Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent.

We recently described a direct spectrophotometric method for unconjugated bilirubin, with caffeine reagent (Clin Chem 1986;32:1389-93). Because this method is independent of the protein matrix we used it for the preparation of bilirubin standards (Clin Chem 1987;33:1817-21). Now, in this paper, we utilize the caffeine reagent in setting up a bilirubin method for serum from neonates. This resulted in a two-wavelength (465 and 528 nm) equation, which fully corrects for HbO2 interferences. In combination with a bilirubin standard, this equation may be transformed into a simple relative formula for use with this simple dilution method. We studied this two-wavelength method with 55 neonates' sera, comparing results with those by both the diazo method of Doumas et al. (Clin Chem 1985;31:1779-89) and the borate method of Hertz et al. (Scand J Clin Lab Invest 1974;33:215-30). We found that this new method is independent of hemolysis and of the matrix of the sera. Therefore, it is very suitable for use in neonatology.

Use of the caffeine reagent in direct spectrophotometry of bilirubin.

The molar absorptivity of unconjugated bilirubin in caffeine reagent is independent of the protein matrix. This finding, together with the simplicity of a dilution step for direct spectrophotometry, will improve the calibration methods of bilirubin and make them more nearly accurate. This is encouraging for the development of a new method for bilirubin determination in neonates; moreover, the caffeine reagent has a clearing influence on the turbidity of human sera. These findings should also be important for standardization, especially because the method of Jendrassik and Gróf is also protein-independent. Therefore, the introduction of one reliable, inexpensive, "universal" standard of bilirubin in bovine serum albumin will be of importance for both methods.