Page 23 - Petelin, Ana, ur. 2021. Zdravje starostnikov / Health of the Elderly. Zbornik povzetkov z recenzijo / Book of Abstracts. Koper: Založba Univerze na Primorskem/University of Primorska Press
P. 23
ited lecture plenarna predavanja | plenary lectures
Bilirubin as a biomarker of disease risk:
addressing the analytical challenge
Sabina Passamonti
University of Trieste, Department of Life Sciences, Trieste, Italy
Introduction. Bilirubin is a lipophilic molecule found in serum at the concentra-
tion of 3.5-20 µM, with < 5 % of that being bilirubin diglucuronide. These val-
ues result from the balance between daily production of about 300 mg from
heme catabolism and the biliary elimination of bilirubin diglucuronide. The clin-
ical value of bilirubinemia has grown from being just a diagnostic biomarker of
haemolysis or liver failure to a predictive one. Mild elevations of bilirubin, as
in Gilbert’s syndrome, are associated with reduced cardiovascular disease and
mortality risk. This has sparked the ambition to find non-genetic factors, such
as drugs, diets, or life styles, driving mild hyperbilirubinemia. The protective ef-
fect of bilirubin is ascribed to the free radical scavenging activity of the redox
couple bilirubin/biliverdin. However, high-throughput methods for its analysis
are so far lacking, which prevents a deeper understanding of bilirubin homeo-
stasis. We have addressed this unmet diagnostic need by developing a simple,
high-throughput method for the analysis of the full set of bile pigments in hu-
man blood (i.e., biliverdin, bilirubin, and bilirubin glucuronide), which requires a
tiny volume (10 microL) of capillary blood sampled by finger puncture.
Methods. We produced a bifunctional synthetic protein (HUG), composed of
a protein scaffold (HELP) fused with UnaG, which binds bilirubin and emits flu-
orescence. The fluorimetric assay is performed in microtiter plates, requires a
multiplate reader, and produces no waste.
Results. We characterised the kinetics of bilirubin binding by HUG. Due to
its very high affinity, HUG enabled the fluorimetric titration of bilirubin in al-
bumin-free, neutral solutions in the range 2-100 nM. When in solution as a
complex with albumin, all albumin-bound bilirubin was captured by HUG. The
HUG method was validated and compared to the standard method based on
the diazo reagent. We have applied it for the direct microanalysis of bilirubin in
experimental hepatology, human and animal blood.
Discussion and conclusions. This method opens the opportunity to analyze the
full set of blood bile pigments in experimental biology and medicine, as well as
in clinical trials and personalized medicine studies. We expect to contribute to
an improved scientific understanding of bilirubin metabolism and its regulation.
Keywords: bilirubin, biomarker, disease risk
21
Bilirubin as a biomarker of disease risk:
addressing the analytical challenge
Sabina Passamonti
University of Trieste, Department of Life Sciences, Trieste, Italy
Introduction. Bilirubin is a lipophilic molecule found in serum at the concentra-
tion of 3.5-20 µM, with < 5 % of that being bilirubin diglucuronide. These val-
ues result from the balance between daily production of about 300 mg from
heme catabolism and the biliary elimination of bilirubin diglucuronide. The clin-
ical value of bilirubinemia has grown from being just a diagnostic biomarker of
haemolysis or liver failure to a predictive one. Mild elevations of bilirubin, as
in Gilbert’s syndrome, are associated with reduced cardiovascular disease and
mortality risk. This has sparked the ambition to find non-genetic factors, such
as drugs, diets, or life styles, driving mild hyperbilirubinemia. The protective ef-
fect of bilirubin is ascribed to the free radical scavenging activity of the redox
couple bilirubin/biliverdin. However, high-throughput methods for its analysis
are so far lacking, which prevents a deeper understanding of bilirubin homeo-
stasis. We have addressed this unmet diagnostic need by developing a simple,
high-throughput method for the analysis of the full set of bile pigments in hu-
man blood (i.e., biliverdin, bilirubin, and bilirubin glucuronide), which requires a
tiny volume (10 microL) of capillary blood sampled by finger puncture.
Methods. We produced a bifunctional synthetic protein (HUG), composed of
a protein scaffold (HELP) fused with UnaG, which binds bilirubin and emits flu-
orescence. The fluorimetric assay is performed in microtiter plates, requires a
multiplate reader, and produces no waste.
Results. We characterised the kinetics of bilirubin binding by HUG. Due to
its very high affinity, HUG enabled the fluorimetric titration of bilirubin in al-
bumin-free, neutral solutions in the range 2-100 nM. When in solution as a
complex with albumin, all albumin-bound bilirubin was captured by HUG. The
HUG method was validated and compared to the standard method based on
the diazo reagent. We have applied it for the direct microanalysis of bilirubin in
experimental hepatology, human and animal blood.
Discussion and conclusions. This method opens the opportunity to analyze the
full set of blood bile pigments in experimental biology and medicine, as well as
in clinical trials and personalized medicine studies. We expect to contribute to
an improved scientific understanding of bilirubin metabolism and its regulation.
Keywords: bilirubin, biomarker, disease risk
21
