Quantification of Biliverdin, Bilirubin and Bilirubin Glucuronide in Micro-Samples of Animal Blood

Circulating bile pigments are routinely measured in human and veterinary medicine, toxicology, pharmacology, and biomedical studies, as they are consolidated biomarkers of liver dysfunction and hemolysis and emerging biomarkers of biological responses to stress. This study was performed to extend bile pigment analysis from plasma to microvolumes of whole blood not only to simplify the pre-analytical steps but also to surmount the bottleneck of limited blood volume available for withdrawals in screening and monitoring. We applied the HUG assay for the nanoscale analysis of biliverdin, bilirubin, and bilirubin glucuronide. HUG is a chimeric protein expressing the UnaG domain that emits fluorescence upon binding bilirubin. This study shows that, under specific conditions, the HUG assay enables quantification of the three bile pigments in rat and fish whole blood. Moreover, quantification is equivalent in blood, plasma and serum. This approach skips pre-analytical steps, reduces errors and time to analysis, thereby facilitating research of circulating bile pigments in animalia while preserving the possibility of comparing new data with historical sets of clinical chemistry data in the literature.