Loss of cell constituents from hepatocytes on centrifugation

In studies of the metabolism of isolated hepatocytes, it is often necessary to measure the concentrations of cell constituents both in cells and medium. When hepatocytes are separated in the special tubes of Hems, Lund & Krebs (1975) (Biochem. J. 150, 47--50), they lose much glucose, urea and Na+, whereas there is no loss of K+, glutamate, aspartate and adenine nucleotides. Cell water is also lost, as measured by the distribution of 3H2O. This loss is mainly due to an exchange of cell water with the aqueous solution in the stems of the tubes through which the cells pass on centrifugation. In general, substances are lost only when the intracellular concentration is equal to, or lower than, the extracellular concentration. Probably solutes are lost because they travel with the water unidirectionally out of the cell. A loss of solute does not occur when the cells are centrifuged in conical tubes with a layer of silicone oil between the cell suspension and the deproteinizing layer. The reasons for the loss occurring in the special separation tubes are discussed.