Unit: Total and Direct Bilirubin

1 MLAB 2401 - Clinical Chemistry Lab Manual C F 113 UNIT: Total and Direct of Total and Direct completion of this exercise, the student will be able formation, excretion, and clinical significance of Direct , indirect and Total a Total Bilirubin a Direct Bilirubin 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 several degradation steps, the free Bilirubin becomes bound by albumin and is transportedthrough the blood to the liver.

2 This Bilirubin is not soluble in water, and is referred to as insoluble,indirect, or unconjugated. 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 methods for Bilirubin estimation were based on measurement of its oxidation product,biliverdin or on assessment of the icteric index.

3 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 that reacts with the diazo reagent without the addition of alcohol is called Direct orconjugated while the form that reacts only in the presence of alcohol is called indirect low concentration of Bilirubin is found in normal plasma, almost all of which is indirect.

4 The sumof the Direct and indirect forms (or conjugated and unconjugated) is termed Total Bilirubin . Routineanalytical procedures exist for the determination of Total Bilirubin and for the measurement ofdirect 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 SignificanceAny 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 may be classified as prehepatic, hepatic, or post-hepatic.

5 In prehepatic jaundice, excessbilirubin production (hemolysis) is responsible. Hepatic jaundice occurs when either the removalof Bilirubin from the blood or conjugation of Bilirubin by the liver is defective. This can haveUNIT: Total and Direct Bilirubin (continued)F 114 C MLAB 2401 - Clinical Chemistry Lab Manualorganic or genetic causes. Post-hepatic jaundice refers to anatomic obstruction of the extra-hepatic biliary tree. The most common causes of jaundice are liver disease and blockage of thecommon bile duct.

6 It is necessary to distinguish between the causes of jaundice early in thedisease prior to the onset of complications, as the course of treatment is dependent on the causeof the jaundice is caused by overproduction of Bilirubin due to excessive hemolysis and theinability of the liver to adequately remove this pigment from the blood. This condition is usuallyassociated with elevated values of serum indirect of the liver and infectious or toxic hepatitis are caused by some type of intrahepaticobstruction, where production of Bilirubin is not increased, but accumulates and is discharged backinto the blood.

7 In these conditions, the indirect form of Bilirubin predominates in the early phase,but as liver damage progresses the Direct form also becomes jaundice, caused by a post-hepatic blockage of the larger bile passages, particularlythe common bile duct, results in a reflux of Bilirubin into the blood. This condition, whenuncomplicated, is associated with elevated serum Bilirubin only of the Direct of Total Bilirubin and determination of the Direct and indirect fractions is importantin routine screening for and the differential diagnosis of for Bilirubin determination should be protected from light, since Bilirubin is light-sensitive and will break down under of den Bergh, Malloy and Evelyn Reaction In an aqueous solution.

8 Ehrlich's diazoreagent reacts with the Direct Bilirubin in the serum to form a pink to reddish-purple coloredcompound (azobilirubin). It is read at one minute. In a 50% methyl alcohol solution, Ehrlich'sdiazo reagent reacts with the Total Bilirubin in the serum to form a pink to reddish-purplecolored compound. (Read at 30 minutes.) of Jendrassik and Grof Serum or plasma is added to a solution of sodiumacetate and caffeine-sodium benzoate. The sodium acetate buffers the pH of the diazoreaction, while the caffeine-sodium benzoate accelerates the coupling of Bilirubin withdiazotized sulfanilic acid.

9 The azobilirubin color develops within 10 minutes. (Anaccelerating agent facilitates the coupling of albumin-bound Bilirubin with the diazo reagent.) The ASTRA System Direct Bilirubin Chemistry Module employs a modification ofthe Jendrassik-Grof rate Bilirubin Conjugated Bilirubin reacts with DSA under acid conditions toform a red chromophore. The absorbance due to the chromophore is directlyproportional to the conjugated Bilirubin in the sample and is measured using a two-filter(540-600 nm) end point Bilirubin + DSA + H 6 Red chromophore+(non-absorbing at 540 nm) (absorbs at 540 nm)UNIT: Total and Direct Bilirubin (continued)MLAB 2401 - Clinical Chemistry Lab Manual C F Bilirubin Total Bilirubin reacts with DSA under acid conditions to form a redchromophore.

10 Lithium dodecyl sulfate (LDS) is employed to solubilize the unconjugatedbilirubin. The absorbance due to the chromophore is directly proportional to the bilirubinin the sample and is measured using a two-filter (540-600 nm) end point + DSA + H Red chromophore+ LDS (non-absorbing at 540 nm) (absorbs at 540 nm) Bilirubin (up to 21 days) The absorbance of the sample, measured using atwo-filter (452-540 nm) differential technique is directly proportional to the bilirubinconcentration.