OBJECTIVES

Liver disease is probably one of the most frequently stated and misunderstood diagnosis made by reptiles clinicians and pathologist alike. Liver diseases may lead to elevations of several enzymes, including ASAT, ALAT, GGT, AP and LDH but unfortunately most of these parameters are widely distributed in other tissues and assessment of liver state and function could be difficult. Bile acids are considered useful in the assessment of liver function in many birds and mammals, but their application in reptile medicine has yet to be conclusively determined.

The purpose of this study was to investigate plasma bile acids concentrations in clinically normal captive tortoises belonging to the more often species evaluated by reptile practitioners. Bile acid assay were determined using in house lab methodology. We included some sick tortoises which were liver biopsied, comparing plasma bile acids concentrations and pathology findings.

MATERIALS

32 Herman´s tortoises (Testudo hermanii), 30 Greek tortoises (Testudo graeca), 7 hinged-back tortoises (Kynixis belliana) and 8 Russians tortoises were included in this study. All animals were in good health status and previously hematology and biochemistry values of each tortoise were determined. Heparinized blood samples used for this investigation were obtained from jugular vein or subcaparacial vein and samples were centrifuged and processed immediately or frozen at -20 ª C until processed (no more than 12 days). Plasma bile acids were measured with a Multistat F.L.S- III Centrifugal analyzer, with an enzymatic kit for spectrometric endpoint determination (Sterognost 3-alpha; Flu, Nyergard &Co, Norway). The enzymatic test is highly specific for free bile acids and their corresponding glycine and taurine conjugates. The sensitivity of the test system is 0.1 umol/L. Liver biopsies were taken under 10 mg/kg propofol IV anesthesia, through the instrument port of the rigid endoscope (2.7mm Hopkins telescope, Karl Storz Endoscopy, Germany) by right inguinal approach. Small tissue samples were placed into histology filters before being submitted in 10 % neutral buffered formalin for histopathology. Biopsies sampling didn't take more than ten minutes.

RESULTS

The results of Plasma bile acids concentrations obtained are shown in table 1. From the four species of tortoises tested there was not significant difference between the sexes of each species.

Not always a relationship between pathology diagnostics and plasma bile acids levels could be established. Propofol IV anesthesia is a good choice for endoscopic liver biopsy in tortoises.

CONCLUSION

Preliminary values published suggest normal values of less than 60 umol/L using commercial enzymatic assays for 3-alpha-hydroxy-bile acids. The major bile acid varies with the taxonomic order of the reptile, and concern therefore must exist as to universal application of a single assay across all reptiles. Practiclas protocols for bile acids stimulation test have not been documented, but, assuming specificity and sensitivity, they may offer an accurate assessment of liver function and a means of identifying those cases of severe liver compromise in which little or no hepatocellular damage exists. On the other hand, there appears to be nothing to surpass the diagnostic value of a hepatic biopsy for both microscopic interpretation and where infectious agents may be present, microbiological culture. The effects of feeding on plasma bile acid concentration may partially explain the range of values obtained. Avian studies have shown that up to 4.5 fold increase in plasma bile acid can be found in postprandial samples from some individual pigeons, while the postprandial sample increases never exceed 1.7 times the upper limit of the reference interval established for the group. In the case of tortoises, sample were obtained within 2 days of fasting but further investigations are need to determined the effects of feeding.