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UNIT: Total and Direct Bilirubin

UNIT: Total and Direct Bilirubin

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clinical chemistry
clinical chemistry

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MLAB 2401 - Clinical Chemistry Lab Manual
F 113
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
free bilirubin
becomes bound by albumin and is transportedthrough the blood to the liver. This bilirubin is not soluble in water, and is referred to as
, or 
. 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.
Clinical Significance
 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
 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 have
UNIT: Total and Direct Bilirubin (continued)
F 114
MLAB 2401 - Clinical Chemistry Lab Manual
organic 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. 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.Hemolytic 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
bilirubin.Cirrhosis 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. In these conditions, the
indirect form of bilirubin predominates in the early phase,but as liver damage progresses the direct form also becomes elevated.
Obstructive 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
type.Measurement of total bilirubin and determination of the direct and indirect fractions is importantin routine screening for and the differential diagnosis of jaundice.Specimens for bilirubin determination should be protected from light, since bilirubin is light-sensitive and will break down under exposure.
Methods of Determination
 Van den Bergh, Malloy and Evelyn Reaction
— In an aqueous solution, 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.)2.
Methods 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. The azobilirubin color develops within 10 minutes. (Anaccelerating agent facilitates the coupling of albumin-bound bilirubin with the diazo reagent.)3.
— The ASTRA System Direct Bilirubin Chemistry Module employs a modification othe Jendrassik-Grof rate method.4.
a.Conjugated 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 technique.Conjugated bilirubin + DSA + H
Red chromophore
(non-absorbing at 540 nm) (absorbs at 540 nm)
UNIT: Total and Direct Bilirubin (continued)
MLAB 2401 - Clinical Chemistry Lab Manual
F 115
b.Total Bilirubin – Total bilirubin reacts with DSA under acid conditions to form a redchromophore. 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 technique.Bilirubin + DSA + H Red chromophore
LDS(non-absorbing at 540 nm) (absorbs at 540 nm)c.Neonatal 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. Absorbance at 452 nm is due to the bilirubin concentration, and, if present, hemoglobin. At 540 nm, bilirubin does not absorb, while hemoglobin exhibitsthe same absorbance as it does at 452 nm. The use of 540 nm as the blankingwavelength thus eliminates any hemoglobin contribution from the total absorbance at452 nm.Bilirubin in newborn babies can be read in this direct spectrophotometric procedure inpart due to the fact that the normal range is much higher than for adults. In addition,carotene and other dietary pigments prevent adult and specimens from older childrenfrom being suitable.
ProcedureTotal and Direct Bilirubin (Sigma #605) Quantitative, Colorimetric
Principle of Reaction
Bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. The color of this derivativeis pH dependent, occurring as pink in acid or neutral medium and blue under alkaline conditions.
(conjugated) bilirubin couples with diazotized sulfanilic acid (p-diazobenzenesulfonic acid),forming a blue color at alkaline pH.> blue color azobilirubinDirect bilirubin (conjugated) + diazotized sulfanilic acid alkaline
(unconjugated) bilirubin is diazotized only in the presence of an “accelerating” agent,caffeine-benzoate-acetate mixture. Thus, the blue azobilirubin produced in mixtures containing“accelerating” agent originates from both the
fractions and reflects the
bilirubin concentration.Total bilirubin + caffeine-benzoate-acetate mixture + diazotized sulfanilic acid
Supplies and Reagents
1.caffeine reagent (caffeine, sodium benzoate, sodium acetate)2.alkaline tartrate
Strong base. Avoid contact with skin and clothing.

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