Abstract:
The present work is concerned with the reactions of the bile pigments-bilirubin and biliverdin in N,N-Dimethylformamide (DMF) as a solvent system. Qualitative experiments
in DMF were performed as a preliminary study to determine the reactivity of bilirubin with a number of oxidizing and complexing agents. It was found to readily react with a few of these reagents. Consequently, some of. the reactions were followed using visible spectroscopy. The solubility and stability of bilirubin in DMF was also qualitatively
compared in a number of other solvents.
Particular attention was focused on the properties of bilirubin under acidic and basic conditions. The bile pigment appeared unreactive to acids. This was confirmed by potentiometric titrations with HCl and picric acid using a glass indicator electrode. The oxidative voltammetry of bilirubin at a platinum indicator electrode was examined
and showed no significant changes in the presence of picric acid. On the contrary, the acidic nature of bilirubin was indicated by its reactivity with basic impurities in the
DMF solvent. It was shown to behave as a diprotic acid as evidenced by quantitative changes occurring in its visible spectra and by potentiometric titration with tetramethylguanidine (TMG). A quantitative shift of oxidation potential from approximately +.7 volts to +.4 volts vs. Ag/AgCl(sat ), NaCl(sat) reference electrode was also observed with the addition of two molar equivalents of TMG. Controlled potential coulometry of bilirubin under these basic conditions indicated that the oxidation at +.4 volts leads to the same two electron oxidation to biliverdin as occurs under neutral conditions. The oxidative potential of biliverdin was also observed to shift to lower potentials with the addition of
excess TMG.
The reactivity of bilirubin with the Zinc(II) ion was also investigated under neutral conditions and in the presence of TMG as a base. Visible spectroscopy indicated
that with the addition of TMG a reversible metal ionbilirubin complex forms with a corresponding shift in maximum absorbance from 450nm to 530nm. An examination of
the dependence of complex formation on the concentrations of TMG and Zinc(II) was also undertaken utilizing visible spectroscopy and potentiometry. The results tend to indicate
that a possible complex equilibrium is established between Zinc(II) as a Lewis acid, bilirubin as a diprotic acid, and TMG as a common base.
The formation of the Zinc(II)-bilirubin complex was also correlated with a shift in the oxidation potential of bilirubin from +.4 vol ts to approximately 0.0 volts using both platinum and vitreous carbon indicator electrodes. An attempt was made to elucidate the oxidative process at 0.0 volts using constant potential coulometry in combination
with visible spectroscopy. The experimental data are discussed and interpreted as resulting from the formation of a mixture of products. The oxidation of the Zinc(II)
complex with I 2 is also followed spectrophotometrically and compared to the results of the electrolysis.
A separate study of the reactivity of biliverdin with TMG and Zinc(II) was necessitated to clarify the voltammetric and spectral changes observed in the bilirubin reactions. Biliverdin was also found to readily complex with Zinc(II) under basic conditions and to undergo significant spectral shifts. Changes in the anodic voltammetry of biliverdin with the addition of TMG and Zinc(II) are reported and are similar to the changes observed with bilirubin.
An electrolysis of the Zinc(II)-biliverdin complex was also performed and the results used to help interpret the spectral data obtained from the Zinc(II)-bilirubin
complex oxidation.
A short study of the reaction of the Copper(II) ion with bilirubin and biliverdin was also undertaken. As in the case with Zinc(II), no reactions occurred until TMG was added. The visible spectra following the course of the reaction of Copper(II) with both bile pigments is presented. Both reactions appeared to progress in a quantitative fashion leading to the formation of the same apparent product The reactions were irreversible and tend to indicate that the bile pigments undergo oxidation.