As sick as a dog: test accurately quantitates inflammation marker in canines

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Ezine

  • Published: Sep 14, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: As sick as a dog: test accurately quantitates inflammation marker in canines

3-BrY

As pathogens chanced upon mutations that would allow them to pillage for their livelihood, their hosts—be they dogs or humans (or their ancestors)—had to adapt to fend off these invaders, or else succumb to the genetic graveyard.

Nature is a dog-eat-dog world—as true for our four-legged canine friends as it is for Homo sapiens. Charles Darwin allegedly coined the following: ‘It is not the strongest of the species that survive, nor the most intelligent, but the one most responsive to change.’ With this in mind, it is easy to imagine why it was necessary for man’s best friend to evolve an intricate immune system.

As pathogens chanced upon mutations that would allow them to pillage for their livelihood, their hosts—be they dogs or humans (or their ancestors)—had to adapt to fend off these invaders, or else succumb to the genetic graveyard. These issues must have been resolved quite early on, and as a result many proteins are conserved across multiple species, forming before our species branched from the phylogenetic tree. Accordingly, the billions invested in medical research for humans could benefit veterinary practices too.

Eosinophil peroxidase (EPO—not to be confused with erythropoietin) is one such conserved protein. The most abundant polypeptide within the granules contained within white blood cells (WBCs), EPO is released when WBCs are activated in response to an infection or a range of inflammatory diseases, including asthma and allergies. Since merely counting the eosinophils in a drop of blood does not give us a picture of how many are activated, scientists have long sought non-invasive markers that could inform clinical decisions and research.

EPO, after many reactions, begets 3-bromotyrosine (3-BrY). 3-BrY is both stable in canine serum and useful for veterinary uses, which therefore makes it a suitable marker of WBC activation—and more loosely of inflammation and infection.

EI-GC/MS

Many methods exist for the determination of 3-BrY in human bodily fluids. Largely based on electron capture (EC) GC-MS, these assays, however, have not been approved for application to dog sera—and nor have the EPO profiles in dogs been studied.

Galvanised by this gap in our knowledge, researchers from Texas A&M University in the USA and Kasetsart University in Thailand collaborated to develop and validate an EI-GC/MS-based assay able to accurately determine levels of 3-BrY in dog sera. They also sought to ‘establish a reference interval (RI) for serum 3-BrY concentrations in healthy control dogs,’ Sattasathuchana, the main author of the paper, writes in Veterinary Clinical Pathology.

To analyse samples, serum samples were first cleaned up with a C18 solid-phase extraction column and derivatised to make them amenable to mass analyses. Next, the derivatised samples were loaded onto a half phenyl/half dimethylpolysiloxane column and sequentially eluted these in order of volatility, from high to low, as the temperature of the helium gas was incrementally ramped from 180 to 310 °C. Volatilised components were then bombarded with electrons and the mass spectrometer was trained to detect the 3-BrY ions with a mass-to-charge ratio of 257.

Pooch profiling

Their simple assay based on EI-GC/MS could detect down to 2.5 pmol of 3-BrY and could quantitate levels from 0.63 to 50 μmol/L. What’s more, the assay was able to determine standards ranging from 0.95 to 37.48 μmol/L six times within the same assay or over six consecutive days with less than 14 % imprecision, whilst being within 79.1 to 126.7% accuracy in quantitating spiked standards ranging from 2.5 to 20 μmol/L.

Having developed the necessary tools, the authors then moved onto their second objective: to profile the baseline levels of 3-BrY in the sera drawn from 41 dogs, ranging from the tiny Shih Tzu to the gentle-giant St. Bernard. While levels below 1.12 μmol/L are considered normal, anything above should be treated with suspect, the authors write.

What was made clear, however, was that peripheral eosinophil counts did not correlate with serum 3-BrY at all, which highlights the usefulness of their developed novel assay. ‘The availability of this assay may provide a new non-invasive method to help diagnose and monitor eosinophil-associated diseases in small animals,’ claimed the authors.

Related Links

Veterinary Clinical Pathology, , 2016, Early View paper. Sattasathuchana et al. Development and analytic validation of an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-bromotyrosine in canine serum.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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