RELEASE OF SMALL AMOUNTS OF FREE FATTY-ACIDS FROM HUMAN ADIPOCYTES ASDETERMINED 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 fatt y acids in the incubation medium of isolated fat cells. The assay invo lves pretreatment with the detergent sodium dodecyl sulfate (SDS) to l iberate the free fatty acids from the bovine serum albumin before acti vation 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 H 2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the i nterval of 0.01-1 nmol in the cuvette corresponding to 2-200 mum in th e sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubati on volume (200 mul) of very diluted (10(4) cells/ml) human adipocyte s uspensions. Samples (25 mul) containing 0.25% BSA from incubates of ad ipose tissue cells did not interfere with the standard curve. The anal ytical interference from different factors that could be used in studi es of lipolysis was investigated. No interference was observed up to t he following concentrations: 5 muM epinephrine, 5 muM norepinephrine, 80 muM isoproterenol, 1 mm insulin, 2.5 mm propranolol, 5 mm phentolam ine, 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 b e used for serial studies of FFA release in diluted cell suspensions u sing 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.