Photoisomers: Obfuscating Factors in Clinical Peroxidase Measurements of Unbound Bilirubin?

OBJECTIVES. The objectives of the study were to measure the effect of 4Z, 15E-bilirubin on peroxidase free bilirubin measurements and to review the literature on this topic. METHODS. 4Z, 15E-Bilirubin was generated in situ in serum or serum albumin solution through controlled irradiation of isomerically pure 4Z, 15Z-bilirubin IX alpha, under conditions in which the total amount of bilirubin remained constant. Reactions were monitored by difference spectroscopy, to ensure that solutions were not irradiated beyond the initial photostationary state and that concentrations of other isomers were kept to a minimum. Prepared in this way, 10% to 25% of the total bilirubin in the final solutions was in the form of the 4Z, 15E-isomer. Free bilirubin in the solutions was measured with a peroxidase method, before and after irradiation. The use of bovine serum albumin as a surrogate for human albumin in in vitro studies also was investigated. RESULTS. The findings of previous studies are not altogether consistent, with a common flaw in several being the failure to measure photoisomer concentrations. For bilirubin in serum albumin solution, conversion of similar to 25% of the 4Z, 15Z-isomer to 4Z, 15E-bilirubin led to a much smaller decrease (<20%) in the apparent free bilirubin concentration; for bilirubin in serum, conversion of similar to 15% of the 4Z, 15Z-isomer to photoisomers resulted in a much larger increase (similar to 40%). Irradiation of bilirubin in bovine serum albumin solution generated a very different array of photoisomers than that observed in human albumin solutions. CONCLUSIONS. The effect of photoisomers on the accuracy and specificity of free 4Z, 15Z-bilirubin measurements remains uncertain. In a clinical setting, free bilirubin measurements need to be interpreted with caution when samples contain photoisomers. Irradiated bovine albumin solutions of isomerically impure bilirubin used in previous studies are poor models for investigating the effects of phototherapy in humans and the albumin binding of photoisomers. Pediatrics 2009; 123: 67-76