Diagnosis of PPID and IR

Equine Cushing's disease, also known as Pituitary Pars Intermedia Dysfunction (PPID), and Insulin Resistance (IR) are distinctly separate conditions. The often overlapping signs and symptoms can make diagnosis difficult.

Pituitary Pars Intermedia Dysfunction (PPID) — Equine Cushing's Disease

PPID/Cushing's Disease is a disorder of the pituitary gland and results in hormonal disturbances. The pituitary gland secretes high levels of the hormone ACTH (adrenocorticotrophic hormone), which stimulates the production of cortisol, a stress hormone.

Recommended Diagnostic Testing for PPID

Endogenous ACTH — a single blood draw to measure the level of endogenous ACTH — is diagnostic for PPID in equines and recommended by the ECIR Group. Horses should not be fasted before ACTH testing. Fasting considerably lowers ACTH. The sample requires special handling for accuracy. Consider TRH Stimulation for suspect horses with normal ACTH. There are seasonal fluctuations to consider for diagnosis of PPID. Please review the section on Seasonal Rise.

In the United States, the ECIR Group suggests:

• Cornell University
  https://www.vet.cornell.edu/animal-health-diagnostic-center/laboratories/endocrinology
• van Eps Laminitis and Endocrinology Laboratory (Penn Vet)
  https://www.vet.upenn.edu/research/research-laboratories/van-eps-laboratory/
• BET Labs
  https://betlabs.com/

From Cornell Endocrinology Submission Guidelines

For ACTH an EDTA plasma tube (usually lavender top) is required for specimen collection. Collect blood to the tube fill volume, gently mix by inversion, and chill immediately by refrigeration or immersion in an ice bath. Separate plasma from cells by centrifugation as quickly within 4 hours for horses. Centrifuge for 5-10 minutes (preferably in a refrigerated centrifuge), transfer the plasma into shipping tube, and freeze immediately. Ship with frozen cold packs using an overnight courier service. Plasma samples should arrive frozen (strongly recommended) or adequately chilled (i.e., kept near 4C at all times).

In Canada, the ECIR Group suggests Guelph University Laboratory.

From Page 28, 2009 Lab Services User Guide for Guelph University, E. Endogenous adrenocorticotropic hormone (ACTH) Assay:

Equine HAC. Endogenous ACTH assays are the most useful test for diagnosing pituitary tumors in horses. One limitation is that ACTH is a fragile hormone that requires meticulous care in handling. Blood should be collected in a chilled silicone coated EDTA tube, centrifuged immediately, plasma collected in a plastic (nylon) tube and kept frozen until assayed.

https://www.uoguelph.ca/ahl

Examples of other labs world-wide are:

In Australia and New Zealand, Gribbles Pathology and Charles Sturt University

Gribbles will do Leptin testing.

http://www.gribblesvets.com.au/

https://www.gribblesvets.co.nz

http://www.csu.edu.au/vetservices/vdl

In the UK, Liphook Equine Hospital Laboratory

http://liphookequinehospital.co.uk/equine-laboratory/

Other Diagnostic Tests for PPID

The Dexamethasone Suppression Test (DST): The DST measures cortisol response to an injection of dexamethasone. In normal horses, dexamethasone causes profound suppression of cortisol release. Once considered the gold standard for testing, this test has largely been replaced by Thyroid-Releasing (TRH) Stimulation Stimulation Test because of the risk of dexamethasone inducing laminitis in insulin resistant horses.

The Domperidone Response Test involves oral, or possibly intravenous, dosing with the drug domperidone, which causes an approximately 300% rise in ACTH in a horse with PPID but not in a normal horse.

The Thyroid-Releasing (TRH) Stimulation Test: Thyroid releasing hormone (TRH) stimulates release of ACTH. In normal horses the rise is very small but in PPID horses it is pronounced. Samples for ACTH are drawn before and 10 minutes after TRH injection. This test has more false positives than endogenous ACTH but because of its higher sensitivity is a good test for horses strongly suspected to be early PPID horses but which test negative by endogenous ACTH. Results are not reliable during the fall seasonal rise.

Urinary Cortisol:Creatinine Ratio is a test done on urine as a screen for PPID/Cushing's disease. It is not 100% diagnostic, but is highly suggestive. It is more accurate than blood cortisol levels in detecting increased production of cortisol.

Cortisol Rhythm is not diagnostic for PPID/Cushing's Disease, and is considered to have 30%+ false-positive readings.

Equine Metabolic Syndrome (EMS)

Most veterinarians now know what EMS is, and that horses may have EMS without having PPID/Cushing's Disease. A lot of confusion, however, remains about how to correctly diagnose it.

Symptomatically, most but not all, EMS horses have a body condition score of 6 or higher (or a history of being "easy keepers"), abnormal fat collections, such as fatty crests, fat in the eye hollows, or other fat pads. These horses are often brought to the vet's attention because of laminitis.

Blood tests for EMS — insulin, glucose, leptin, and adiponectin

The hallmark of EMS is high levels of insulin — hyperinsulinemia. This is caused by the failure of insulin-sensitive cells to respond to "normal" levels of insulin.

There are many controversies over how best to test for EMS in equines. There is a dynamic interaction between glucose and insulin that changes over the course of the day. Insulin levels change when the horse eats, sleeps, or exercises. A low-sugar/starch diet can influence insulin results. A normal level will depend on the conditions of the test. 

The fat-derived hormone, leptin, is also usually abnormally elevated in insulin resistance. There are many other things that can lower or increase leptin:  age, weight, sudden changes in nutrient requirements such as foaling, starting an exercise program, and differences between breeds. ECIR does not recommend routine testing for this hormone.

Most university and commercial labs offer serum insulin and glucose tests:

• https://www.vet.upenn.edu/research/research-laboratories/van-eps-laboratory/
• https://www.vet.cornell.edu/animal-health-diagnostic-center/laboratories/endocrinology
• https://betlabs.com/
• https://www.gribblesvets.com.au/veterinarians/our-tests/equine-testing/
• https://liphookequinehospital.co.uk/laboratory/

Adiponectin is used in the UK and the EU and may also be tested in the US; however, there is no additional value to adiponectin if insulin testing is performed. Adiponectin helps to regulate glucose and fat burning, and to maintain insulin sensitivity. Low levels are associated with EMS. It has come to be preferred over leptin because it is not influenced by things like weight or exercise, and also because it was the only factor other than insulin levels that predicted laminitis risk.

• https://www.ncbi.nlm.nih.gov/pubmed/27363591

Testing in an acutely laminitic horse. It is probably best to refrain from testing for EMS until the acute phase of laminitis has been controlled. There's a good chance that an acutely laminitic horse is going to test positive for EMS, regardless of the cause, because of the pain and stress.

Horses should not be fasted before testing. This is done only for human tests. If a horse is meal fed — periods in excess of 6 hours when the horse has nothing to eat — the first meal after the fast will generate a larger insulin spike than subsequent closer-together feedings, even if the meal is exactly the same. Recent research has confirmed the 20-year recommendation of non-fasted testing from the ECIR Group. For equines on SGLT2i drugs, please see SGLT2i Use for EMS.

To avoid a false-positive result, the horse should have hay available at all times the night before and day of the testing. If that's not possible (e.g., a ravenous horse that will inhale as much of anything you put out as quickly as possible), make sure the blood is drawn at least 4 hours after the first meal of the day, keeping some hay in front of the horse until the testing is done.

The horse should not be fed anything except hay prior to testing. The horse also should not be exercised within four hours of the test, and avoid shipping before testing if at all possible. Stress and exercise influence glucose, insulin, and leptin dynamics.

About the proxies and EMS Calculator. For decades ECIR Group members successfully used the EMS Calculator to guide diagnosis and monitoring. Since developed by Virginia Tech, the use of the proxies that informed the calculator has been affirmed by many research publications. In ensuing years, laboratories in the US have instituted changes in assays. In addition, a wide variety of labs around the globe are now offering testing using various assays. These facts make the application of the proxies for diagnosis or monitoring more difficult to assess correctly. The ECIR Group no longer recommends use of the calculator as an individual diagnostic or monitoring tool.

Insulin levels the ECIR Group supports for diagnosis. Three publications support what ECIR Group has recommended and has followed for over two decades:

1. Ollie, et al., (2019), determined the upper limit of normal for a fasting insulin should be 5.2 uIU/mL.
2. Lindase, et al., (2021), recommended a cutoff for fasting insulin of 9.5 uIU/mL using a euglycemic hyperinsulinemic clamp as the reference method.
3. Trieber, et al., (2005), established data of an upper limit of 13 uIU/mL for normal ponies on pasture at the height of the laminitis season. 

If the equine is tested exactly as ECIR Group protocol states, the following would apply:

1. under 13 uIU/mL: negative if fed prior to testing.
2. under 9.2: negative if fasted prior to testing.
3. over 13: likely EMS*.

*Breed and exact test method used could influence the result. Insulin over mid-teens should be highly suspicious if tested under ECIR protocols.

Most EMS horses do not have elevated blood glucose. Tests resulting in a diabetic range will depend on specific laboratory and assay, dietary intake and timing of last meal.

If the equine is tested exactly as ECIR Group protocol states, the following would apply:

1. under 100 mg/dL: normal on an EMS-appropriate diet.
2. 101 to 115 mg/dL: suspicious on an EMS-appropriate diet.
3. over 115 mg/dL: diabetic on an EMS-appropriate diet.

Important facts to remember:

1. Duplicate testing by ECIR Group members at laboratories using different insulin assays has shown insulin results may vary significantly between laboratories. 
2. Some assays are more accurate at lower values, others at higher values.  For this reason, it is important to keep the "big picture" in mind when there is a questionable diagnosis. Consider clinical signs as well as the laboratory numbers. When laboratory results do not match the clinical picture, consider repeat testing at another facility. 
3. For purposes of monitoring response to therapy: Once a diagnosis is made, follow-up testing should be performed at the same laboratory.
4. A horse can have EMS and still have an insulin level that is within the laboratory reference range. See Lab Reference Ranges and Fasting.

Information Contained On This Page Adapted From
Equine Cushing's & Insulin Resistance Course
Offered by Eleanor M. Kellon, VMD www.drkellon.com