1518 ³H BILIRUBIN UPTAKE IN NEUROBLASTOMA CELLS (N115)

Abstract

Inspite of an understanding of the biochemistry of bilirubin and the clinical picture associated with hyperbilirubinemia, no studies have defined the target of bilirubin at the cellular level. To determine this, ³H-bilirubin is prepared biosynthetic-ally in the rat by injection of ³H-δ aminolevulinic acid. ³H-bilirubin extracted from the pooled bile of 6 rats had specific activity of 3.6 × 10⁶cmp/mMol. The N115 cells seeded at a concentration of 10⁵ cells in 2 cm tissue culture dishes are adapted to grow in a protein free media. To determine the effect of time and temperature on ³H-bilirubin uptake, 0.2μCi/24nM of ³H-bilirubin was added to the media at bilirubin-albumin molar ratio (B/A) of 0-1.5 and incubated from 0 min to 24 hrs at 37°C, 24°C and 4°C. The amount of bilirubin uptake plateaus at 3hrs, is affected by the concentration of albumin, but is not sharply temperature dependent. The bilirubin uptake at 3hrs in the absence of albumin was 89.3cpm/μg DNA, decreased to 42.5cpm/μg DNA at B/A of 2.5 and to 31.2cpm/μg DNA for a B/A of 0.8. To determine the effect of bilirubin concentration on uptake, ³H-bilirubin ranging from 0.005μCi to 0.040μCi were added to the media and incubated for 3 hrs at 37°C in the absence of albumin. Uptake is linearly related to the concentration of ³H-bilirubin added. The results show ³H-bilirubin uptake peaks by 3hrs, is concentration dependent and there is no sharp temperature dependence. Although the uptake mechanism has not been determined, it is limited by the amount of albumin present, and is probably a passive process.

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