Urine Test Developed for Vitamin-E-Related Disease Screening
Veterinarians could soon determine which horses are at risk of certain neurologic diseases through a simple urine test that reveals how a horse breaks down vitamin E.

By counting and analyzing compounds left in urine (and blood) when a horse metabolizes vitamin E, researchers say they have found a way to measure the vitamin’s presence in—and its impact on—a horse’s body. The findings could also lead to a way to diagnose a debilitating neurologic disease—equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM)—which veterinarians can currently only test for and diagnose postmortem, said Carrie Finno, PhD, DVM, Dipl. ACVIM, associate professor and Gregory L. Ferraro Endowed Director of the Center for Equine Health at the University of California, Davis, School of Veterinary Medicine.

“This was the first study to actually measure vitamin E metabolites in horse blood and urine as an initial step toward measuring vitamin E metabolism in horses,” she said.

Vitamin E in Nature and in the Equine Body

Provided primarily to horses through fresh green pasture grass, vitamin E plays an important role in musculoskeletal and neurologic health. Low levels of the vitamin could lead to health issues in horses, especially those who already have a genetic predisposition for diseases such as eNAD/EDM. But clinical signs of such conditions often look very much like other neurologic diseases, and veterinarians struggle to make a clear diagnosis, Finno said. Usually, the diagnosis only becomes definite during postmortem examinations when scientists find telltale degenerative lesions in the brainstem and spinal cord.

A urine (or blood) test that identifies how quickly a horse metabolizes vitamin E could potentially point to eNAD/EDM, allowing veterinarians to initiate treatment and make informed decisions with the owner about the horse’s future, she said.

Such a test could also further research that could help explain how and why the disease develops, possibly leading to better therapeutic options.

The Challenges of Screening for Vitamin E—and the Solutions

Vitamin E is difficult to find in urine because the body metabolizes (digests) it before it gets excreted into the urine. “So, while we cannot identify vitamin E itself, we can identify and quantify the metabolites of vitamin E,” Finno said.

“A urine vitamin E test will detect the metabolite levels of the vitamin,” she continued. “Since you can also measure these metabolites in blood, it is possible to determine the rate of vitamin E metabolism in a particular horse. Then, if certain horses (such as those with eNAD/EDM), have increased vitamin E metabolism rates, we can understand why those particular horses require more vitamin E during development than an unaffected horse.”

Finno and her fellow researchers developed an ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method for horse urine, and they validated it by measuring the amount of vitamin E and counting its metabolites in the animals’ urine through special processes known as acidic hydrolysis and solid phase extraction.

Then they evaluated the new test by spiking normal horse urine and synthetic human urine with different concentrations of vitamin-E-related compounds. This allowed them to determine that their method was more than 90% accurate. Specifically, the test picked up three major vitamin E metabolites: alpha‐carboxyethyl hydroxychroman (α‐CEHC), alpha‐carboxymethylbutyl hydroxychromans (α‐CMBHC) and gamma‐carboxyethyl hydroxychroman γ‐CEHC.

The team then tested the urine of six healthy horses living on green pasture to establish baseline “normal” levels of vitamin E metabolites in equine urine.

It’s an exciting first step, Finno said. But further research is necessary to understand the metabolites’ link with eNAD/EDM.

Fortunately, that research is already well advanced, Finno said. “We actually have a follow-up paper (currently under [peer] review) that examines the rate of vitamin E metabolism in horses with eNAD/EDM,” she told The Horse. “So stay tuned for those results.”