Chemical, chromatographic, or spectrometric methods are generally unsuitable for the detection of molecules in the nano and subnanogram region because of their low sensitivity. The radioimmunoassay (RIA) developed by Yalow and Berson in 1959 combined the high sensitivity of radioactively labeled substances with the high specificity of immunological reactions for the first time. In this way it was possible to detect quantitatively the tiniest traces of substances in the presence of an excess of other, in some cases, similar foreign substances without prior enrichment. Immunoassays have certainly developed to become the most valuable analytical tool of in vitro diagnostics and are today routinely employed for the detection of endogenous and exogenous substances (e.g. hormones, tumor-associated proteins, bacteria, viruses, toxins, drugs, etc). The many disadvantages of radioactivity such as the required handling licenses, disposal costs, precautions necessary to prevent risks to health, short shelf-life, and limited sensitivity soon led to the search for other nonradioactive labeling methods. Encouraged by the development of light measuring techniques and the commercial availability of highly sensitive apparatus, radioactive isotopes as labels are today being replaced increasingly by enzymes, fluorophores, or luminophores. Some of the new luminescent labels have, however, not only facilitated replacement of radioisotopes, but also a breakthrough into what has until now been unattainable levels of sensitivity. The following article reviews the methods of luminescent labeling and their applications mainly in the area of immunoassays.

Luminescent Labels—More than Just an Alternative to Radioisotopes?

6 – Bile Pigments: Bilatrienes and 5,15-Biladienes

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivatives, 4-methyl-5-oxo-3-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2000.01812.x

Oxidative degradation of bilirubin produces vasoactive compounds

Bilirubin-IX alpha photooxidation products were detected by high performance liquid chromatography in the urine of neonates undergoing phototherapy for hyperbilirubinemia. The in vivo photoproducts were identified by chromatographic comparison with authentic synthetic standards using two complementary methods. Bilirubin photooxidation products were not detected in urine from jaundiced infants not receiving phototherapy. The specific photoproducts identified in the urine include propentdyopents, hematinic acid imide and its hydrolysis product (3-carboxy-2-methyl-2-hexenedioic acid), and the hydrolysis product (2-vinyl-3-methyl-maleic acid) of methylvinylmaleimide. Their total urinary concentrations were low (0.2-0.9 mg/dl) during phototherapy. These observations show that photooxidation of bilirubin clearly does occur during phototherapy. They are consistent with the view that, although photooxidation is not the major photochemical event associated with phototherapy, it can and clearly does occur concurrently with photoisomerization.

/pdf/bilirubin-photooxidation-products-in-the-urine-of-jaundiced-36q2d9ls9k.pdf

Bilirubin Photooxidation Products in the Urine of Jaundiced Neonates Receiving Phototherapy

Analysis of bilirubin and pyrromethenone N—H 1H-NMR chemical shifts indicates that in CDCl3 solvent, where intermolecularly H-bonded dimers or intramolecularly H-bonded conformations are involved, the lactam N—H groups are more deshielded than the broader pyrrole N—H groups. In d6-DMSO, disaggregation and unfolding occurs with H-bonding to the solvent and the sharper lactam N—H resonances becoming more shielded than the pyrrole N—H signals.

Y-T. Park

Papers

On the role of singlet oxygen in the self-sensitized photo-oxygenation of bilirubin and its pyrromethenone models

Dye-sensitized photooxygenation of oxopyrromethenes related to Bilirubin

On the syntheses and singlet oxygen reactivity of oxodipyrromethene models for bilirubin