Glenys Noble

Evaluation of commercially available assays for the measurement of equine insulin

Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lins concordance coefficient (ρ(c)) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ(c) of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.

Effect of surgeon experience on postoperative plasma cortisol and C-reactive protein concentrations after ovariohysterectomy in the dog: a randomised trial.

OBJECTIVE To determine if postoperative C-reactive protein (CRP) or cortisol concentrations were significantly changed between dogs undergoing ovariohysterectomy by an experienced or inexperienced surgeon. As part of the Charles Sturt University teaching program, 45 bitches from an animal shelter were surgically sterilised between March and October 2010. METHODS The dogs were randomly assigned to surgeons, with 37 sterilised by veterinary undergraduates and 8 by experienced surgeons. Blood samples were collected preoperatively and at 2, 4 and 6 h postoperatively. A standard midline ovariohysterectomy was performed and detailed records kept. RESULTS The median surgery time for experienced surgeons was 17 min versus 87 min for inexperienced surgeons. Anaesthesia time and blood loss were greater among the inexperienced surgeons. The CRP concentration increased significantly postoperatively for all animals (P < 0.001). Bitches sterilised by inexperienced surgeons had a significantly greater rise in CRP at 4 and 6 h post-surgery (P = 0.046). Serum cortisol concentrations were found to increase significantly over time for all animals (P < 0.001), but were not affected by surgeon experience. CONCLUSION The results suggest that inexperienced surgeons affect their patients differently to experienced surgeons, potentially through tissue trauma or anaesthetic duration. The lack of difference in the cortisol concentrations reflects the large number of triggers for cortisol release and, potentially, that there was little difference between the groups in terms of perceived pain in the presence of good analgesia. Serum CRP concentration may be a more sensitive measure than serum cortisol of differences in surgical trauma.

Pharmacokinetics and Safety of Single and Multiple Oral Doses of Meloxicam in Adult Horses

BACKGROUND Safety of meloxicam, a potent NSAID with selective COX-2 inhibition, has not been evaluated in horses. OBJECTIVES To evaluate pharmacokinetics and safety of single and repeated oral doses of meloxicam in adult horses. ANIMALS Forty-nine healthy, university-owned adult lightbreed horses. METHODS Study conducted in 2 parts. Part I addressed pharmacokinetics of single oral dose meloxicam (0.6 mg/kg) in 16 horses. Part II, 33 horses were randomly assigned to 5 treatment groups to assess prolonged administration (0.6 mg/kg PO q24h for 6 weeks, n = 7) or higher doses (1.8 mg/kg, n = 7, or 3.0 mg/kg PO q24h, n = 7) of meloxicam for 2 weeks, compared with control horses (placebo, n = 7, or phenylbutazone, 4.4 mg/kg q12h on day 1, 2.2 mg/kg q12h for 4 days, then 2.2 mg/kg q24h for 9 days, n = 5). RESULTS Maximum plasma concentration following a single oral dose of meloxicam was 915.1 ± 116.9 ng/mL and elimination half-life 10.2 ± 3.0 hours. Meloxicam (0.6 mg/kg, q24h, PO for 6 weeks) yielded plasma concentrations between 100 and 1000 ng/mL and was well tolerated by healthy adult horses. Administration of 3-5 times the recommended dose of meloxicam was associated with decreased total serum protein and albumin concentrations, gastrointestinal damage, renal damage, or bone marrow dyscrasia. PBZ administration was associated with the development right dorsal colitis, gastric ulceration, and protein losing enteropathy in 2 horses. CONCLUSIONS AND CLINICAL IMPORTANCE Administration meloxicam at 0.6 mg/kg q24h was well tolerated for 6 weeks, without drug accumulation in plasma. Higher doses were associated with dose-dependent adverse effects typical of class of drugs.

Effects of meloxicam and phenylbutazone on equine gastric mucosal permeability.

BACKGROUND Newer NSAIDs that more selectively target the induced isoform of the cyclooxygenase enzyme (COX2) activity might reduce adverse effects while preserving therapeutic benefits of these drugs. OBJECTIVES To compare the effect of oral administration of multiple dose rates of meloxicam and phenylbutazone (PBZ) on gastric mucosal integrity in horses. ANIMALS Twenty-five light breed horses. METHODS In vivo toxicity study. Horses were randomly assigned to 5 treatment groups, receiving placebo, PBZ (4.4 mg/kg PO q12h day 1, 2.2 mg/kg PO q12h for 4 days, 2.2 mg/kg PO q24h for 9 days), or 3 dose rates of meloxicam (0.6 mg/kg q24h, 1.8 mg/kg q24h, 3.0 mg/kg q24h) for 14 days. Sucrose permeability testing was performed on Day 0 (before treatment) and on Day 13. All personnel involved with data collection or analysis were blinded to treatment. RESULTS Administration of PBZ at the above dose rate significantly increased gastric permeability to sucrose, evidenced by increased peak serum sucrose concentrations (280-1,580 pg/μL, P = .001) after treatment. Similar changes were not evident after administration of meloxicam at any dose rate tested, or in control horses (P > .05). Treatment was not associated with significant differences in ulceration of the squamous or glandular mucosa. Peak sucrose concentrations were not correlated with serum total protein or albumin concentrations (R(2) = -0.07, P = .61, R(2) = -0.08, P = .58, respectively). CONCLUSION AND CLINICAL IMPORTANCE These results suggest that PBZ was associated with greater compromise to gastric mucosal integrity than meloxicam.

Pharmacokinetics of metformin after enteral administration in insulin-resistant ponies

OBJECTIVE To determine pharmacokinetics and plasma steady-state kinetics of metformin after oral or nasogastric administration in insulin-resistant (IR) ponies. ANIMALS 8 IR ponies. PROCEDURES Metformin (30 mg/kg) was administered to 8 ponies via nasogastric tube Blood samples were collected at intervals for 24 hours. Plasma concentrations of metformin were measured via liquid chromatography-electrospray tandem mass spectroscopy Pharmacokinetic variables were determined via noncompartmental analysis. Metformin (15 mg/kg, PO, twice daily [8 am and 5 pm]) was administered to 4 ponies for an additional 20 days, and blood samples were obtained every 2 days. Plasma concentration at steady state (Css) was determined. RESULTS Mean±SD elimination half-life (t1/2) of metformin was 11.7±5.2 hours, maxima plasma concentration was 748±269 ng/mL at 54±32 minutes, mean area under the curve was 355±92 microg.h/mL, and apparent clearance was 90.6±28.1 mL/min/kg. The Css was 122±22 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE Metformin reportedly enhances insulin sensitivity of peripheral tissues without stimulating insulin secretion, but bioavailability in horses is low. The t1/2 of metformin in IR ponies was similar to that in humans. Actual clearance of metformin adjusted for bioavailability in IR ponies was similar to that in humans; however, during chronic oral administration at dosages reported in efficacy studies, the Css of metformin was less than values associated with therapeutic efficacy in humans The apparent lack of long-term efficacy of metformin in horses is likely attributable to low bioavailability, rather than to rapid clearance.

Effects of a commercial dose of L‐tryptophan on plasma tryptophan concentrations and behaviour in horses

REASON FOR PERFORMING STUDY L-tryptophan is a common ingredient in equine calmative products, but its effectiveness has not been demonstrated in horses. HYPOTHESIS To determine whether a commercial dose of L-tryptophan increases plasma tryptophan and alters behaviour in horses fed a roughage or concentrate meal. METHODS L-tryptophan (6.3 g) or placebo (water) was administered per os in a cross-over design, to 12 Thoroughbred horses (503 +/- 12.1 kg bwt), just before a meal of lucerne hay or oats. Plasma tryptophan was measured by gas chromatography. Horse behaviour was observed in an empty enclosure, then in the presence of an unfamiliar person and a novel object. RESULTS Total plasma tryptophan increased 3-fold in both studies, peaking 1.5-2 h after dosing. After the peak, tryptophan remained high for several hours if the horses had been fed hay, but fell sharply if fed oats, consistent with the glycaemic responses to these meals. However, the ratio of tryptophan to 4 large neutral amino acids (phenylalanine, tyrosine, leucine and isoleucine) increased in the tryptophan-treated horses to a similar extent and for a similar duration, with both diets. The presence of a stranger or novel object increased heart rate (P<0.05), but caused no behavioural effects that were altered by tryptophan, regardless of the diet. CONCLUSIONS Plasma tryptophan increases when tryptophan is administered at a dose used in some commercial products, but this is not reflected by marked behavioural changes in the horse. POTENTIAL RELEVANCE Further work is required to refine behavioural tests and identify an effective dose of L-tryptophan in the horse.

Potential treatments for insulin resistance in the horse: a comparative multi-species review.

Insulin resistance and hyperinsulinaemia increase the risk of laminitis and horse owners and veterinarians should attempt to enhance insulin sensitivity in at-risk groups. In obese animals this may be achieved, in part, by promoting weight loss and increasing exercise, but such intervention may not be appropriate in non-obese insulin-resistant animals, or where exercise is contra-indicated for clinical reasons. An alternative approach to controlling insulin sensitivity in obese and non-obese horses may be the use of certain herbal compounds that have shown promise in humans and laboratory animals, although little is known of the effects of these compounds in horses. The herbs can be grouped according to their primary mechanism of action, including activators of the peroxisome proliferator-activated receptors, anti-obesity compounds, anti-oxidants, compounds that slow carbohydrate absorption, insulin receptor activators and stimulators of glucose uptake, with some herbs active in more than one pathway. Certain herbs have been prioritised for this review according to the quality and quantity of published studies, the reported (or extrapolated) safety profile, as well as potential for efficacy, all of which will hopefully motivate further research in this field.

Prevalence of equine adenovirus antibodies in horses in New South Wales, Australia.

There are currently two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV1) and equine adenovirus type 2 (EAdV2); EAdV1 is predominantly associated with upper respiratory tract infections while EAdV2 appears to have a higher association with gastrointestinal infection, however, very little is known about the prevalence of these viruses in horse populations in Australia. In this study we tested 122 serum samples obtained from horses in New South Wales, Australia, using a standard serum neutralization (SN) assay and ELISA. Ninety-seven of the 122 sera displayed had moderate to high titers of antibodies to EAdV1 and/or EAdV2. Eighteen of the 122 sera were positive for both EAdV1 and EAdV2. In contrast, only thirty-seven positive samples were detected using the ELISA. These results suggest that EAdV1 and EAdV2 infections are widely prevalent in the horse population tested and that SN is currently the most suitable assay for the detection of EAdV1 and EAdV2 antibodies in equine serum.

Diurnal rhythm and effects of feeding, exercise and recombinant equine growth hormone on serum insulin concentrations in the horse

REASONS FOR PERFORMING THE STUDY As growth hormone increases lean body mass, it could be a therapy for obese horses. However, growth hormone use induces hyperinsulinaemia in some species, so further investigation is warranted. OBJECTIVES To investigate the effects of feeding, exercise and growth hormone therapy on basal insulin concentrations in healthy horses. STUDY DESIGN In vivo experimental study. METHODS Blood samples were obtained every 30 min from 12 geldings over 24 h, to establish basal serum insulin concentrations, before they underwent a 3-week exercise programme. Horses were allocated into 2 groups and exercised for another 4 weeks. Group A received daily i.m. injections of recombinant equine growth hormone; 5 mg/day for 5 days, then 12.5 mg/day for 16 days. Blood samples were taken daily before feeding. Insulin vs. time area under curve of Groups A and B were compared using a Students unpaired t test. RESULTS Horses demonstrated insulin peaks within 2 h of feeding of 577 ± 108.3 pmol/l at 09.30 h and 342.4 ± 75.7 pmol/l at 17.30 h, despite receiving the same meal. The nadir was between midnight and 07.30 h. Exercise had no effect on basal insulin concentrations prior to equine growth hormone administrations. The equine growth hormone injections increased serum insulin concentrations (P = 0.01) within Group A, from 44.4 ± 15.3 pmol/l initially to 320.9 ± 238.2 pmol/l by Day 12. Exogenous growth hormone caused variable hyperinsulinaemia, which was alleviated once equine growth hormone administration ceased. CONCLUSIONS Single serum samples taken prior to the morning meal provide basal insulin concentrations. Exercise did not change basal insulin concentrations. However, equine growth hormone injections increased basal insulin concentrations, which were not ameliorated by exercise. POTENTIAL RELEVANCE This therapy is not recommended to address obesity in insulin-resistant equids.

Acepromazine pharmacokinetics: a forensic perspective.

Acepromazine (ACP) is a useful therapeutic drug, but is a prohibited substance in competition horses. The illicit use of ACP is difficult to detect due to its rapid metabolism, so this study investigated the ACP metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS) as a potential forensic marker. Acepromazine maleate, equivalent to 30mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7days post-administration and analysed for ACP and HEPS by liquid chromatography-mass spectrometry (LC-MS). Acepromazine was quantifiable in plasma for up to 3h with little reaching the urine unmodified. Similar to previous studies, there was wide variation in the distribution and metabolism of ACP. The metabolite HEPS was quantifiable for up to 24h in plasma and 144h in urine. The metabolism of ACP to HEPS was fast and erratic, so the early phase of the HEPS emergence could not be modelled directly, but was assumed to be similar to the rate of disappearance of ACP. However, the relationship between peak plasma HEPS and the y-intercept of the kinetic model was strong (P=0.001, r(2)=0.72), allowing accurate determination of the formation pharmacokinetics of HEPS. Due to its rapid metabolism, testing of forensic samples for the parent drug is redundant with IV administration. The relatively long half-life of HEPS and its stable behaviour beyond the initial phase make it a valuable indicator of ACP use, and by determining the urine-to-plasma concentration ratios for HEPS, the approximate dose of ACP administration may be estimated.