Ultra Turrax system

The candidate compound is added to the perfusate before the isolated liver is transferred to the perfusion setup. After 5 minutes recirculation in the system without the organ a sample of the perfusate is collected for determination of the starting concentration of the candidate compound (value at time 0). Then the isolated liver is connected to the perfusion chamber and subsequently samples of the perfusate are taken in 10 to 15 minute intervals as well as bile samples are taken in 15 minutes intervals for the determination of compound levels. The volume of excreted bile per 30 minutes is determined gravimetrically (difference between tube weight without and with bile per collection period) with the assumption that 1 g is equivalent to 1 ml of bile. If radioactive-labelled candidate compounds are used the intervals for bile collection can be much shorter (1 to 2 minutes). The liver is perfused for 2 hours and at the end of the perfusion experiment the liver is removed and immediately frozen for determination of tissue level of the compound in liquid nitrogen and later on stored at -20 °C until analysis. For analysis of tissue levels of the candidate compound the frozen livers are homogenized in water (1 g tissue in 1 ml water (or solvent to extract the candidate compound from the tissue)) using an ultra-turrax. 

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