Occasionally I get a question about exactly what forms of bilirubin our assays are measuring, or what a direct bilirubin (DBili) measures versus a total bilirubin (TBili). This post is a short discussion on bilirubin.
Bilirubin is the degradation product of heme, which is the oxygen carrying group found in hemoglobin. Every hemoglobin molecule has four heme groups. Each of those heme groups will degrade into a bilirubin, so anything causing red blood cell destruction or increased turnover will generally result in increased bilirubin levels. This bilirubin is called unconjugated bilirubin because nothing is bound to it. Unconjugated bilirubin is extremely water insoluble and it is carried to the liver, mostly by albumin. In the liver, an enzyme called UDP-glucuronyltransferase (UDP-GT) adds glucuronic acid molecules to the bilirubin. This bilirubin is now called conjugated bilirubin, and it is water soluble.
There are four basic forms of bilirubin found in blood, unconjugated, mono-conjugated (one glucuronic acid added), di-conjugated (2 glucuronic acids added) and protein bound. They are also referred to as alpha, beta, gamma and delta bilirubins, respectively. How much of each form is measured depends on the assay used to measure it.
Most of the currently available wet chemistry assays for bilirubin use diazotized salts of sulfanilic acid to react with bilirubin and form a colored compound. The initial reaction occurs with the conjugated, water soluble forms, and is referred to as the “direct-reacting” or “direct” bilirubin. Then an accelerant is added to the assay, and the rest of the bilirubin reacts, giving you a total bilirubin. Therefore DBili is a measure of most of the conjugated forms but usually also includes any protein-bound forms that may be present. Unconjugated bilirubin is part of the total, but is not measured directly by diazo reactions.
A few assays give a direct spectrophotometric measurement of the conjugated and unconjugated forms themselves, specifically the dry slide technology available on Ortho Diagnostics instruments. Other than these few assays, to directly measure the unconjugated bilirubin requires an HPLC method which measures all the various forms separately. Transcutaneous bilirubin instruments give a measure of total bilirubin.
In disease states, monitoring the TBili concentration is important, however knowing whether the bilirubin present is conjugated or unconjugated will give you an idea of what the underlying problem may be. Elevated unconjugated bilirubin (high total with low direct) suggests increased hemolysis, or inability of the liver to conjugate bilirubin. Neonatal jaundice is usually caused by unconjugated bilirubin due mainly to immature liver enzymes, ie not enough UDP-GT to conjugate all the bilirubin present. Additionally, high unconjugated bilirubin will move into the tissues because of its water insolubility, and can cause brain damage if the concentration is high enough for long enough, a condition known as kernicterus. Elevated conjugated bilirubin (high total and high direct) suggests conditions causing inability of the liver to properly drain bilirubin into the bile (cholestasis). Prolonged high unconjugated bilirubin is a more serious condition than prolonged high conjugated bilirubin, because conjugated bilirubin can be excreted in urine and tends not to accumulate in the tissues.
-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.