Bioanalysis of Bilirubin: State of Our Art and Future Developments (Review Article)

Abstract The study comprised several co-investigations, each of which formed an original contribution to bilirubin research. Variation in the bioanalytical concentration of the unconjugated specimen and its reversion to other products was examined in each case. The contributions of the different components of the study are broadly summarized as follows: (i) photolysis by sunlight and phototherapy illumination; (ii) the effect of ionizing radiation; (iii) solvent effects and chromatographic studies; (iv) kinetic studies; (v) thermal and chemical effects. Initially, in vitro studies in dilute aqueous NaOH compared the efficiency of sunlight with illumination from a phototherapy unit. The data indicated that at comparable light intensities the phototherapy unit was as effective as sunlight in reducing the bilirubin levels to less than 10% of its original value. This work was extended by subjecting dilute non-aqueous solutions (chloroform) of bilirubin to 137Cs gamma irradiation. It was found that the ionizing radiation caused the bilirubin levels to decrease progressively with increased dose, which led to the formation of biliverdin. When the solvent was changed to deuterated chloroform, biliverdin was observed as a product of the chemical reaction between the solvent and bilirubin. Isolation of the components of the reaction mixture was done by adsorption chromatography. The separated fractions were confirmed spectrophotometrically. A more refined separation was accomplished in a preliminary study under conditions of reverse-phase HPLC, implementing isocratic elution with a solvent composition of 5:95 (v/v) CH3COOH and CH3OH. This produced capacity factors of 3.2 and 6.5 for biliverdin and bilirubin, respectively, for an optimum flow rate of 1 mL/min. The kinetics involving the CDCl3 reaction and the photochemical reaction in sunlight were subsequently examined, and respective rate constants of 0.17 s−1 and 0.15 s−1 were attained. The temperature dependence of the conversion of bilirubin (in aqueous NaOH) to biliverdin revealed that a considerable drop in the bilirubin concentrations was encountered with progressive increases in temperature in the range 25–68°C. This led to improved yields of biliverdin. It was further observed that the application of temperature in the presence of a CuSO4 spike intensified the phenomenon. The scope of our research is discussed.