INVESTIGATIONS OF EQUINE ENDOCRINOLOGY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0206299
• Project Start Date: Jan 1, 2006
• Project End Date: Jan 1, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
VETERINARY CLINICAL SCIENCES

Non Technical Summary
Tumors of the pituitary gland and other hormonal abnormalities are an emerging problem in equine medicine. This project will develop and validate a new way to evaluate horses with pituitary tumors using domperidone.

Animal Health Component

60%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3810	1020	40%
311	3810	1160	60%

Knowledge Area
311 - Animal Diseases; 305 - Animal Physiological Processes;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1160 - Pathology; 1020 - Physiology;

Keywords

horse

endocrine

hormone

cushing's

pars intermedia

domperidone

cortisol

acth

metabolic disease

Goals / Objectives
Equine pituitary pars intermedia dysfunction (PPID) is the most common endocrinopathy of horses and ponies. What is more, as the number of aged horses in hospital populations increases, the number of equids diagnosed with PPID can be expected to increase as well. It is our objective to make investigation of equine pituitary disease a research focus. We will begin this line of investigation by performing an experiment to verify that the dopamine D2 receptor antagonist domperidone induces an exaggerated increase in endogenous adrenocorticotropin (ACTH) and, subsequently, cortisol in horses with PPID. At the end of the study we will be able to provide a diagnostic testing protocol that will use the effect of domperidone on endogenous ACTH and cortisol concentrations to accurately differentiate horses with and without PPID. Being able to accurately identify horses with PPID will then enable us to evaluate different treatment modalities, and to assess the efficacy of various dietary and pharmacologic interventions. The objective of this proposal is to devise a domperidone response test. This will be a provocative diagnostic test that can differentiate horses with PPID from normal horses and those with other diseases. It is the overall objective of this project to provide veterinary internists and practitioners with the means to reliably and safely identify and monitor horses with PPID, both those with advanced cases and otherwise asymptomatic horses shortly after pars intermedia dysfunction occurs. This objective can be further broken down to these specific research aims: 1) To determine endogenous ACTH and cortisol levels in normal and PPID horses over an 8 hour span after they receive one of 3 dosage levels of domperidone. 2) To use the time of maximal ACTH and cortisol difference after domperidone administration as determined in objective 1 to test additional horses, both with and without PPID, using a two-time-point testing technique.

Project Methods
Six horses will be used in the initial study; four with a clinical diagnosis of PPID, and 2 age-matched controls. Horses will be considered PPID positive if they have hirsutism or a history of not shedding out properly, and have an abnormal dexamethasone response test. All horses will be housed on pasture or in dirt lots with free access to hay and water at all times. Forty-eight hours prior to testing, horses will be moved into box stalls Twenty four hours prior to testing, an indwelling catheter will be aseptically placed in the jugular vein. This will be done to eliminate the stress of repeated venipuncture. Concentrated heparin solution will be used to keep the catheter patent until the following day. Blood will be collected via the jugular catheter every other hour for 10 hours. In each instance, ten ml of blood will be withdrawn and half will be placed into a silicone-lined tube which contains EDTA as an anticoagulant and then placed on ice, and half will be collected into an evacuated sterile tube with no additive. Plasma will be separated and placed into a polypropylene tube within 30 minutes of collection. Samples will be frozen at -70oC until assay. Immediately after the first sample is collected, domperidone (Equidone, Equi-Tox, Inc.) will be administered per os. One of four doses will be given. The order of dosage will be randomized for each horse. Doses that will be administered are: 0 (carrier alone), 0.5 mg/kg, 1.0 mg/kg, and 5 mg/kg. The ten hour endogenous ACTH and cortisol curves will be compared for all four doses of domperidone in the six test animals. The time point that is consistently the most elevated over baseline samples in PPID horses and produces the largest difference between PPID and control animals will be selected as the post domperidone time point to be used for future studies. Hormone concentrations will be determined using a chemiluminescent immunoassay using an Immulite machine (DPC-Cirrus). After the final testing period all horses will be euthanized (Beuthanasia 2ml/kg I.V.) and a complete postmortem performed. Statistical and data analysis: The optimum domperidone dose and response time will be selected by determining the time point and dose with the greatest median ACTH response in affected horses. Seasonal variation in the curves will be compared by Friedman two-way ANOVA by ranks for nonparametric data with repeated measures. In year two, comparisons of affected and non-affected horses at different seasons will also be made by Friedman two-way ANOVA by ranks. To make pairwise comparisons, the Wilcoxon signed rank test with adjusted degrees of freedom will be used. These calculations will also be performed for cortisol measurements.

Progress 01/01/06 to 01/01/10

Outputs
OUTPUTS: Results have been disseminated orally to communities of interest via presentations at scientific meetings, continuing education presentations at meetings of veterinarians and user groups, and to members of the School of Veterinary Medicine community via resident seminars. Results have been disseminated to the larger scientific community via publication of manuscripts in peer-reviewed journals and via the publication of scientific proceedings. PARTICIPANTS: Dr. Barack Pressler - collaborator, expert in renal physiology Dr. George Moore - collaborator, biostatistician Dr. Ben Uberti - graduate student TARGET AUDIENCES: Equine veterinarians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
It was found that urinary protein/creatinine ratio can be quantitated in horses and used to predict renal disease. In addition, it was discovered that heparin administration prevents alterations in the urinary protein/creatinine ratio, but does not prevent experimentally-induced laminitis. Furthermore, it was found that ovary removal has no effect on either insulin sensitivity or the hypothalus/pituitary/adrenal axis in mares.

Publications

• Uberti B, *Pressler BM, Alkabes SB, Chang C-Y, Moore GE, Lescun TB, Sojka JE. Effect of heparin administration on urine protein excretion during the developmental stage of experimentally-induced laminitis in horses. Am J Vet Res. In press, 2009.
• Uberti B, Eberle DB, *Pressler BM, Moore GE, Sojka JE. Determination of 24-hour protein excretion and urine protein:creatinine ratio and correlation with random sample urine protein:creatinine ratio in healthy horses and ponies. Am J Vet Res. 2009 Dec;70(12):1551-6

Progress 01/01/09 to 01/01/10

Outputs
OUTPUTS: Research supported by the Hatch funds has been completed. It has been disseminated via publication: two manuscripts describing the work have been accepted and are in press in peer-reviewed research journals. The work has also been discussed at national meetings as a research abstract. Finally, the work has been presented both as poster and oral presentations at local meetings. PARTICIPANTS: Purdue University: Dr. Janice Sojka, Principal Investigator Dr. Barrack Pressler, Co-investigator Dr. George Moore, Co-investigator Dr. Laurent Couetil, Co-investigator Dr. Benjamin Uberti - Graduate student, Received Master's Degree in 2009 Bernard Eberle - undergraduate research fellow Indiana Racing Commission - Dr. Ursula Jedra, Co-Investigator Opportunities were provided on this project for graduate instructors in the school of veterinary medicine and to undergraduates in the School of Agriculture. TARGET AUDIENCES: The target audience for this research are equine veterinarians and horse owners. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The research results described in the peer-reviewed manscripts has advanced the field of equine endocrinology in important ways. We now understand normal equine urinary protein excretion levels and how that is influenced by systemic inflammatory response syndrome. In addition, the domperidone-response test has advanced the field of diagnosis of equine pituitary conditions.

Publications

• Uberti B, Eberle DB, Pressler BM, Moore GE, Sojka JE. 2009 Determination of 24-hour protein excretion and urine protein:creatinine ratio and correlatation with random sample urine protein:creatinine ratio in healthy horses and ponies. Am J Vet Res In press
• Uberti B, Pressler BM, Alkabes SB, Chang C-Y, Moore GE, Lescun TB, Sojka JE. 2010 Effect of heparin administration on urine protein excretion during the developmental stage of experimentally-induced laminitis in horses. Am J Vet Res In press
• Jackson LP, Sojka JE, Moore GE, Denton BD, Miller MA. 2010 Effect of season and domperidone dose on domperidone-stimulated change in adrenocorticotrophic hormone concentrations in horses with pituitary pars Intermedia dysfunction. In preparation
• Sojka JE, Jedra U, Hester N, Jackson LP, Juarez A. Topical dexamethasone and dimethyl sulfoxide solutions do not result in detectable blood levels of dexamethasone.2008 J Eq Vet Sci 2008;28:739-742

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The information generated in this area has been disseminated to large animal veterinarians via talks at state and national veterinary meetings. It has been discussed at Purdue University via lectures to junior veterinary students, senior veterinary students, and veterinarians via didactic lectures and research seminars. We are currently in discussion with a drug manufacturer to include the research findings into a diagnostic testing and treatment protocol. PARTICIPANTS: All individuals are employees of Purdue University. Dr. Janice Sojka, Dr. Michel Levy, Dr. Laurent Couetil, Dr. George Moore, Dr. Margaret Miller, Dr. Brenda Pardo, Dr. Paige Jackson, and Dr. Benjamin Uberti The project allowed 3 graduate students (Dr. Pardo, Uberti, and Jackson) to perform research required by their degree programs). TARGET AUDIENCES: The primary target audience for this work is equine veterinarians and internal medicine specialists. Other audiences included the horse owner, veterinary students, veterinary technicians, and generalists. PROJECT MODIFICATIONS: The current research has moved beyond investigating diagnostic methods for PPID to investigating the hormonal factors influencing laminitis.

Impacts
The main impact to date is the development of a new and unique method to diagnose equine pars intermedia dysfunction (PPID) using oral domperidone. PPID is the most common endocrinopathy in horses and there was a large need for this information.

Publications

• Miller MA, Pardo ID, Jackson LP, Moore GE, Sojka JE, Correlation of pituitary histomorphometry with adrenocorticotrophic hormone response to domperidone administration in the diagnosis of equine pituitary pars intermedia dysfunction. Vet Pathol. 2008 Jan;45(1):26-38.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The objective of the project - to plan, conduct, and report on projects involved with equine endocrinologic disease have been met. These have been shared via presentations at national meetings and research publications. PARTICIPANTS: Dr. Paige Jackson Dr. Ben Uberti TARGET AUDIENCES: The target audience for this work is veterinary practitioners, both mixed animal practice, large animal practice, and equine practitioners.

Impacts
Outcomes were developed using statistical analysis of obtained data. One such outcome was conducting a Special Interest Group seminar and case discussion at the 2006 American College of Veterinary Internal Medicine Forum. An internal grant based on this work was funded in 2007. Grant: Normal urinary protein-creatinine ratio in the horse and in insulin resistant ponies with and without experimentally-induced laminitis. PI Sojka JE, Co-I: Pressler B, Uberti B. Indiana Racing Commission ($8000) 2007

Publications

• Publications Couetil LL, Sojka Je. 2007. Le dysfonctionment de la pars intermedia de l hypophyse chez le cheval. Pratique Veterinaire Equine 39:19-24
• Sojka JE, Jackson LP, Moore GE, Miller M. 2006. Domperidone causes an increase in endogenous ACTH concentration in horses with pituitary pars intermedia dysfunction (Equine Cushings Disease). Proc 52th Conv AAEP. 320
• In Press: Sojka JE, Jedra U, Hester N, Jackson LP, Juarez A. 2007. Topical dexamethasone and dimethly sulphoxide (DMSO) solutions do not result in detectable blood levels of dexamethasone. Accepted Journal of Equine Veterinary Science
• Miller MA, Pardo ID, Jackson LP, Moore GE, Sojka JE. 2007. Correlation of pituitary histomorphometry with adrenocorticotrophic hormone response to domperidone administration in the diagnosis of equine pituitary pars intermedia dysfunction Accepted Veterinary Pathology

Progress 10/01/05 to 09/30/06

Outputs
Research is now ongoing to develop a dose response curve to determine the optimum dose and timing interval in a Domperidone-response test. This evocative test will be used to diagnose Pituitary Pars Intermedia Dysfunction (PPID)in horses. Currently we are testing 6 horses - 3 with PPID and 3 control using 4 doses of domperidone (0, 0.5,1, and 5 mg/kg) during 4 times of the year. This is important as there is a marked seasonal rise in ACTH levels in the autumn. Three of the 4 testing periods have been passed, and the data collection will be completed in January of 2007. This work is sponsered by the American Quarter Horse Association and the ACVIM Foundation. Additional efforts: Pituitary glands of aged horses that are posted are being systematically evaluated and graded for abnormalities. A 5 grade scale has been devised ranging from normal (1) to gross adenoma (5). It has been found that some degree of pituitary change is the rule in aged horses, not the exception. It is possible that all geriatric horses suffer from some degree of pituitary dysfunction. Additional agents that alter dopaminergic tone will be tested in order to devise a parenteral compound capable of evoking an exaggerated response in ACTH concentrations in PPID horses. Candidate agents include: apomorphine, cisapride, and perphenazine.

Impacts
It is expected that results of this study will enable early and accurate diagnosis of PPID in horses. This will allow earlier intervention in many horses and the prevention of laminitis, a life-threatening complication. In addition, early identification of PPID horses will allow us to follow the course of this disease over many years. This will result in a quantum increase in our knowledge in regards the importance of inciting events and the natural history of the disease process.

Publications

• No publications reported this period