Formation and disposition of newly synthesized heme in adult rat hepatocytes in primary culture.

Studies with the intact liver have suggested that newly synthesized heme exists transiently in a small pool before its incorporation into tissue heme proteins. The same or a closely related pool may regulate synthesis of heme and serve as the precursor of "early peak" bilirubin. To delineate this postulated pool by a direct approach, we have utilized primary cultures of adult rat hepatocytes. Cultures pulse-labeled with delta-amino[3H]levulinic acid at various time points were fractionated into 105,000 X g supernatant and pellet. Labeled heme appeared within 1 to 2 min in the cytosol fraction, followed by transfer to the pellet. The kinetics of heme formation and transfer and of labeled bilirubin production were analyzed by computer simulation utilizing the least squares method. The experimental findings conformed best to a four-compartment model that includes a second cytosolic heme compartment exchanging with the initially labeled compartment but not serving as a direct precursor of bilirubin. Calculation of apparent rate coefficients indicated that, in cultured hepatocytes, 20% of newly formed heme is converted directly to bile pigment, whereas 80% is utilized for formation of cellular heme proteins (64% in the pellet, 16% in the second cytosol compartment). This experimental approach has provided direct evidence for a rapidly formed cytosolic heme fraction which appears to be identical with the previously postulated regulatory or "unassigned" heme pool of the liver.