Nakamura H, Uetani Y, Komura M, Takada S, Sano K, Matsuo T
Department of Pediatrics, Kobe University School of Medicine, Japan.
Biol Neonate. 1987;52(5):273-8. doi: 10.1159/000242720.
To assess the interaction of bilirubin with albumin and to determine the site of bilirubin toxicity in cells, a study was made of the O2- production of neonatal neutrophils (PMNs) by two different stimulators: (1) concanavalin A (Con A) plus cytochalasin D (Cyt D), which acts on the cell surface, and (2) phorbol myristate acetate, which acts intracellularly. PMNs that had been separated from cord blood were incubated for 60 min at 37 degrees C in the solution with different molar ratios of bilirubin/albumin (unbound bilirubin, ranging from 0.35 to 3.92 micrograms/dl). The unbound bilirubin was determined by peroxidase oxidation method. A PMN viability of more than 96% was maintained after the incubation in each of solutions. The O2- production rate of PMNs stimulated by Con A plus Cyt D was inhibited in the presence of unbound bilirubin levels as low as 1.12 micrograms/dl, and the rate decreased as the levels of unbound bilirubin rose. The O2- production rate stimulated by Con A plus Cyt D was more remarkably inhibited than that by phorbol myristate acetate, which directly activates intracellular protein kinase C. These findings suggest that bilirubin toxicity to PMN can be shown at levels of unbound bilirubin as low as those in hyperbilirubinemic sera, and the critical site at which bilirubin exerts its toxicity is mainly in membrane level rather than on intracellular functions.
为评估胆红素与白蛋白的相互作用，并确定胆红素在细胞中的毒性作用位点，我们使用两种不同的刺激剂对新生儿中性粒细胞（PMN）的超氧阴离子生成进行了研究：（1）刀豆蛋白A（Con A）加细胞松弛素D（Cyt D），其作用于细胞表面；（2）佛波酯，其作用于细胞内。将从脐带血中分离出的PMN在37℃下于含有不同摩尔比胆红素/白蛋白（未结合胆红素，范围为0.35至3.92微克/分升）的溶液中孵育60分钟。未结合胆红素通过过氧化物酶氧化法测定。在每种溶液中孵育后，PMN的活力维持在96%以上。在未结合胆红素水平低至1.12微克/分升时，Con A加Cyt D刺激的PMN超氧阴离子生成率受到抑制，且随着未结合胆红素水平的升高，该生成率降低。Con A加Cyt D刺激的超氧阴离子生成率比直接激活细胞内蛋白激酶C的佛波酯刺激的超氧阴离子生成率受到更显著的抑制。这些发现表明，胆红素对PMN的毒性在未结合胆红素水平低至高胆红素血症血清中的水平时即可显现，且胆红素发挥毒性作用的关键位点主要在细胞膜水平而非细胞内功能上。
