For approximately two decades, bilirubin has not been regarded solely as a toxic product of haem catabolism destined for elimination, but rather as a biologically active molecule with antioxidant properties. Owing to its ability to scavenge oxygen free radicals, bilirubin may play a protective role against various chronic diseases in which oxidative stress is a major pathogenetic factor. In this context, the analysis of fractionated bilirubinemia has progressively expanded its diagnostic value, extending from traditional liver assessment to the study of cardiovascular risk and other chronic degenerative conditions.
Even modest variations in plasma bilirubin levels can significantly influence cardiovascular risk, highlighting the need to develop analytical methods that are more sensitive and specific than the traditional diazo method. In particular, it is essential to directly measure unconjugated bilirubin, the main bioactive fraction responsible for the redox properties attributed to the pigment.
In this context, our research group uses the HUG biosensor (HELP-UnaG), a fusion protein developed from the elastin-like polypeptide expression platform, for the advanced study of bile pigments in biological systems and medicine. The combination of the thermoresponsive properties of the HELP domain and UnaG's ability to bind bilirubin and emit fluorescence enables direct, highly sensitive and specific detection of bilirubin, even in complex biological matrices, without the need for extraction with organic solvents. Furthermore, the ability to jointly quantify bilirubin and biliverdin through enzymatic conversion makes HUG a particularly versatile tool for assessing the activity of heme oxygenase, a key enzyme in regulating cellular redox status.
The laboratory's main lines of research are developed on this basis, focusing both on the study of the molecular mechanisms involved in bile pigment metabolism and redox regulation, and on clinical and translational applications aimed at identifying new disease biomarkers.
1. Study of Molecular Mechanisms of Redox Status
A significant portion of our laboratory’s research focuses on in vitro studies using cellular models, aiming to clarify the molecular mechanisms underlying physiological and pathological processes.
Molecular evidence indicates that both bilirubin and certain acylated anthocyanins can be transported from the intestine to the blood via OATP1A2, a membrane transporter located on the luminal surface of the intestinal epithelium, which is involved in the transport of drugs, bile acids, and flavonoids. The beneficial effects of anthocyanins on human health have been demonstrated in a substantial body of studies conducted on in vitro and in vivo experimental models, as well as in clinical trials, which show anti-inflammatory, anti-cancer, anti-hypertensive, anti-arteriosclerotic, anti-obesity, and anti-diabetic effects, along with improved blood lipid parameters. Anthocyanins act through complex mechanisms involving various molecular targets. Theoretically, anthocyanins could modulate bilirubinemia by interfering with the intestinal reabsorption of bilirubin excreted in bile. A pilot study aims to determine whether anthocyanins inhibit the transport of bile pigments and acids in in vitro models of intestinal and biliary epithelium, where it is relatively straightforward to use various molecules, including pharmacological agents, to characterise the function of different membrane transporters.
In parallel, the biosensor is being used to study the modulation of heme catabolism and the associated redox state, integrating the measurement of specific oxidative stress markers. This approach allows us to delineate the role of bile pigments as active mediators in physiological processes and pathological conditions.
2. Clinical Applications and Disease Biomarkers
Another line of research in the laboratory is oriented towards the clinical and translational field, aiming to explore the potential of bile pigments as biomarkers in various pathological conditions. In this context, the use of the HUG biosensor enables high-precision quantification and characterisation of bile pigments (bilirubin and biliverdin) in different biological matrices, including those difficult to analyse with conventional methods. The aim is to assess whether physiological changes in pigments, both locally and systemically, correlate with parameters associated with cellular stress, inflammatory processes, and pathophysiological alterations. This integrated approach allows the identification of novel relationships between bile pigment metabolism, redox status, and disease, contributing to the understanding of underlying mechanisms and the development of innovative diagnostic tools. Potential applications range from neurodegenerative diseases to male infertility – where bile pigments reflect local redox processes – to metabolic disorders such as obesity and various forms of cancer.
