RELEASE OF SMALL AMOUNTS OF FREE FATTY-ACIDS FROM HUMAN ADIPOCYTES AS DETERMINED BY CHEMILUMINESCENCE

A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) released from human adipocytes in vitro is described. Bovine serum albumin (BSA) is used as acceptor of free fatty acids in the incubation medium of isolated fat cells. The assay involves pretreatment with the detergent sodium dodecyl sulfate (SDS) to liberate the free fatty acids from the bovine serum albumin before activation by acyl-CoA synthetase (ACS) (EC 6.2.1.3). This is followed by oxidation of the resulting thioesters by acyl-CoA oxidase (ACO). The H2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the interval of 0.01-1 nmol in the cuvette corresponding to 2-200 mum in the sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubation volume (200 mul) of very diluted (10(4) cells/ml) human adipocyte suspensions. Samples (25 mul) containing 0.25% BSA from incubates of adipose tissue cells did not interfere with the standard curve. The analytical interference from different factors that could be used in studies of lipolysis was investigated. No interference was observed up to the following concentrations: 5 muM epinephrine, 5 muM norepinephrine, 80 muM isoproterenol, 1 mm insulin, 2.5 mm propranolol, 5 mm phentolamine, and 5 muM ascorbate. Results obtained with the present assay were highly correlated (r = 0.997) with those obtained by a 260-times less sensitive spectrophotometric kit method. The present assay can thus be used for serial studies of FFA release in diluted cell suspensions using small amounts of human adipose tissue obtained by needle biopsy. At least 250 separate determinations of FFA release can be made with 1 g of tissue.