500 mg/day

Hepatocytes were isolated from male Sprague-Dawley rats. Fatty acids initially were dissolved in serumfree culture medium containing 5% fatty acid-free bovine serum albumin (BSA) by stirring at 50 C and adding the minimum quantity of NaOH required to achieve a solution. These stock solutions (approximately 2-5 mM) were sterilized by passage through a 0.22 mkm filter. After deterruination of the actual fatty acid concentrations by gas liquid chromatography (GLC), stock solutions were diluted with culture medium. Hepatocytes were seeded at 2.5 x 10^6 viable cells per three ml culture medium (RPMI 1640 containing 5% fetal calf sermn, 50 mkg/ml gentamicin, 1 pM insulin and 0.1 mM hydrocortisone-21-sodium succinate) in 60 mm petri dishes and maintained at 37 C in a humidified atmosphere of 5% CO2/95% air. Exposure to fatty acids was commenced after two hr by replacing the culture medium with medium containing the fatty acids. Subsequently, the medium was changed and the cells were redosed every 24 hr.