Influence of glucuronosyl bilirubin and (EZ)-cyclobilirubin on determination of serum unbound bilirubin by UB-analyser.

Abstract

In the enzyme reaction for the determination of the unbound (free) bilirubin concentration by glucose oxidase and peroxidase, materials with low affinity for serum protein are reactive. The influence of these materials on the determination of serum unbound bilirubin was investigated. METHODS Serum samples from patients with neonatal hyperbilirubinaemia were analysed by high-performance liquid chromatography for total glucuronosyl bilirubin concentration (TGC) and (EZ)-cyclobilirubin concentration [(EZ)-C]. Based on these measurements, the samples were classified into three groups: group I [13 samples, TGC <2 micromol/L and (EZ)-C < 2.5 micromol/L]; group II [four samples, TGC < 2 micromol/L and (EZ)-C > or = 2.5 micromol/L]; and group III (five samples, TGC > or = 2 micromol/L). The concentrations of total bilirubin and unbound bilirubin were measured in these same samples with a UB-analyser. When the absorbance at 460nm was monitored, the decrease in absorbance was non-linear (concave curve). The degree of concavity was estimated (D15 value) as the deviation from linearity at 15s. The D15 value was significantly higher in groups II and III than in group I. D15 value correlated significantly with TGC, (EZ)-C and unbound bilirubin concentration, and the unbound bilirubin concentration correlated significantly with TGC and (EZ)-C. These results indicated that determination of serum unbound bilirubin concentration using the UB-analyser could be positively skewed by high concentrations of TGC and (EZ)-C.