Measurement of Serum Bilirubin The terms direct- and indirect-reacting bilirubin are based on the original van den Bergh reaction. This assay, or a variation of it, is still used in most clinical chemistry laboratories to determine the serum bilirubin level. In this assay, bilirubin is exposed to diazotized sulfanilic acid, splitting into two relatively stable dipyrrylmethene azopigments that absorb maximally at 540 nm, allowing for photometric analysis. The direct fraction is that which reacts with diazotized sulfanilic acid in the absence of an accelerator substance such as alcohol. The direct fraction provides an approximate determination of the conjugated bilirubin in. | Chapter 043. Jaundice Part 2 Measurement of Serum Bilirubin The terms direct- and indirect-reacting bilirubin are based on the original van den Bergh reaction. This assay or a variation of it is still used in most clinical chemistry laboratories to determine the serum bilirubin level. In this assay bilirubin is exposed to diazotized sulfanilic acid splitting into two relatively stable dipyrrylmethene azopigments that absorb maximally at 540 nm allowing for photometric analysis. The direct fraction is that which reacts with diazotized sulfanilic acid in the absence of an accelerator substance such as alcohol. The direct fraction provides an approximate determination of the conjugated bilirubin in serum. The total serum bilirubin is the amount that reacts after the addition of alcohol. The indirect fraction is the difference between the total and the direct bilirubin and provides an estimate of the unconjugated bilirubin in serum. With the van den Bergh method the normal serum bilirubin concentration usually is 17 iimol l 1 mg dL . Up to 30 or iimoi l mg dL of the total may be direct-reacting conjugated bilirubin. Total serum bilirubin concentrations are between and iimol L and mg dL in 95 of a normal population. Several new techniques although less convenient to perform have added considerably to our understanding of bilirubin metabolism. First they demonstrate that in normal persons or those with Gilbert s syndrome almost 100 of the serum bilirubin is unconjugated 3 is monoconjugated bilirubin. Second in jaundiced patients with hepatobiliary disease the total serum bilirubin concentration measured by these new more accurate methods is lower than the values found with diazo methods. This suggests that there are diazo-positive compounds distinct from bilirubin in the serum of patients with hepatobiliary disease. Third these studies indicate that in jaundiced patients with hepatobiliary disease monoglucuronides of bilirubin predominate over .
