Kinetics of the formation of biliverdin during the photochemical oxidation of bilirubin monitored by column liquid chromatography.

In the photochemical oxidation of bilirubin, biliverdin is formed as the primary product and is further degraded. This photooxidation is especially significant in the presence of riboflavin. Column liquid chromatography was used to monitor the kinetics of this reaction. The biliverdin concentration amounts to a maximum of ca. 38% of the total loss of bilirubin in experiments in vitro. It is probable that this mechanism is also operative during phototherapy. The formation of a product of the photooxidation of biliverdin that has not yet been identified has been observed; the product behaves as a dimer. A method for the determination of biliverdin in the blood of newborn infants has been developed. It has been found that the biliverdin content increases during hyperbilirubinaemia.

Reversible photoreduction of flavin with bilirubin II.

Bilirubin react with flavins under both aerobic and anaerobic conditions. Under anaerobic conditions these substances behave as a redox-system in solution, riboflavin functioning as an electron acceptor and bilirubin as an electron donor. The reaction products are the leuco-form of riboflavin and biliverdin. On the basis of the dependence of the optical spectrum and of the reaction rate on pH it can be assumed that bilirubin reacts in the anionic form. Under anaerobic conditions a reversible reaction occurs leading to a dynamic equilibrium of the reactants. The products were identified and determined by optical spectroscopy, chromatography and potentiometry. Excretion of biliverdin in the stool of new-born infants was orientatively monitored during the phototherapy of hyperbilirubinemia, after the administration of small doses of riboflavin. It has been found that excretion of biliverdin increases during the phototherapy. The possibility of utilizing the described reaction for an improvement in the phototherapy of new-born infant hyperbilirubinemia is discussed.

Reversible photoreduction of flavin with bilirubin I.

In the photochemical degradation of bilirubin under anaerobic condition, the bilirubin riboflavin-system behaves as an equilibrium electrochemical system characterized by a redox-potential. Bilirubin decomposes irreversibly and riboflavin functions as a reversible electron acceptor. This equilibrium is not established under aerobic conditions, probably due to reoxidation of the riboflavin leuco-form by oxygen. Riboflavin accelerates the bilirubin photodegradation about 30 times at equimolar concentrations of the two components and in the presence of a buffer at pH 7.3. The present results of a study on a model system are applicable in the phototherapy of newborn infant hyperbilirubinemia.

Sectrofluorimetric micromethod for determining riboflavin in the blood of new-born babies and their mothers.

A microdetermination of riboflavin on the blood of new-born babies and their mothers was developed on the basis of hydrolysis of the blood in a trichloroacetic acid medium, separation of riboflavin and FMN on a Florisil column and measurement of the content spectrofluorimetrically by the standard addition method after elution with collidine buffer. The sensitivity of the method is 0.01 microgram/ml, the blood sample size 0.5--1.0 ml. The normal level in new-born babies in the first day of life was found to have an average value of 17.1+/-2.4 microgram/100 ml, in women 14.2+/-2.5 microgram/100 ml; the contents in cord blood and maternal vein blood were also determined.