Sensitive kinetic bioluminescent assay of glycerol release from human fat cells

A sensitive and accurate assay of lipolysis has been developed, measuring the rate of glycerol release from fat cells with a bioluminescent assay. The rate of glycerol-dependent ATP-consumption in a system consisting of glycerokinase, ATP, luciferin, and luciferase was determined kinetically as a decrease of the ATP-induced luminescence and used for calculation of the concentration of glycerol. Under the conditions employed, it was possible to measure the concentration of glycerol down to a level of about 0.5 micron mol/l. Under the same conditions, the detection limit of the usual fluorometric method was about 15 micron mol/l. The coefficient of variation obtained with the bioluminescent assay was 11% at a level of 1.0 micron mol of glycerol, 2-6% at a level of about 5 micron mol/l, and 1-3% at a level of about 20 micron mol/l. Satisfactory results were obtained in different recovery experiments. Using human fat cells, it was possible to determine the rate of glycerol release with a cell concentration in the medium of only 5,000-10,000 cells/ml. It is concluded that the bioluminescent assay of glycerol release should be preferred when there is a demand for sensitivity, e.g., when the rate of lipolysis is low and when only a small amount of biopsy material is available.