David Rendle

Type 2 diabetes mellitus with pancreatic β cell dysfunction in 3 horses confirmed with minimal model analysis.

REASONS FOR PERFORMING STUDY Type 2 diabetes mellitus (T2DM) is diagnosed rarely in equine practice although it may be under-recognised. A greater awareness of the condition and therapeutic considerations would be to the benefit of such cases presenting in practice. More investigation into the pharmacological management of these cases is needed. OBJECTIVES Three cases of diabetes mellitus were investigated using a specific test for insulin sensitivity and pancreatic beta cell function in order to define accurately and characterise the existence of T2DM in all 3 subjects. METHODS The insulin-modified frequently sampled i.v. glucose tolerance test was performed in each case and the data so obtained were subject to minimal model analysis of insulin-glucose dynamics. Cases were then monitored following treatment using a combination of dietary modification, metformin, glibenclamide and pergolide. RESULTS Marked insulin resistance was identified in each case and, furthermore, severe pancreatic beta cell dysfunction was present therefore classifying each case as end stage T2DM. Treatment was nevertheless associated with restoration of normoglycaemia in all cases. CONCLUSIONS T2DM in horses may be more common than generally considered.In some cases individuals may respond to therapy aimed at restoring insulin sensitivity and pancreatic function. Drugs used in other species for the treatment of T2DM have not yet been adequately tested in horses. POTENTIAL RELEVANCE T2DM should be considered as an important differential diagnosis in mature to elderly horses and ponies suffering from weight loss, polydipsia and polyuria. Clinicians should be encouraged to offer treatment and management advice when such cases are encountered.

Investigation of rhythms of secretion and repeatability of plasma adrenocorticotropic hormone concentrations in healthy horses and horses with pituitary pars intermedia dysfunction

REASONS FOR PERFORMING STUDY There is little published information on whether measurement of plasma ACTH concentration at a single timepoint is a repeatable indicator of pituitary pars intermedia dysfunction (PPID). OBJECTIVES To determine whether ultradian or circadian fluctuations in ACTH production influence plasma ACTH concentration in normal horses and horses with PPID. STUDY DESIGN Prospective observational study. METHODS Plasma ACTH concentration in 8 non-PPID horses and 8 horses with PPID was measured at 08.00, 11.00, 14.00 and 17.00 h on 5 nonconsecutive days within a 3 week period. In addition, at 08.30 h on one day, 6 samples were collected from each horse at precisely 5 min intervals over a period of 25 min. Descriptive and graphical analysis was performed and a linear mixed effects model was fitted to assess the effect of time of day on ACTH concentration in non-PPID and PPID horses. RESULTS Evidence of ultradian fluctuation in ACTH production was not identified in either non-PPID or PPID horses. Evidence for circadian fluctuation was identified in non-PPID horses; plasma ACTH concentrations were highest at 08.00 h and decreased through the day. There was no evidence of circadian fluctuation in PPID horses. In non-PPID horses, the magnitude of circadian changes in ACTH concentration was smaller than variations in concentration that occurred at random. Intrahorse variability of ACTH concentration was greater in PPID horses than in non-PPID horses. CONCLUSIONS Ultradian and circadian fluctuations in ACTH concentration are unlikely to influence clinical decision making; however, variations of potential clinical relevance do occur in individual horses, for reasons that remain to be determined, and increase in magnitude with progression of PPID. Results of the current study indicate that when an ACTH concentration between 19 and 40 pg/ml is measured, further testing should be considered to increase the accuracy of PPID diagnosis.

Stability of common biochemistry analytes in equine blood stored at room temperature

REASONS FOR PERFORMING STUDY Time delays between collection of blood samples and biochemical analysis of equine blood are unavoidably common in equine practice. The effect that delays may have on the accuracy of results of blood biochemical analyses is not well established. HYPOTHESIS Delays in processing of blood of up to 72 h results in alterations in measured levels of common biochemical analytes that are of potential clinical relevance. Separation of serum prior to storage is protective against the effects of time delays. METHODS Samples of clotted blood, separated serum and oxalate fluoride plasma from 20 horses were stored and analysed at 0, 24, 48 and 72 h. Graphical exploration of each analyte was undertaken. General linear models with fixed effects were fitted for the whole blood data. The mean bias and 95% limits of agreement were calculated, using bootstrapped data, to assess agreement between pairs of samples analysed at 0 h and other time points. Bland-Altman plots were used to explore general trends in the data. Paired t tests were used to compare the results from whole blood and separated serum. RESULTS Delays in processing equine blood resulted in significant increases in measured concentrations of aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total bile acids and magnesium. A significant decrease in concentration was identified for glucose (serum and oxalate fluoride preserved plasma). Separation of serum immediately following clot formation resulted in nonsignificant increases in accuracy for some analytes. CONCLUSIONS AND PRACTICAL SIGNIFICANCE: Delays in processing of blood samples may result in biochemical changes of clinical relevance in individual cases; however, in the majority of cases, where delays are only a few days and a number of analytes are assessed concurrently, delays are unlikely to have an effect on the interpretation of results. Separation of serum following clot formation is of limited benefit. Clinical samples in which a delay in processing has occurred may be interpreted with reference to the data presented.

Multicentric T-cell lymphoma presenting as inferior palpebral swelling in a Standardbred mare

A 6-year-old Standardbred mare that presented with bilateral palpebral swelling and a left corneal ulcer was diagnosed with a multicentric T-cell lymphoma. Other clinical findings included submandibular lymphadenopathy and oedema, pharyngeal oedema and rhinitis. Prior to euthanasia the horse developed keratomalacia, the onset of which was coincidental with the use of high doses of topical and systemic corticosteroids. Although an unusual presentation, palpebral swelling should be considered as a clinical sign of lymphoma.

The effects of sample handling and N‐phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma

REASONS FOR PERFORMING STUDY Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. OBJECTIVES This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. STUDY DESIGN Experimental study. METHODS Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemiluminescent assay. Plasma samples were then: 1) centrifuged immediately, 2) centrifuged immediately with the addition of maleimide, or 3) allowed to separate by gravity followed by the addition of maleimide, before all samples were stored at 22°C and analysed at 4, 8, 24 and 48 h post collection. A linear mixed effects model and Bland-Altman analyses were performed. Significance was set at P < 0.05. RESULTS No significant effect of plasma treatment (P = 0.1) on change in ACTH concentration was identified. However, significant effects of horse health status (P < 0.001) and time (P < 0.001) on change in ACTH concentration were identified. No significant interactions were found. Significant decreases in ACTH concentration occurred in horses with PPID between 4 and 8 h after blood collection. In non-PPID horses, the decrease in ACTH concentration over time was not significant. Agreement with baseline values decreased over time and was greater for non-PPID horses than for PPID horses. CONCLUSIONS Clinically useful results are still obtained if ACTH concentration is measured up to 48 h after sample collection. Allowing samples to separate by gravity rather than centrifugation did not have a significant effect on ACTH concentration, and the addition of maleimide was of no benefit.

Determination of pergolide in horse plasma by UPLC–MS/MS for pharmacokinetic applications

Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC-MS/MS assay was developed with a simple sample preparation involving methanol protein precipitation and injection of supernatant. The assay was applied to samples from a horse dosed with 10mg pergolide (as the mesylate salt) by nasogastric intubation. Plasma samples were collected over a 48h period. The assay demonstrated performance sufficient to enable application to low level PK studies. Within-batch precision and accuracy were within acceptance criteria; precision was less than 10% RSD (n=5) and accuracy was -7.3% at 0.014ng/mL, the lower limit of quantification was 0.006ng/mL and the method detection limit was 0.002ng/mL. In the treated horse, Cmax was 0.40ng/mL and the assay easily allowed determination of plasma levels in the elimination phase to 48h. In conclusion, this assay using UPLC-MS/MS and methanol protein precipitation easily meets the challenging demands of pergolide analyses in plasma.

Pharmacokinetics of pergolide after intravenous administration to horses

OBJECTIVE To determine the pharmacokinetics of pergolide after IV administration to horses. ANIMALS 8 healthy adult horses. PROCEDURES Pergolide mesylate was administered IV at a dose of 20 μg/kg (equivalent to 15.2 μg of pergolide/kg) to each horse, and blood samples were collected over 48 hours. Pergolide concentrations in plasma were determined by means of high-performance liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined on the basis of noncompartmental methods. RESULTS After IV administration of pergolide, mean ± SD clearance, elimination half-life, and initial volume of distribution were 959 ± 492 mL/h/kg, 5.64 ± 2.36 hours, and 0.79 ± 0.32 L/kg, respectively. CONCLUSIONS AND CLINICAL RELEVANCE With an elimination half-life of approximately 6 hours, twice-daily dosing may be more appropriate than once-daily dosing to reduce peak-trough fluctuation in pergolide concentrations. Further pharmacodynamic and pharmacokinetic studies of pergolide and its metabolites will be necessary to determine plasma concentrations that correlate with clinical effectiveness to determine the therapeutic range for the treatment of pituitary pars intermedia dysfunction.