bearing constituents. Methods: Cartilage, subchondral- and trabecular bone samples from two differently loaded sites (site 1, dorsal joint margin; site 2, central area) of the proximal articular surface of 30 macroscopically normal equ...
The diagnostic and prognostic value of alkaline phosphatase activity in serum and peritoneal fluid from horses with acute colic. Alkaline phosphatase (ALP) is an enzyme present in intestinal mucosa, bile, bone, and renal tubule cells. We sought to assess the diagnostic and prognostic relationships of total ALP (ALPt) activity and that of intestine-derived ALP (ALPi) in serum and peritoneal fluid of 126 horses with colic. ALPt and ALPi activities were measured in both serum and peritoneal fluid by using both standard and L-phenylalanine-based buffers, respectively. Neither ALPt nor ALPi activity were useful in classifying type or severity of intestinal damage. ALPt and ALPi activities in peritoneal fluid were lowest in h...
Red blood cell deformability and aggregation behaviour in different animal species. Comparative animal studies showed the wide variation of whole blood and plasma viscosity, and erythrocyte aggregation among mammalian species. Whole blood viscosity and red blood cell aggregation is influenced by red cell fluidity. To evaluate differences in erythrocyte deformability in mammals, three species were investigated, whose erythrocytes have a different aggregation property: horse, as a species with high, dog with medium, and sheep with almost unmeasurable aggregation tendency. Erythrocyte deformability was tested ektacytometrically (Elongation Index [EI], LORCA, Mechatronics, Hoorn,...
Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes. Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recen...
Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving sta...
Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells. Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and e...
Effects of feeding meals with various soluble-carbohydrate content on muscle glycogen synthesis after exercise in horses. To determine effects of feeding diets with various soluble-carbohydrate (CHO) content on rates of muscle glycogen synthesis after exercise in horses. Methods: 7 fit horses. Methods: In a 3-way crossover study, horses received each of 3 isocaloric diets (a high soluble CHO [HC] diet, a low soluble CHO [LC] diet, or a mixed soluble CHO [MC] diet). For each diet, horses were subjected to glycogen-depleting exercise, followed by feeding of the HC, LC, or MC diet at 8-hour intervals for 72 hours. Results: Feeding the HC diet resulted in a significantly higher glycemic response for 72 hours and sign...
Electrospray ionization mass spectrometric characterization and quantitation of xanthine derivatives using isotopically labelled analogues: an application for equine doping control analysis. Isotope-dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance-enhancing drugs. Theobromine has to exceed a threshold level of 2 microg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodome...
Investigation of the expression and localization of glucose transporter 4 and fatty acid translocase/CD36 in equine skeletal muscle. To investigate the expression and localization of glucose transporter 4 (GLUT4) and fatty acid translocase (FAT/CD36) in equine skeletal muscle. Methods: Muscle biopsy specimens obtained from 5 healthy Dutch Warmblood horses. Methods: Percutaneous biopsy specimens were obtained from the vastus lateralis, pectoralis descendens, and triceps brachii muscles. Cryosections were stained with combinations of GLUT4 and myosin heavy chain (MHC) specific antibodies or FAT/CD36 and MHC antibodies to assess the fiber specific expression of GLUT4 and FAT/CD36 in equine skeletal muscle via indirect immunofl...
Expression of the full-length and alternatively spliced equine luteinizing hormone/chorionic gonadotropin receptor mRNAs in the primary corpus luteum and fetal gonads during pregnancy. The full-length equine luteinizing hormone/chorionic gonadotropin (LH/CG) receptor (eLH/CG-RA) cDNA and two alternatively spliced isoforms (eLH/CG-RB,C) were isolated from luteal tissue and characterized using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends. The 680-amino acid full sequence of eLH/CG-RA displayed 87-92% homology with other mammalian LH/CG-Rs. The eLH/CG-RB and eLH/CG-RC cDNA isoforms were truncated from the 3'-end of exon X: eLH/CG-RB spliced out of frame into the last exon whereas eLH/CG-RC contained an in-fram...
Effect of matrix depleting agents on the expression of chondrocyte metabolism by equine chondrocytes. This study was carried out to investigate the effect of two enzymes (collagenase and chondroitinase) and two cytokines/metabolites (interleukin-1beta and retinoic acid) of known catabolic activity on the expression of cartilage metabolism/phenotype in equine articular cartilage. Articular cartilage explants from 11 horses (5-13 years old) were treated for 48 h and assayed for total sulphated glycosaminoglycan (GAG), the incorporation of 35S-sulphate, collagen degradation and mRNA expression of the proteoglycans collagen II, collagen IIA, collagen III, collagen IX, collagen X, collagen XI and g...
Proteomic tools to characterize the protein fraction of Equidae milk. The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, wit...
