Effects by heme, insulin, and serum albumin on heme and protein synthesis in chick embryo liver cells cultured in a chemically defined medium, and a spectrofluorometric assay for porphyrin composition

Abstract

Primary chick embryo liver cells, which had been previously cultured in Eagle's medium containing 10% fetal bovine serum, had the same characteristics (inducibility of delta-aminolevulinic acid synthetase and synthesis of plasma proteins) when cultured in a completely defined Ham F-12 medium containing insulin. Insulin was active in the physiological range; 2 to 3 nM were sufficient to increase the induced delta-aminolevulinic acid synthetase to 50% of the maximum effect obtained with a saturating amount of insulin (30 nM). Serum albumin added to the Ham-insulin medium caused protoporphyrin but not uroporphyrin, generated in the cultured liver cells, to be transferred to the medium. As little as 10 mug of human serum albumin per ml caused the transfer of one-half of the protoporphyrin. Bovine serum albumin was only about 1/30 as effective. A spectrofluorometric method and calculation procedure are described for quantitation, in the nanomolar range, of total porphyrin and the percentage of this that is protoporphyrin or uroporphyrin plus coproporphyrin. The method is satisfactory for the measurement of porphyrins generated by 1 mg wet weight of cells in culture in 20 hours. Heme (0.1 to 0.3 muM), when added to the medium as hemin, human hemoglobin, or chicken hemoglobin, specifically inhibited the induction of delta-aminolevulinic acid synthetase by one-half. This high sensitivity for heme was observed under conditions in which the defined medium was free of serum and where a chelator of iron was added to the medium to diminish the synthesis of endogenous heme. Heme endogenously generated from exogenous delta-aminolevulinic acid also inhibited the induction; chelators of iron prevented this inhibition. The migration of heme from the mitochondria to other portions of the cell is discussed in terms of the affinities of different proteins for heme. A hypothesis of a steady state of liver heme metabolism, controlled by the concentration of "free" heme, is presented. The different effects of heme on the synthesis of a number of proteins are summarized.