Subfractionation of serum bilirubins by alkaline methanolysis and thin-layer chromatography. An aid in the differential diagnosis of icteric diseases.

The determination of direct and indirect-reacting bilirubin fractions by diazo procedures does not allow a definite diagnosis of icteric diseases. Therefore, the clinical relevance of serum bilirubin subfractionation by alkaline methanolysis and subsequent thin-layer chromatography (AM-TLC) was evaluated. Esterified bilirubins could be detected and quantitated in all serum samples investigated. The ratio of serum esterified to total bilirubin was 10-28% in 60 healthy adults (mean 17 +/- 5% S.D.), 1-11% in 77 patients with Gilbert's syndrome (mean 6 +/- 2%), and 2 and 3%, respectively, in two patients with Crigler-Najjar disease type II. The difference was highly significant (p less than 0.001) and the overlap was restricted to three of 139 individuals. The ratio of esterified to total bilirubin was similar to that obtained with HPLC when corrected for with a blank run. The absolute concentration of bilirubin esters in serum from Gilbert's syndrome patients was similar to that from healthy controls, but the unconjugated pigment was increased. In patients with chronic haemolysis (n = 9) and chronic persistent hepatitis (n = 12), the hyperbilirubinaemia consisted of a proportional increase of both unconjugated and esterified bilirubin. As such, the ratio of conjugated to total bilirubin was not significantly different from control values. Patients with acute hepatitis during the first (n = 18) and third ('remission') week of the disease (n = 15), liver cirrhosis (n = 34), and extrahepatic cholestasis (n = 20) predominantly showed an increase in bilirubin conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)