Short-term effects of canagliflozin on glucose and insulin responses in insulin dysregulated horses: A randomized, placebo-controlled, double-blind, study.

BACKGROUND: Decreasing hyperinsulinemia is crucial in preventing laminitis in insulin dysregulated (ID) horses. Complementary pharmacological treatments that efficiently decrease postprandial hyperinsulinemia in ID horses are needed. OBJECTIVES: Compare short-term effects of canagliflozin vs placebo on glucose and insulin responses to an oral sugar test (OST) as well as the effects on body weight and triglyceride concentrations in horses with ID. ANIMALS: Sixteen privately-owned ID horses. METHODS: A single-center, randomized, double-blind, placebo-controlled, parallel design study. The horses were randomized (ratio 1:1) to either once daily PO treatment with 0.6 mg/kg canagliflozin or placebo. The study consisted of an initial 3-day period for obtaining baseline data, a 3-week double-blind treatment period at home, and a 3-day follow-up period similar to the initial baseline period but with continued double-blind treatment. Horses were subjected to an 8-sample OST in the morning of the third day on both visits. RESULTS: Maximal geometric least square (LS) mean insulin concentration (95% confidence interval [CI]) during the OST decreased after 3 weeks of canagliflozin treatment compared with placebo (83.2; 55.4-125.0 vs 215.2; 143.2-323.2 µIU/mL). The geometric LS mean insulin response (insulin AUC0-180 ) for canagliflozin-treated horses was >66% lower compared with placebo. Least square mean body weight decreased by 11.1 (4-18.1) kg and LS mean triglyceride concentrations increased by 0.99 (0.47-1.5) mmol/L with canagliflozin treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Canagliflozin is a promising drug for treatment of ID horses that requires future studies.

Carbohydrate pellets to assess insulin dysregulation in horses.

BACKGROUND: A glycemic challenge test is used for the diagnosis of insulin dysregulation (ID) in horses and ponies. Different forms of the test exist where the administrative route and dose of glucose vary, which makes interpretation of results challenging. HYPOTHESIS/OBJECTIVES: To evaluate the palatability of, and blood glucose and insulin responses to, carbohydrate pellets fed as an oral glucose test (OGT), and to establish the diagnostic threshold for ID when using the pellets. ANIMALS: University and privately-owned horses and ponies (n = 157) comprised of 31 breeds and both sexes. METHODS: Multicenter cohort study. A custom-produced glycemic pellet was offered for free intake at 0.5 g/kg BW soluble carbohydrate and serum insulin and blood glucose concentrations measured before and after (60, 120, and 180 minutes) the pellets were offered. Pellet acceptance and intake time (those that finished within 10 minutes) were determined to assess palatability. RESULTS: The pellets were palatable to 132/157 animals, and ponies found the pellets more (P = .004) palatable than horses. The median intake time (4 [3-6] minutes) was positively correlated with acceptance grade (r = .51; P < .0001). Consumption of the pellets elicited peak blood glucose (6.6 [5.8-7.8] mmol/L) and serum insulin (40.5 [19-99.8] µIU/mL) responses at 120 minutes. At 120 minutes the optimal cut-off was 83 µIU/mL (95% CI: 70-99 µIU/mL) for the IMMULITE 2000XPi assay. CONCLUSIONS AND CLINICAL IMPORTANCE: The pellets were palatable and a suitable, novel carbohydrate source for the OGT.

The disposition of trimethoprim and sulfadiazine in neonatal foals after intravenous administration.

BACKGROUND: Septicaemia in the neonatal foal is caused by both Gram positive and Gram negative bacteria. The life-threatening nature of this condition requires treatment to be initiated with broad spectrum antimicrobial drugs pending antimicrobial susceptibility testing. Potentiated sulphonamides, for example, trimethoprim combined with sulfadiazine, could be clinically relevant options but their pharmacokinetics in the neonatal foal are unknown. OBJECTIVES: To describe the plasma disposition of trimethoprim and sulfadiazine in neonatal foals and to relate the results to patterns in the minimum inhibitory concentration (MIC) for Escherichia coli, a recognized pathogen in neonatal foal sepsis. METHOD: A total of five doses of trimethoprim (2.5 mg/kg) and sulfadiazine (12.5 mg/kg) were administered intravenously every 12 h to eight neonatal foals that were 3 days old at inclusion. A non-linear mixed effects model was fitted to the trimethoprim and sulfadiazine experimental data. The 24 h area under the free plasma trimethoprim and sulfadiazine concentration-time curves (fAUC) and the pharmacokinetic/pharmacodynamik (PK/PD)-index fAUC/MIC was calculated to evaluate the potential clinical benefits of the administered dose. RESULTS: For trimethoprim, the typical values were 1.99 L/kg, 0.33 L/h·kg and 4.2 h for the apparent volume of distribution, clearance and terminal half-life, respectively. The 24 h fAUC for trimethoprim was 11.3 µg·h/ml (7.2-15.2) and the fAUC/MIC ratio for E. coli was 23 (16.4-29.2) (population mean (range)). For sulfadiazine, the typical values were 0.61 L/kg, 0.09 L/h·kg and 5.3 h for the apparent volume of distribution, clearance and terminal half-life, respectively. The 24 h fAUC for sulfadiazine was 246.8 µg·h/ml (175.6-335.4). CONCLUSION: For trimethoprim, the plasma exposure is insufficient in some foals to successfully treat bacterial infections with an MIC-value of 0.5 µg/ml using the studied dosing regimen.

Evaluation of fasting plasma insulin and proxy measurements to assess insulin sensitivity in horses.

BACKGROUND: Proxies are mathematical calculations based on fasting glucose and/or insulin concentrations developed to allow prediction of insulin sensitivity (IS) and ß-cell response. These proxies have not been evaluated in horses with insulin dysregulation. The first objective of this study was to evaluate how fasting insulin (FI) and proxies for IS (1/Insulin, reciprocal of the square root of insulin (RISQI) and the quantitative insulin sensitivity check index (QUICKI)) and ß-cell response (the modified insulin-to-glucose ratio (MIRG) and the homeostatic model assessment of ß-cell function (HOMA-ß)) were correlated to measures of IS (M index) using the euglycemic hyperinsulinemic clamp (EHC) in horses with insulin resistance (IR) and normal IS. A second objective was to evaluate the repeatability of FI and proxies in horses based on sampling on consecutive days. The last objective was to investigate the most appropriate cut-off value for the proxies and FI. RESULTS: Thirty-four horses were categorized as IR and 26 as IS based on the M index. The proxies and FI had coefficients of variation (CVs) ≤ 25.3 % and very good reliability (intraclass correlation coefficients ≥ 0.89). All proxies and FI were good predictors of the M index (r = 0.76-0.85; P < 0.001). The proxies for IS had a positive linear relationship with the M index whereas proxies for ß-cell response and FI had an inverse relationship with the M index. Cut-off values to distinguish horses with IR from horses with normal IS based on the M index were established for all proxies and FI using receiver operating characteristic curves, with sensitivity between 79 % and 91 % and specificity between 85 % and 96 %. The cut-off values to predict IR were < 0.32 (RISQI), < 0.33 (QUICKI) and > 9.5 µIU/mL for FI. CONCLUSIONS: All proxies and FI provided repeatable estimates of horses' IS. However, there is no advantage of using proxies instead of FI to estimate IR in the horse. Due to the heteroscedasticity of the data, proxies and FI in general are more suitable for epidemiological studies and larger clinical studies than as a diagnostic tool for measurement of IR in individual horses.

The effect of diet-induced obesity and pasture on blood pressure and serum cortisol in Standardbred mares.

BACKGROUND: Obesity is associated with insulin resistance, vascular dysfunction and altered cortisol metabolism both in humans and in horses. OBJECTIVES: Evaluate the effect of weight gain induced by a haylage diet low in nonstructural carbohydrates (NSC) on insulin sensitivity, blood pressure and serum cortisol concentrations. STUDY DESIGN: In vivo experiment. METHODS: Nine adult Standardbred mares fed a fat supplemented haylage diet at 250% of the horses' daily metabolisable energy requirements for 22 weeks. Horses were then turned out on pasture for 4 weeks. Insulin sensitivity (SICLAMP ) was measured before and after weight gain and after 4 weeks of pasture using the euglycemic hyperinsulinaemic clamp (EHC) method. Body condition score (BCS), blood pressure and serum cortisol were monitored throughout the study. All data were analysed using the linear mixed model procedure. Values of P < 0.05 were considered as statistically different. RESULTS: All horses became obese during the weight gain period (BCS> 7). Mean arterial blood pressure (MAP) increased during the weight gain period and was significantly higher than initial values at the end of the weight gain period (78 ± 3 mm Hg vs 92 ± 3 mmHg). MAP remained increased on pasture (93 ± 3 mmHg). SICLAMP was unaffected by weight gain 0.9 ± 0.1 vs 1.0 ± 0.1 ([mg/kg/min × 103 ]/[µIU/mL × mmol/L])) but improved after pasture (1.6 ± 0.1 ([mg/kg/min × 103 ]/ [mU/L]). Serum cortisol concentrations increased during the weight gain period (80 ± 9 nmol/L vs 112 ± 9 nmol/L) and remained increased during pasture. MAIN LIMITATIONS: Limited number of horses and no control group. CONCLUSIONS: Obesity was associated with a linear increase in blood pressure and an increase in serum cortisol that was not associated with insulin sensitivity.

Hematologic, prostaglandin F2α -metabolite, serum amyloid A, and serum iron changes in horses with experimentally induced endotoxemia.

BACKGROUND: Endotoxemia is a common and severe disease of horses. Most previous studies have monitored changes caused by a bolus dose of endotoxin over short time periods. OBJECTIVES: We aimed to describe inflammatory responses to endotoxin with inflammatory and hematologic markers monitored over a longer time than has been performed in the past using more prolonged endotoxin exposures. METHODS: Escherichia coli O55:B5 endotoxin was administered as a 6-hour continuous intravenous infusion of lipopolysaccharide (LPS) to eight horses. Blood cell counts, and prostaglandin F2α -metabolite (PGM), serum amyloid A (SAA), and serum total iron concentrations were monitored for up to 3 or 6 days. RESULTS: An immediate and severe decrease in neutrophils and monocytes occurred in all horses, which subsequently changed to a moderate to strong neutrophilia and monocytosis that persisted for more than 78 hours postinfusion (PI) of LPS. Lymphocyte and eosinophil numbers decreased gradually and then normalized after 66- and 78-hours PI, respectively. Mild to moderate, biphasic thrombocytopenia occurred. A pronounced, transient increase in PGM occurred between 1 and 7 hours, peaking at 2 hours. Serum amyloid A began to increase after 6 hours PI and remained elevated after 72 hours PI. Serum iron was decreased between 6 and 48 hours. The clinical signs were most prominent during the first 24 hours PI and subsided within 48 hours PI. CONCLUSIONS: Neutrophilia, monocytoses, and high SAA concentrations were present in horses even after the clinical signs had subsided. Serum iron normalized before SAA. Knowledge of these findings is imperative when interpreting laboratory results in horses with possible endotoxin exposure.

A modified oral sugar test for evaluation of insulin and glucose dynamics in horses.

BACKGROUND: An oral sugar test (OST) using Karo® Light Corn Syrup has been developed in the USA as a field test for the assessment of insulin dysregulation in horses but the syrup is not available in Scandinavian grocery stores. The aim of the study was to compare the results of a modified OST between horses with equine metabolic syndrome (EMS) and healthy horses using a Scandinavian commercially available glucose syrup (Dansukker glykossirap). In addition, the effect of breed and the repeatability of the test were evaluated. In the present study, clinically healthy horses (7 Shetland ponies, 8 Icelandic horses, 8 Standardbred horses) and 20 horses of various breeds with EMS underwent the modified OST test. The Icelandic horses and Shetland ponies underwent the OST twice. Insulin and glucose data from the OST were used to calculate peak insulin concentration (PeakINS), time to peak insulin concentration (T-peakINS), area under the curve for insulin (AUCINS) and glucose (AUCGLU) as well as whole body insulin sensitivity index (ISICOMP). RESULTS: Compared to the healthy group, the EMS group had 6-7 times higher geometric mean for PeakINS and AUCINS and 8 times lower geometric mean for ISICOMP. The EMS group had a delayed T-peakINS compared to the healthy group. There was no effect of breed in the group of healthy horses on PeakINS, T-peakINS, AUCINS, AUCGLU and ISICOMP. Coefficient of variation for repeated tests was 19.8, 19.0 and 17.6 % for PeakINS, AUCINS and ISICOMP respectively. CONCLUSIONS: The results of the present study demonstrate that the modified OST appears to be a practical and useful diagnostic tool for assessment of insulin dysregulation in the horse. However, to make it possible to establish the most appropriate sampling interval and to evaluate the accuracy of the modified OST, further studies in horses with a variable degree of insulin resistance are needed, where results from the modified OST are compared with quantitative measurements for IS.

Blood pressure in Warmblood horses before and during a euglycemic-hyperinsulinemic clamp.

BACKGROUND: Insulin resistance (IR) in humans is related to hypertension and impaired vasodilation. Insulin administration has been shown to lower blood pressure both in insulin resistant as well as in insulin sensitive individuals. The aim of the study was to investigate the association between insulin sensitivity and alterations in blood pressure in healthy horses before and after a euglycemic-hyperinsulinemic clamp (EHC). A 3-h EHC was performed in 13 healthy horses (11 mares, 2 geldings). Blood samples for measurement of plasma glucose and insulin were collected before the start of the EHC, every 10 min during the EHC and immediately after the EHC. Mean, systolic- and diastolic blood pressure was measured before and during the final 10 min of the EHC using an indirect high-definition oscillometric monitor (HDO, horse model) applied to the middle of the coccygeal artery. Five consecutive measurements were made in each horse and on each occasion. Insulin and glucose data from the EHC were used to calculate the mean rate of glucose disposal per unit of insulin during steady state (M/I ratio). Insulin resistance was defined as a M/I ratio <5 mg/kg/min/mUL (Lindåse et al. in Am J Vet Res 77:300-309, 2016). RESULTS: Insulin administration decreased systolic, diastolic and mean arterial pressure in all horses. The M/I ratio for all horses was negatively correlated with the decrease in systolic blood pressure (r2 = 0.55, P = 0.004) and mean arterial pressure (r2 = 0.31, P = 0.048) but not diastolic blood pressure (r2 = 0.12, P = 0.26). Eight horses were defined as insulin resistant (IR) and five horses had normal insulin sensitivity. The five horses with normal insulin sensitivity showed a greater decrease in systolic blood pressure (-17.0 ± 7.4 vs. -3.4 ± 4.6 mmHg, P = 0.001) and MAP (19.2 ± 14.7 vs. 6.9 ± 8.7 mmHg, P = 0.04) than IR horses. There was no difference in the decrease in diastolic blood pressure between groups (16 ± 12.8 vs. 8.9 ± 12.1 mmHg, P = 0.17). CONCLUSIONS: This study indicates that there is a relationship between insulin sensitivity and systolic and MAP in horses. However, studies on a larger number of horses are needed to confirm this association.

Time-related changes in equine neutrophils after experimental endotoxemia: myeloperoxidase staining, size, and numbers.

BACKGROUND: Neutrophil myeloperoxidase content is determined by the Advia 2120 hematology system by staining characteristics. Changes in myeloperoxidase staining are shown by location of neutrophils on Advia peroxidase dot plots and as myeloperoxidase index (MPXI). Significant changes in MPXI have been reported during severe inflammation in horses, dogs, and people but conclusions were inconsistent. OBJECTIVES: Infusion of endotoxin was used to initiate an inflammatory stimulus under controlled conditions and over a longer time period than in previous studies to document kinetics of changes in neutrophil numbers, morphology, and myeloperoxidase staining. Identification of consistent time-related changes may allow better interpretation of changes in neutrophil characteristics during inflammation. MATERIALS: Five Standardbred trotting horses received an intravenous infusion over a 6-hour period with Escherichia coli endotoxin. Neutrophil count, MPXI, neutrophil characteristics in Advia 2120 Perox dot plots and neutrophil morphology in blood smears were monitored with repeated sampling for up to 10 days. RESULTS: Endotoxin infusion immediately caused severe neutropenia which converted to neutrophilia 14 hours after start of endotoxin infusion. Neutrophilia was still present 78 hours after start of infusion. Large "giant" neutrophils first appeared in blood smears and Advia Perox dot plots after 36-48 hours. A marked and consistent decrease in MPXI was seen in all horses 6 days (150 hours) after endotoxin exposure. CONCLUSIONS: Endotoxemia caused prominent, time-related changes in equine neutrophil characteristics including emergence of giant neutrophils and markedly decreased MPXI several days after endotoxin infusion.

Effects of diet-induced weight gain and turnout to pasture on insulin sensitivity in moderately insulin resistant horses.

OBJECTIVE: To quantify insulin sensitivity and monitor glucose, insulin, and lipid concentrations in a group of moderately insulin-resistant horses during induction of obesity by use of a forage diet supplemented with fat and during subsequent turnout to pasture. ANIMALS 9 adult Standardbred mares (11 to 20 years old). PROCEDURES: Weight gain of horses was induced during 22 weeks by use of a forage diet supplemented with fat fed in gradually increasing amounts, followed by feeding of that fat-supplemented diet at 2.5 times the daily maintenance requirements. Horses were then turned out to pasture. Insulin sensitivity was measured with the euglycemic hyperinsulinemic clamp method before and after weight gain and after 4 weeks at pasture. Body weight, body condition score, and cresty neck score as well as fasting and postprandial concentrations of plasma insulin, plasma glucose, serum triglyceride, and serum nonesterified fatty acids were measured during the study. RESULTS: Body weight typically increased by 10%, and body condition score (scale, 1 to 9) increased by > 1.5 from the start to the end of the weight-gain period. There was no difference in insulin sensitivity or metabolic clearance rate of insulin during the weight-gain period. Four weeks at pasture generally improved insulin sensitivity and metabolic clearance rate of insulin by 54% and 32%, respectively, but there was no change in body weight or body condition score. CONCLUSIONS AND CLINICAL RELEVANCE: Findings indicated that dietary composition played a more important role than did short-term weight gain on alterations in insulin sensitivity of horses.

Intramuscular administration of sodium benzylpenicillin in horses as an alternative to procaine benzylpenicillin.

The aim was to supply information about the possibility of replacing the procaine salt with the sodium salt for benzylpenicillin IM treatment in horse in order to diminish the risk for procaine adverse effects. In a crossover study eight horses were given 15 mg/kg sodium benzylpenicillin (Na-pc) twice daily or procaine benzylpenicillin (control) once daily IM for four days. The half-life of Na-pc was 1.9h, peak concentration was 14,600 ng/mL reached after about 23 min. Trough plasma concentration was 281 ng/mL and protein binding 62.8%. The fT>MIC for Staphylococcus aureus was 63% and 100% for Streptococcus equi subsp. equi and Streptococcus zooepidemicus, indicating an adequate antimicrobial therapy. However, Na-pc cannot be recommended from a welfare point of view since the horses showed more pain related behaviour and more pain and swelling compared to the control treatment.

Interday variation and effect of transportation on indirect blood pressure measurements, plasma endothelin-1 and serum cortisol in Standardbred and Icelandic horses.

BACKGROUND: Systemic hypertension is a prominent feature in humans with metabolic syndrome (MS) and this is partly caused by an enhanced endothelin-1 (ET-1) mediated vasoconstriction. There are indications that systemic hypertension might be a feature in equine metabolic syndrome (EMS) but if ET-1 is involved in the development of hypertension in horses is not known. Increased levels of cortisol have also been found in humans with MS but there are no reports of this in horses. Before blood pressure, plasma ET-1 and serum cortisol can be evaluated in horses with EMS, it is necessary to investigate the interday variation of these parameters on clinically healthy horses. The aims of the present study were therefore to evaluate the interday variation and influence of transportation on systemic blood pressure, plasma ET-1 and serum cortisol in healthy Standardbred and Icelandic horses, and to detect potential breed differences. METHODS: Nine horses of each breed were included in the study. Blood pressure was measured and blood samples were collected between 6 and 9 am on two separate days. Eight of the horses (four of each breed) were transported to a new stable were they stayed overnight. The next morning, the sampling procedure was repeated. RESULTS: The interday variation was higher for plasma ET-1 (37%) than for indirect pressure measurements (8-21%) and serum cortisol (18%). There were no differences in systemic blood pressure between the two breeds. The Icelandic horses had significantly lower serum cortisol and significantly higher plasma ET-1 concentrations compared to the Standardbred horses. Plasma ET-1 was significantly elevated after transportation, but systemic blood pressure and serum cortisol did not differ from the values obtained in the home environment. CONCLUSIONS: Indirect blood pressure, plasma ET-1 and serum cortisol are of interest as markers for cardiovascular dysfunction in horses with EMS. The elevated plasma ET-1 concentrations recorded after transportation was likely caused by a stress response. This needs to be considered when evaluating plasma ET-1 in horses after transportation. The differences detected in plasma ET-1 and serum cortisol between the two breeds might be related to differences in genetic setup, training status as well as management conditions.

Effect of repeated oral administration of glucose and leucine immediately after exercise on plasma insulin concentration and glycogen synthesis in horses.

OBJECTIVE: To determine whether repeated oral administration of glucose and leucine during the period immediately after intense exercise would increase the release of insulin and thereby enhance glycogen synthesis in horses. ANIMALS: 12 Standardbred horses. PROCEDURES: In a crossover study design, after glycogen-depleting exercise, horses received oral boluses of glucose (1 g/kg at 0, 2, and 4 hours) and leucine (0.1 g/kg at 0 and 4 hours) or boluses of water (10 mL/kg at 0, 2, and 4 hours; control treatment). Blood samples for determination of glucose, insulin, and leucine concentrations were collected prior to and during a 6-hour period immediately after exercise. Biopsy specimens of a gluteus muscle were obtained before and immediately after exercise and at 3, 6, and 24 hours after exercise for measurement of glycogen concentration. RESULTS: When glucose and leucine were administered to the horses, plasma insulin concentration was significantly higher during the 6 hours immediately after exercise than it was when water was administered to the horses. Serum glucose concentration during the 4 hours immediately after exercise was significantly higher when glucose and leucine were administered than the serum glucose concentration when water was administered. Muscle glycogen concentrations did not differ between the 2 treatments during the 24 hours after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Synthesis of muscle glycogen after intense intermittent exercise was not enhanced by oral boluses of glucose and leucine after exercise despite pronounced increases in plasma insulin and serum glucose concentrations.

Repeated post-exercise administration with a mixture of leucine and glucose alters the plasma amino acid profile in Standardbred trotters.

BACKGROUND: The branched chain amino acid leucine is a potent stimulator of insulin secretion. Used in combination with glucose it can increase the insulin response and the post exercise re-synthesis of glycogen in man. Decreased plasma amino acid concentrations have been reported after intravenous or per oral administration of leucine in man as well as after a single per oral dose in horses. In man, a negative correlation between the insulin response and the concentrations of isoleucine, valine and methionine have been shown but results from horses are lacking. This study aims to determine the effect of repeated per oral administration with a mixture of glucose and leucine on the free amino acid profile and the insulin response in horses after glycogen-depleting exercise. METHODS: In a crossover design, after a glycogen depleting exercise, twelve Standardbred trotters received either repeated oral boluses of glucose, 1 g/kg body weight (BW) at 0, 2 and 4 h with addition of leucine 0.1 g/kg BW at 0 and 4 h (GLU+LEU), or repeated boluses of water at 0, 2 and 4 h (CON). Blood samples for analysis of glucose, insulin and amino acid concentrations were collected prior to exercise and over a 6 h post-exercise period. A mixed model approach was used for the statistical analyses. RESULTS: Plasma leucine, isoleucine, valine, tyrosine and phenylalanine concentrations increased after exercise. Post-exercise serum glucose and plasma insulin response were significantly higher in the GLU+LEU treatment compared to the CON treatment. Plasma leucine concentrations increased after supplementation. During the post-exercise period isoleucine, valine and methionine concentrations decreased in both treatments but were significantly lower in the GLU+LEU treatment. There was no correlation between the insulin response and the response in plasma leucine, isoleucine, valine and methionine. CONCLUSIONS: Repeated post-exercise administration with a mixture of leucine and glucose caused a marked insulin response and altered the plasma amino acid profile in horses in a similar manner as described in man. However, the decreases seen in plasma amino acids in horses seem to be related more to an effect of leucine and not to the insulin response as seen in man.

Cardiac troponin I and the occurrence of cardiac arrhythmias in horses with experimentally induced endotoxaemia.

The aim of this study was to determine whether experimentally-induced endotoxaemia induced elevations in plasma cardiac troponin I (cTnI) concentrations in horses and how this might affect the incidence of cardiac arrhythmias. Eight Standardbred horses received an intravenous continuous rate infusion of endotoxin (total dose 500 ng/kg) for 6 h while being monitored using electrocardiography (ECG). Blood samples were collected before the start of the endotoxin infusion, every 60 min during the infusion, then 1, 2, 3, 8, 10 and 24 h post-infusion, and analysed for cTnI concentrations. One horse was excluded from the study owing to a high initial cTnI concentration. Endotoxin infusion induced an increase in cTnI concentrations in all horses, reaching mean peak concentration of 0.135±0.094 µg/L by 1 h post-infusion. The cTnI concentrations then decreased and were no longer significantly different from pre-infusion concentrations at 6, 10 and 24 h post-infusion. The number of ventricular events was generally low during the infusion period, but increased during the first 3 h post-infusion in 6/7 horses. In conclusion, elevated cTnI concentrations could be detected early after an endotoxaemic insult using an ultrasensitive cTnI assay, with peak cTnI concentrations preceding the occurrence of ventricular events on ECG.

Radiographic parameters for diagnosing sand colic in horses.

BACKGROUND: Ingestion of sand can cause colic, diarrhoea and weight loss in horses, but these signs are unspecific and can have many other causes. The amount of sand that induces disease may vary between individuals. To avoid over-diagnosing, it is important to determine the amount of sand that can be found in horses without clinical signs of gastrointestinal disease. The aim of this study was to use previously suggested parameters for establishing a radiographic diagnosis of sand colic, and compare these findings between a sand colic group and a control group. METHODS: Abdominal radiographs were obtained in 30 horses with a complaint unrelated to the gastrointestinal tract. In addition, archived abdominal radiographs of 37 clinical cases diagnosed with sand impaction were investigated. The size of the mineral opacity indicative of sand in the abdomen was measured and graded according to a previously published protocol based on height and length. Location, homogeneity, opacity and number of sand accumulations were also recorded. RESULTS: Twenty out of 30 control horses (66%) had one or more sand accumulations. In the present study; height, length and homogeneity of the accumulations were useful parameters for establishing a diagnosis of sand colic. Radiographically defined intestinal sand accumulation grades of up to 2 was a common finding in horses with no clinical signs from the gastrointestinal tract whereas most of the clinical cases had much larger grades, indicating larger sand accumulations. CONCLUSION: Further work to establish a reliable grading system for intestinal sand content is warranted, but a previously proposed grading system based on measurements of height and length may be an alternative for easy assessment of sand accumulations in the meantime. The present study indicates that a grade 1 - 2 sand accumulation in the intestine is a frequent finding in horses. When working up a case with clinical signs from the gastrointestinal tract, one or more accumulations of this grade should not be considered the cause until other possibilities have been ruled out.

Proglycogen, macroglycogen, glucose, and glucose-6-phosphate concentrations in skeletal muscles of horses with polysaccharide storage myopathy performing light exercise.

OBJECTIVE: To determine concentrations of proglycogen (PG), macroglycogen (MG), glucose, and glucose-6-phosphate (G-6-P) in skeletal muscle of horses with polysaccharide storage myopathy (PSSM) before and after performing light submaximal exercise. ANIMALS: 6 horses with PSSM and 4 control horses. PROCEDURES: Horses with PSSM completed repeated intervals of 2 minutes of walking followed by 2 minutes of trotting on a treadmill until muscle cramping developed. Four untrained control horses performed a similar exercise test for up to 20 minutes. Serum creatine kinase (CK) activity was measured before and 4 hours after exercise. Concentrations of total glycogen (G(t)), PG, MG, G-6-P, free glucose, and lactate were measured in biopsy specimens of gluteal muscle obtained before and after exercise. RESULTS: Mean serum CK activity was 26 times higher in PSSM horses than in control horses after exercise. Before exercise, muscle glycogen concentrations were 1.5, 2.2, and 1.7 times higher for PG, MG, and G(t), respectively, in PSSM horses, compared with concentrations in control horses. No significant changes in G(t), PG, MG, G-6-P, and lactate concentrations were detected after exercise. However, free glucose concentrations in skeletal muscle increased significantly in PSSM horses after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of the results suggests that glucose uptake in skeletal muscle is augmented in horses with PSSM after light exercise. There is excessive storage of PG and MG in horses with PSSM, and high concentrations of the 2 glycogen fractions may affect functional interactions between glycogenolytic and glycogen synthetic enzymes and glycosomes.

Effect of extraction time and acid concentration on the separation of proglycogen and macroglycogen in horse muscle samples.

The objective of this study was to determine whether the concentrations of proglycogen (PG) and macroglycogen (MG) in biopsy samples of horse muscle are influenced by extraction time or perchloric acid (PCA) concentration. In study 1, individual muscle-biopsy samples from 10 horses were divided into 4 parts each and then randomly subjected to 4 periods of extraction (10, 20, 60, or 120 min) with 1.5 M PCA. In study 2, individual muscle-biopsy samples from 6 horses were divided into 24 pieces each and then randomly subjected to 12 combinations of extraction time (10, 20, 30, or 40 min) and PCA concentration (0.5, 1.5, or 3.0 M). The results from study 1 indicated that PG and MG concentrations are affected only after extraction for 120 min; the PG concentration decreased significantly (P < 0.05), and the MG concentration increased (not significantly). In study 2, extraction in 3.0 M PCA yielded significantly lower PG and higher MG concentrations (P < 0.05) than extraction in 0.5 or 1.5 M PCA with each of the extraction times. The results of this study further support the existence of 2 glycogen pools and demonstrate that they are not an extraction artifact. The study also suggests that the 2 pools are stable during extraction over a range of extraction times and acid concentrations. However, if the exposure to acid is very long and, or, the acid concentration is high, some of the insoluble PG appears to be hydrolyzed and to enter the MG pool.

Analysis of proglycogen and macroglycogen content in muscle biopsy specimens obtained from horses.

OBJECTIVE: To determine proglycogen (PG) and macroglycogen (MG) content in equine skeletal muscle and to compare 2 analytical methods (acid hydrolysis [AC] and PG plus MG determination) for measurement of total muscle glycogen content (Gly(tot)) in biopsy specimens. SAMPLE POPULATION: Muscle biopsy specimens obtained from 41 clinically normal horses. PROCEDURE: Forty-five muscle biopsy specimens obtained from the middle gluteal (n = 31) or triceps (14) muscle were analyzed, using AC and MG plus PG determination for Gly(tot). Variability within muscle biopsy specimens for each method was calculated from duplicate analyses of muscle specimens. In a second experiment, variation in MG and PG content between muscle biopsy specimens and the effect of sample collection depth on the concentration of MG and PG in the middle gluteal muscle was evaluated. RESULTS: There was a strong correlation (r = 0.99) between Gly(tot) values obtained by use of AC and MG plus PG determination. Coefficients of variation for within- and between-specimen variability of Gly(tot) were approximately 4% for each method. The PG fraction was always in excess of the MG fraction. Biopsy specimens obtained from the superficial part of the middle gluteal muscle contained significantly more Gly(tot) and PG than specimens obtained from deeper parts. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirms that MG and PG exist in equine skeletal muscle and can be measured reliably in biopsy samples. This technique could be applied in future studies to investigate glycogen metabolism in exercising horses and horses with glycogen-storage diseases.