Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase

Itoh, S.; Kusaka, T.; Imai, T.; Isobe, K.; Onishi, S.

Annals of Clinical Biochemistry 37: 452-456


ISSN/ISBN: 0004-5632
PMID: 10902860
DOI: 10.1177/000456320003700404
Accession: 010546517

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We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3.5-7.4) in 0.1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4.5 or 5.5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3.7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.