MLAB 2401 - Clinical Chemistry Lab Manual
UNIT:Total and Direct Bilirubin
Determination of total and direct bilirubin.
Upon completion of this exercise, the student will be able to:1.Explain formation, excretion, and clinical significance of direct, indirect and total bilirubin.2.Perform a total bilirubin determination.3.Perform a direct bilirubin determination.
Like so many other substances measured in clinical chemistry laboratories, bilirubin is a wasteproduct. Bilirubin, the principle pigment in bile, is derived from the breakdown of hemoglobin. After several degradation steps, the
becomes bound by albumin and is transportedthrough the blood to the liver. This bilirubin is not soluble in water, and is referred to as
. In the liver, bilirubin is rendered soluble by conjugation withglucuronide. The water-soluble bilirubin, called
direct or conjugated
, is transported along withother bile constituents into the bile ducts, then to the intestines. In the intestines, bacterial enzymeaction converts bilirubin to several related compounds, collectively referred to as urobilinogen.Early methods for bilirubin estimation were based on measurement of its oxidation product,biliverdin or on assessment of the icteric index. Introduction of the diazo reaction for bilirubin byvan den Bergh in 1918 led to its widespread adoption for quantitating the pigment in serum. Vanden Bergh and Muller found that bilirubin in normal serum reacted with Ehrlich's diazo reagent(diazotized sulfanilic acid) when alcohol was added. Their observation that bile pigment reactedwith the diazo reagent without the addition of alcohol led to the recognition that some change inbilirubin had been affected by the liver.Bilirubin that reacts with the diazo reagent without the addition of alcohol is called “direct” or conjugated while the form that reacts only in the presence of alcohol is called “indirect” or unconjugated. A low concentration of bilirubin is found in normal plasma, almost all of which is indirect. The sumof the direct and indirect forms (or conjugated and unconjugated) is termed
bilirubin. Routineanalytical procedures exist for the determination of
bilirubin and for the measurement of
bilirubin. The indirect fraction is obtained by subtracting the direct value from the totalvalue.
The determination of direct as well as total bilirubin is used in differentiating certain typesof jaundice.
Any increase in formation or retention of bilirubin by the body may result in jaundice, a conditioncharacterized by an increase in the bilirubin level in the serum and the presence of a yellowishpigmentation in the skin.Jaundice may be classified as prehepatic, hepatic, or post-hepatic. In
, excessbilirubin production (hemolysis) is responsible.
occurs when either the removalof bilirubin from the blood or conjugation of bilirubin by the liver is defective. This can have