Physico-chemical characterization of bilirubin-10-sulfonate and comparison of its acid–base behavior with unconjugated bilirubin

Unconjugated bilirubin (UCB) is the end-product of heme catabolism in the intravascular compartment. Although beneficial for human health when mildly elevated in the body, when present at greater than a critical threshold concentration, UCB exerts toxic effects that are related to its physico-chemical properties, particularly affecting the central nervous system. The aim of the present study was to characterize bilirubin-10-sulfonate (ranarubin), a naturally occurring bile pigment, including determination of its mixed acidity constants (p K a * ). Thanks to the presence of the sulfonic acid moiety, this compound is more polar compared to UCB, which might theoretically solve the problem with an accurate determination of the UCB p K a * values of its propionic acid carboxylic groups. Bilirubin-10-sulfonate was synthesized by modification of a previously described procedure; and its properties were studied by mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and circular dichroism (CD) spectroscopy. Determination of p K a * values of bilirubin-10-sulfonate and UCB was performed by capillary electrophoresis with low pigment concentrations in polar buffers. The identity of the synthesized bilirubin-10-sulfonate was confirmed by MS, and the pigment was further characterized by NMR, IR, and CD spectroscopy. The p K a values of carboxylic acid moieties of bilirubin-10-sulfonate were determined to be 5.02, whereas those of UCB were determined to be 9.01. The physico-chemical properties of bilirubin-10-sulfonate were partially characterized with low p K a * values compared to those of UCB, indicating that bilirubin-10-sulfonate cannot be used as a surrogate pigment for UCB chemical studies. In addition, using a different methodological approach, the p K a * values of UCB were found to be in a mildly alkaline region, confirming the conclusions of a recent critical re-evaluation of this specific issue.