Bilirubin: an autofluorescence bile biomarker for liver functionality monitoring

Abstract

Excitation at 366-465 nm of bilirubin in aqueous solution with solubilizing agents results in emission spectra composed by two main bands. The variation of their relative contributions as shown by changes in the spectral shape are consistent with the bilirubin bichromophore nature. This latter accounts for an exciton-coupling phenomenon, intramolecular interchromophore energy transfer efficiency being affected by microenvironment. Excitation at 366 nm, despite the poor absorption of bilirubin, gives rise to appreciable emission signals from both pure compounds and bile - collected from functionally altered rat livers - favouring the spectral shape response to environment and molecular conformation changes. As compared to the merely bile flow estimation, real-time detection of fluorescence, revealing composition variations, improves near-UV optical-biopsy diagnostic potential in hepatology.

Fatty liver oxidative events monitored by autofluorescence optical diagnosis: Comparison between subnormothermic machine perfusion and conventional cold storage preservation

“…Attention was then given to the bilirubin emission spectrum profile. This depends on the balance between the two main bands peaking at 530 nm and 570 nm, and is strictly influenced by bile composition, in a close relationship with liver functionality . Therefore, the AF area values of the two bands centered at 530 and 570 nm were estimated by means of spectral fitting analysis (Fig.…”

“…The contribution of each endogenous fluorophore to the overall AF spectral area was estimated by a curve‐fitting procedure (PeakFit; SPSS Science, Chicago, IL, USA), based on the Marquardt–Levenberg algorithm . To this end, half‐Gaussian modified Gaussian functions describing the emission profile of each individual spectral component were combined through subsequent adjustments to match the best fit between the curve representing their sum and the measured spectra . To directly compare our redox ratio data, calculated from the relative area values of NAD(P)H and flavins, with those reported by Sato et al ., flavin AF was corrected for the dependence of its emission yield on excitation wavelength (1:5 for 366 nm and 460 nm).…”

Autofluorescence‐based optical biopsy: An effective diagnostic tool in hepatology

“…All spectra were recorded from pure compounds in solution, except for the spectra of lipopigments and proteins, recorded under epi‐illumination by microspectrofluorometry, respectively, from the material remaining after organic extraction of liver tissue homogenates, or from a selected region of a portal area rich in connective tissue in a liver tissue section cut at cryostat. Details are described in the related literature…”

“…In hepatology, some interest has been reserved for the AF of PPIX produced following the administration of its precursor 5‐aminolevulinic acid (5‐ALA), for the detection of liver tumour or bile leakage . Another haem‐related product is bilirubin, which has recently emerged as a promising AF biomarker of bile composition and liver functionality . The diagnostic value of bile bilirubin is strengthened by the fact that, besides haemocatheresis, it derives from the catabolism of liver haem proteins, and in particular cytochrome P‐450 which accounts for about 70% of haem turnover in the liver.…”

“…Bilirubin AF consists of a dominating band, centred at about 530 nm, and a minor, variable contribution at longer wavelengths. The balance between the 2 emission regions depends on an interchromophore energy transfer phenomenon, which is favoured by shortening the excitation wavelength and is influenced by bilirubin concentration and microenvironment . The latter includes bile proteins and biliary acids responsible for a blue AF band, causing changes in overall bile AF that reflect bile composition and provide diagnostic information on liver functionality (Supplemental Figure ) …”