Jansen P L, Peters W H, Janssens A R
J Hepatol. 1986;2(3):485-94. doi: 10.1016/s0168-8278(86)80060-5.
The clinical value of serum bilirubin subfractionation, using high-performance liquid chromatography (HPLC), was studied in 26 patients with primary biliary cirrhosis (PBC) from whom 59 serum samples were obtained. Total bilirubin (TB) levels were determined by alkaline methanolysis and HPLC (TBHPLC) as well as by the conventional diazo method (TB diazo). The correlation between TBHPLC and TBdiazo was significant (P less than 0.001) but bilirubin concentrations measured by HPLC amounted to only about 30% of those by diazo method. This difference is probably caused by tightly albumin-bound bilirubin, so called 'delta bilirubin' or 'bilirubin-albumin', which is measured by the diazo but not by the HPLC method. Bilirubin conjugates were present in 16 sera of 26 patients; 14 of these had normal total bilirubin levels. There was an excellent correlation between bilirubin conjugates (BcHPLC) and TB diazo (P less than 0.001). In all conjugate-containing samples, mono- and diconjugates were present in about equal proportions. Our results suggest that the biochemical methods of choice in the follow-up of patients with primary biliary cirrhosis are determination of total serum bilirubin by conventional diazo technique and determination of serum alkaline phosphatase activity. Both are simple methods which correlate well with liver histology. For the detection of patients with liver disease, such as asymptomatic PBC, the more elaborate HPLC technique has the distinct advantage of being a reliable method to detect bilirubin conjugates at normal total bilirubin levels. However, one should realize that with the presently used HPLC method only free conjugates are measured whereas covalently bound 'bilirubin-albumin' may represent up to 70% of all bilirubin species in cholestatic serum. Both free conjugates and 'bilirubin-albumin' start to appear in serum at total bilirubin levels as low as 5 mumol/l. The presence of bilirubin conjugates and 'bilirubin-albumin' in serum suggests liver disease. Reliable methods to quantify 'bilirubin-albumin' are needed.
采用高效液相色谱法（HPLC）对26例原发性胆汁性肝硬化（PBC）患者进行血清胆红素亚组分分析的临床价值研究，共采集了59份血清样本。总胆红素（TB）水平通过碱性甲醇ysis和HPLC（TBHPLC）以及传统重氮法（TB重氮法）测定。TBHPLC与TB重氮法之间的相关性显著（P小于0.001），但HPLC测定的胆红素浓度仅约为重氮法的30%。这种差异可能是由与白蛋白紧密结合的胆红素，即所谓的“δ胆红素”或“胆红素-白蛋白”引起的，重氮法可测量该物质，而HPLC法无法测量。26例患者中有16例血清中存在胆红素结合物；其中14例总胆红素水平正常。胆红素结合物（BcHPLC）与TB重氮法之间存在极好的相关性（P小于0.001）。在所有含结合物的样本中，单结合物和双结合物的比例大致相等。我们的研究结果表明，原发性胆汁性肝硬化患者随访中首选的生化方法是采用传统重氮技术测定血清总胆红素和测定血清碱性磷酸酶活性。这两种方法都很简单，且与肝脏组织学相关性良好。对于检测肝脏疾病患者，如无症状PBC，更精细的HPLC技术具有明显优势，它是一种在总胆红素水平正常时检测胆红素结合物的可靠方法。然而，应该认识到，使用目前的HPLC方法仅能测量游离结合物，而共价结合的“胆红素-白蛋白”可能占胆汁淤积性血清中所有胆红素种类的70%。游离结合物和“胆红素-白蛋白”在总胆红素水平低至5μmol/L时就开始出现在血清中。血清中胆红素结合物和“胆红素-白蛋白”的存在提示肝脏疾病。需要可靠的方法来定量“胆红素-白蛋白”。
