MEASUREMENT OF APOLIPOPROTEIN-B IN VARIOUS CELL-LINES - CORRELATION BETWEEN INTRACELLULAR LEVELS AND RATES OF SECRETION

We have standardized simple but sensitive enzyme-linked immunoassays t o understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibo dies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts o f low density lipoproteins to obtain a standard curve. Conditioned med ia were added to other wells in parallel, and the amount of apoB was q uantitated from a linear regression curve. To standardize renditions f or the measurement of intracellular apoB, cells were homogenized and s olubilized with different concentrations of taurocholate. We found tha t 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2 , Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular leve ls of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were p ositively correlated (r(2) = 0.81, P = 0.0009) in HepG2 cells. Further more, a positive correlation (r(2) = 0.88, P < 0.0001) was found betwe en intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and a poB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but no t the lipid-free apoB, was tightly controlled.