Clinical value of serum bilirubin subfractionation by high-performance liquid chromatography and conventional methods in patients with primary biliary cirrhosis

Abstract

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.