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Home » Archive » 2022

TDK conference 2022

Canine Serum & Urine Hepcidin Measurements using LCMS & ELISA Techniques
Hotchkiss David - year 6
University of Veterinary Medicine Budapest, Department of Internal Medicine
Supervisors: Dr. Ágnes Sterczer, Dr. Zsuzsanna Vizi

Abstract:

Hepcidin-25 is the key peptide hormone controlling vertebrate iron metabolism. There has been debate however in the last two years about the structure of canine hepcidin-25 and this project set out to establish whether more than one isoform of canine hepcidin-25 exists. By means of LCMS analysis of both urine and serum samples from a random sample of 25 healthy dogs, it was possible to demonstrate that indeed two isoforms of canine hepcidin-25 do exist; these have been christened hepcidin-25α (DTHFPICIFCCGCCKTPKCGLCCKT) and hepcidin-25β (DTHFPICIFCC

GCCKTPKCGFCCRT). This research project also investigated the relative reliability of ELISA versus LCMS as an assay technique for measurement of hepcidin-25 concentrations in urine. ELISA was found to have a reliability of only 73%, so measurement of hepcidin-25 concentrations by LCMS remains the “gold standard” approach. As a part of this study, it was also demonstrated that a significant natural variation exists in hepcidin-25/creatinine ratio in the urine of healthy animals with time, and it is recommended therefore that the mean of at least 3, but preferably 5, samples should be taken, evenly spaced over a period of at least 3 days, in order to determine reliably the hepcidin-25/creatinine ratio in a patient’s urine. No statistically significant correlations were found at all between the measured concentrations of the two canine hepcidin-25 molecules and the measured SeFe, TIBC, MCH, MCHC or MCV parameters in the sampled dogs, whether measurements were made in serum or in urine. The mean urinary total hepcidin-25/creatinine ratio in healthy dogs was found to be 1.08 ± 0.10 and the mean serum total hepcidin-25 concentration was measured at 79.8 ± 4.9 ng/ml, with around 65% of this being hepcidin-25β. Inflammation seen in a second sample of sick dogs resulted in a statistically significant increase in the concentration of both serum hepcidin-25α and hepcidin-25β.



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