Reactivity of bilirubin photoisomers on the measurement of direct bilirubin using vanadic acid method

Objective Investigation of the reactivity of fractions of bilirubin photoisomers with the vanadic acid oxidation method. Methods Bilirubin photoisomers were prepared by irradiating a bilirubin/human serum albumin solution with blue light emitting diode. Direct bilirubin and bilirubin fractions were measured using the vanadic acid oxidation method and high-performance liquid chromatography in the sample before and after irradiation. Results Direct bilirubin was increased in the solution containing bilirubin photoisomers. ( EE)-/( EZ) -cyclobilirubin-IXα and ( ZE)-/( EZ)-bilirubin-IXα completely disappeared after the addition of vanadic acid. Conclusion Bilirubin photoisomers reacted as direct bilirubin in the vanadic acid oxidation method.

Radioimmunoassay of bound and free melatonin in plasma

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.