engleski

Microanalysis of bile pigments in fish blood

In vertebrates, heme released from the heme proteins is oxidized by heme oxygenase to release biliverdin (BV), which is reduced by biliverdin reductase to bilirubin (BR). In the liver, BR is conjugated to bilirubin glucuronide (BRG) and excreted into the bile. BV can be excreted through the bile or, in fish, through the gills and skin. In some fish species, BV causes the blue-green color of the blood and color changes associated with sexual dimorphism. The bile pigments BV and BR act as a redox couple that can scavenge oxygen free radicals. In farmed fish, infection or contamination can trigger an oxidative stress response leading to hemolysis and liver failure, with abnormal BV, BR and BRG levels in the blood. In addition, starvation increases the BR/BR ratio in some fish species. Therefore, analysis of bile pigment levels in the blood of farmed fish can integrate multiparameter monitoring of fish wellness. Routine analysis of BR and BRG is performed using automated methods, which, however, cannot detect BV. We addressed this unmet diagnostic need by developing a high-throughput fluorometric method based on high-affinity BR binding by the bifunctional recombinant protein HUG. This fusion protein, which can be readily purified by exploiting the thermoreactive properties of the elastin-like scaffold, binds bilirubin via the UnaG domain. The stable and very specific complex formation results in a strong fluorescent signal. The assay of BR in whole blood, serum or plasma can be performed in microtiter plates using a multiplate fluorescence reader. Both BV and BRG can be enzymatically converted to BR, allowing their simultaneous detection. This analysis requires <0.1 mL of blood collected from the tail vein of the fish without sacrificing the donor. We applied this method to sea breams reared at Friskina Farm (Croatia) and to sea breams and sea bass reared at the experimental farm of the University of Udine (Italy). In all samples studied, BV was significantly higher than BR or BRG, and differences were observed according to fish species, feed treatment and rearing location. This approach can help filling knowledge gaps in the pathophysiology of biliary pigments in fish species.

bile pigments ; billiverdin ; billirubin ; sea bream ; sea bass ; aquaculture

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