BACKGROUND:Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. MATERIALS AND METHODS:Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4 h incubation at pH 7.4, with increasing molar ratios of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. RESULTS:Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 degrees C vs. 4 degrees C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P < 0.01). The loss of membrane lipids was remarkable at molar ratios > or = 0.5, both at 4 degrees C and 37 degrees C (P < 0.01), although correlation with bilirubin concentration was only significant at 37 degrees C (r = 0.971; P < 0.01). CONCLUSIONS:These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocyte-bound bilirubin during severe hyperbilirubinaemia.

译文

背景:新生儿高胆红素血症仍然是最常见的需要治疗干预的临床疾病之一。然而,仍然缺乏可靠的胆红素毒性指标。这促使我们研究(a)胆红素浓度增加所产生的细胞毒性事件的进展; (b)膜脂质包装与胆红素与红细胞结合的相关性; (c)氯仿提取与白蛋白提取的可靠性,以评估与红细胞结合的胆红素和细胞毒性。
材料与方法:在pH值下孵育4小时后,在4摄氏度或37摄氏度下测定了人类红细胞的形态学改变,游离胆红素,红细胞结合胆红素(白蛋白和氯仿提取物),溶血和膜释放的脂质7.4,随着胆红素与白蛋白的摩尔比增加(0.5-5)。通过形态分析评估白蛋白洗涤引起的细胞毒性的可逆性。
结果:在摩尔比下,观察到37℃vs. 4℃观察到游离胆红素降低,白蛋白提取(表面/非聚集胆红素)降低的红细胞结合胆红素浓度和氯仿提取(深层/聚集胆红素)的较高值。 > 1.胆红素浓度和温度升高时,胞浆菌增多,白蛋白洗涤不能完全逆转。摩尔比为1时,溶血作用已经很明显,摩尔比为3和5时,溶血作用因温度而增强(P <0.01)。摩尔比>或= 0.5时,膜脂的损失在4摄氏度和37摄氏度时均显着(P <0.01),尽管与胆红素浓度的相关性仅在37摄氏度时才显着(r = 0.971; P <0.01 )。
结论:这些结果表明增加脂质流动性和高胆红素浓度促进膜胆红素易位和毒性。他们还显示白蛋白不能取代位于膜内深处或聚集的胆红素,而胆红素又被氯仿去除。因此,在严重高胆红素血症期间,用氯仿提取的胆红素而不是白蛋白提取的胆红素是评估红细胞结合胆红素的更准确的参数。

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