ANALYTICAL CHEMISTRY OF BILIRUBINS: NEW METHODS, CAFFEINE COMPLEXATION, STABILITY, AND BIOSYNTHESIS OF CONJUGATES

Flow injection analysis (FIA) was applied to the diazo reaction for the determination of total bilirubin. The main advantage of the FIA technique, compared to manual and some automated methods, is the speed of analysis, which may overcome some time-dependent interferences. FIA also offers better accuracy and reproducibility. Sixty determinations can be made per hour and the precision for each determination is within 3%. Calibration curves exhibit good linearity over a range of 1.0-27.0 mg/dL. The reproducibility of the slopes of these curves is 1% for determinations carried out within a day or over three days. Flow injection values for the determination of total bilirubin in patient samples correlate well (r=0.998) with Technicon SMAC results. Another FIA system is used for the determination of total bilirubin at normal levels and has a linear dynamic range of 0.25-5.0 mg/dL. This system has potential for the determination of total concentration of conjugated bilirubins in serum samples.;A "highly sensitive method" based on the diazo reaction was developed which can be used to determine bilirubin levels down to approximately 9 nM. In conjunction with phase solubility and spectral techniques, this method was used to determine the formation constants for caffeine-unconjugated bilirubin complexes. TLC and UV-VIS spectrophotometry were also used to investigate chromatographic and spectral properties of these complexes. Results from caffeine complexation studies indicate that two complexes (1:1 and 1:2) are formed between bilirubin IX-(alpha) and caffeine with absorbance maxima at 428 and 454 nm, respectively. The formation constants for the complexes are 205 M('-1) and 9.0 M('-1). The reactivity of these two complexes with the diazo reagent is comparable to that of bilirubin with the diazo reagent. In addition to caffeine complexation, caffeine-benzoate complexation with bilirubin IX-(alpha) is also possible. Caffeine has a greater tendency than dyphylline to complex with unconjugated bilirubin. Other accelerators were also tested. These results are useful in terms of evaluating the relative effectiveness of caffeine as an accelerator in the diazo methods. Furthermore, the approach used for this study provides a general model for studies of other complexation phenomena of bilirubins.