以结合性胆红素制备胆红素测定校准物的研究

Using Purified Conjugated Bilirubin as a Calibrator for Bilirubin Measurement

目的 以结合性胆红素制备胆红素测定校准物 ,改善胆红素尤其是直接胆红素测定的准确度和精密度。方法 对结合性胆红素 (Bc)和二牛磺酸胆红素 (DTB)及其重氮产物进行吸收光谱分析 ,并比较其 DoumasJ- G法总胆红素、直接胆红素反应特征。结果 结合性胆红素和 DTB及其重氮产物具有相似的吸收光谱 ,最大吸收峰也在相同位置 ,两者的总胆红素标准曲线几乎完全重合。它们的直接胆红素测定值具有显著性差异 (P<0 .0 5 ,n=5 ) ,但直接胆红素标准曲线斜率无显著性差异 (Y结合性胆红素 =0 .0 0 36 6 X+0 .0 0 933,r结合性胆红素 2 =0 .9977,P<0 .0 1;YDTB=0 .0 0 391X+0 .0 0 0 2 3,r DTB2 =0 .9987,P<0 .0 1;Pb 1 - b2 >0 .0 5 ,n1 =n2 =5 )。结合性胆红素在不同基质(牛血清白蛋白、人血清白蛋白、人血清 )中的直接胆红素 /结合性胆红素值有显著性差异 (P<0 .0 1,n=5 ) ,而不同浓度间无明显差异 (P>0 .0 5 ,n=5 ) ,其不同浓度下结合性胆红素、DTB与相应的直接胆红素测定值均呈线性趋势 ,但这两者斜率同样无显著性差异 (Y结合性胆红素 =0 .830 0 X结合性胆红素 +1.946 3,r结合性胆红素 2 =0 .9977,P<0 .0 1;YDTB=0 .885 3XDTB- 0 .0 2 5 1,r DTB2 =0 .9986 ,P<0 .0 1;n1 =n2 =5 ,Pb1 - b2 >0 .0 5 )。

Objectives To improve the accuracy and precision of the determination of bilirubin, especially direct bilirubin (DB), and the standardization of that as well. Methods Purified conjugated bilirubin (Bc)and ditaurobilirubin(DTB) and their diazo products were subjected to absorption spectrum analysis. The diazo reaction characters of their calibration solutions were compared by the method of Doumas J G(TB & DB). Results Bc, DTB and their azopigments were found to have the similar absorption spectra with the same λ max . Their TB standard curves almost superposed together all over. Although the slopes of their DB standard curves were not markedly different ((Y Bc =0.00366X+0.00933, r Bc 2=0.9977, P <0.01; Y DTB =0.00391X+0.00023, r DTB 2=0 9987, P <0.01; P b1 b2 >0.05, n 1=n 2 =5), the DB value measured for Bc differed from that for DTB( n =5, P <0 05). In addition, the calibrators made from Bc based different matrices, such as HSA, BSA and human serum, were significantly different in DB/Bc, but no difference was seen among the concentrations. Furthermore, the DB values determined for DTB or Bc increased linearly with the corresponding concentrations, respectively, with no difference between the slopes (Y Bc =0.8300X Bc +1.9463, r Bc 2=0.9977, P <0.01; Y DTB =0.8853X DTB -0.0251, r DTB 2=0.9986, P <0.01; n 1=n 2 =5, P b1 b2 >0.05).Conclusions The results demonstrate that the diazo reaction characters of Bc are identified with those of DTB. However, under the condition of DB, Bc reacts differently from DTB. This study also indicates that as a calibrator of DB based human serum, Bc has the similar constant effect of HCl as serum samples do, so it is a more reliable calibrator to eliminate the matrix effects.