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Optimization of an Isolated Perfused Rainbow Trout Liver Model: Clearance Studies with 7-Ethoxycoumarin
Citation:
NICHOLS, J. W., A. D. HOFFMAN, AND P. N. FITZSIMMONS. Optimization of an Isolated Perfused Rainbow Trout Liver Model: Clearance Studies with 7-Ethoxycoumarin. AQUATIC TOXICOLOGY. Elsevier Science Ltd, New York, NY, 95(3):182-194, (2009).
Impact/Purpose:
The extended viability of isolated trout livers achieved in this study creates new opportunities for research on hepatic function if fish.
Description:
Isolated trout livers were perfused using methods designed to preserve tissue viability and function. Liver performance was evaluated by measuring O2 consumption (VO2), vascular resistance, K+ leakage, glucose flux, lactate flux, alanine aminotransferase (ALT) leakage, and metabolic clearance of 7-ethoxycoumarin (CLH,7-EC). VO2 immediately following isolation was increased by the addition of pyruvate and EDTA to the liver clearing solution. Maximal rates of VO2 during the post-recovery period were observed at perfusion rates of 1.0 mL/min/g-liver or higher. Livers perfused with solutions containing 1.0, 2.5, or 10.0 g/L bovine serum albumin (BSA) exhibited relatively stable physiological performance for up to 10 h. CLH,7-EC appeared to decrease rapidly during the first 1-2 h, probably due in part to non-steady-state conditions. CLH,7-EC declined slowly thereafter, decreasing by 30 to 40% between 2 and 10 h. A linear equation was subsequently developed to correct measured levels of clearance for this decrease in metabolic activity over time. To illustrate the value of this preparation, experiments were conducted to examine the effects of protein binding on hepatic clearance of 7-EC. Clearance rates corrected for declining metabolic activity (CLH,7-EC,CORR) changed in nearly direct proportion to changes in the free concentration of 7-EC efferent to the liver, as predicted by theoretical models of liver function. Additional studies were performed to characterize the concentration-dependence of 7-EC metabolism. Metabolic activity increased linearly with the total concentration of 7-EC afferent to the liver resulting in constant levels of CLH,7-EC,CORR? CLH,7-EC,CORR values for four livers averaged 12.0 ± 5.0 mL/h/g-liver (mean ± SD, n= 60 individual determinations) and were in good agreement with an estimate of hepatic clearance obtained by extrapolating published in vitro data from isolated trout hepatocytes. The extended viability of isolated trout livers achieved in this study creates new opportunities for research on hepatic function if fish.