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    Purification and Partial Characterization of Canine Glutathione Transferase Alpha and the Development of an Immunoassay for the Measurement of Canine Glutathione Transferase Alpha Concentration in Serum (2016)

    Art
    Hochschulschrift
    Autor
    Mischel, Madeline (WE 20)
    Quelle
    Berlin: Mensch und Buch Verlag, 2016 — VIII, 119 Seiten
    ISBN: 978-3-86387-756-9
    Sprache
    Englisch
    Verweise
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000103394
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    Abstract / Zusammenfassung

    Glutathione transferase alpha (GSTA) belongs to a diverse family of isoenzymes involved in the detoxification of a range of xenobiotic compounds by conjugation to glutathione. In humans, GSTA has been identified as an earlier, more specific, and more sensitive indicator of hepatocellular injury than the commonly used aminotransferases, regardless of the cause of hepatic injury. Canine GSTA (cGSTA) has also been shown to detect hepatic injury induced by warm ischemia in the dog.

    The aim of this study was to develop a simple and reproducible protocol for the purification of cGSTA from canine liver, to partially characterize cGSTA, to produce anti-cGSTA antibodies in order to develop and analytically validate an RIA for the measurement of cGSTA in serum, and to compare serum cGSTA concentrations between healthy and dogs with spontaneous hepatocellular disease.

    An efficient, simple, and reproducible protocol for the extraction and subsequent purification of glutathione transferase alpha from canine liver was established and some of the biochemical properties of this protein were evaluated.

    Amino acid analysis as well as liquid chromatography–mass spectrometry (LC/MS/MS) analysis showed a high homology to the isoenzymes cGSTA2 and cGSTA3, respectively. The molecular weight of cGSTA was estimated by SDS-PAGE to be 25 kDa. By MALDI-TOF mass spectrometry, the mass of cGSTA was more precisely determined to be 25,651 Da. Isoelectric focusing of cGSTA revealed an isoelectric point between 9.1 and 9.3 and the specific absorbance at 280 nm was determined to be approximately 1.05.

    Antibodies against cGSTA were raised in rabbits and an RIA for the measurement of cGSTA was developed and analytically validated. However, the assay did not show acceptable linearity, accuracy, precision, or reproducibility, which was assumed to be due to a lack of sensitivity of the assay leading to most clinical samples falling towards the lower limit of the assay’s working range. The working range of this assay was determined to be 6.25 to 400 μg/l. In order to determine whether there would be merit in developing a more sensitive assay for the measurement of cGSTA a preliminary reference interval of <14.35 μg/L was established. Finally, serum concentrations of cGSTA were measured in 49 healthy and 45 diseased animals and compared between the two groups of dogs to assess if cGSTA may have any utility as a biomarker for hepatocellular injury in dogs. Serum cGSTA concentrations were measurable in 39 of 45 dogs (86.7%) with hepatic disease. The median serum concentration of cGSTA was significantly higher in dogs with liver disease (6.25 – 400 μg/L, median: 36.4 μg/L; p<0.0001) than in healthy dogs (6.25 – 400 μg/L, median: 6.25 μg/L; p<0.0001). When separating the diseased dogs into 4 groups, cGSTA was detectable in 3 of the 7 dogs (42.9%) with congenital portosystemic shunts (6.25 – 46 μg/L, median: 6.25 μg/L; p<0.0001), with 4 dogs having serum cGSTA concentrations below the lower detection limit. It was also detectable in 14 of the 15 dogs (93.3%) with chronic hepatitis (13 – 400 μg/L, median: 60 μg/L; p<0.0001), except of one dog that had a serum cGSTA concentration above the upper detection limit of the assay. Serum cGSTA concentrations were furthermore measurable in all 7 dogs (100%) with hepatic tumors (21 – 126 μg/L, median: 30 μg/L; p<0.0001) and in 15 of the 16 dogs (93.75%) with other liver diseases (15 – 400 μg/L, median: 36 μg/L; p<0.0001), with one dog also having a serum cGSTA concentration above the upper detection limit. This suggests that the measurement of serum cGSTA may be able to distinguish between healthy dogs and dogs with hepatic disease, though it may not be useful to distinguish between different types of liver disease, nor as a marker for CPSS.

    In conclusion, serum cGSTA appears to be a promising marker for hepatic disease and warrants further assessment as a serum marker of hepatocellular injury in dogs. However, before this work can begin a more sensitive assay for its measurement must be developed and analytically validated.