Biliverdin: The blue-green pigment in the bones of the garfish (Belone belone) and eelpout (Zoarces viviparus)

Abstract

Little is known about the bluish-green color of the skeleton and bones of the garfish (Belone belone) and eelpout (Zoarces viviparus). Most of the few, contradictory reasons given favor either the iron(II) phosphate, vivianite, or the heme degradation product, biliverdin, but all lack documented spectroscopic confirmation. Our aim was to identify and quantify this blue-green substance and to investigate more precisely its location within the colored parts of these fish species. First, we found that vivianite was not indicated by the iron and phosphate levels in fish samples, whereas biliverdin was confirmed in the blue-green matrices both by UV-Vis (maxima at 376 and 666 nm) and FTIR spectroscopy, and by HPLC-MS/MS (molecular ion m/z [M + H]+ = 611) after sample preparation involving solvent extraction, methylation (yielding biliverdin dimethyl ester), and solid-phase extraction. The overall average garfish biliverdin recovery rate by HPLC-UV was 72.3 %; the average biliverdin content of the vertebral column (including the periosteum) was 23.49 μg/g; periosteum, 61.70 μg/g; and spinal process (Processus spinosus), 24.3 μg/g. Analysis of amino acid distribution in colored and non-colored fish matrices conformed high proportions of hydroxyproline in the former (periosteum 7 %, spinal processes 7.6 %), suggesting that biliverdin has a specific affinity for the structural protein collagen, and not for the bone itself. Finally, all methods confirmed the presence of biliverdin in collagen-rich tissues (periosteum and spinal processes) of garfish and eelpout, and we established a new method for the detection of biliverdin at low concentrations in structural proteins. © 2013 The Author(s).