Task
Determination of total and direct bilirubin.
Objectives
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.
Introduction
Like so many other substances measured in clinical chemistry laboratories, bilirubin is a waste product. 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 transported through the blood to the liver. 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 with glucuronide. The water-soluble bilirubin, called direct or conjugated, is transported along with other bile constituents into the bile ducts, then to the intestines. In the intestines, bacterial enzyme action 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 by van den Bergh in 1918 led to its widespread adoption for quantitating the pigment in serum. Van den 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 reacted with the diazo reagent without the addition of alcohol led to the recognition that some change in bilirubin 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 sum of the direct and indirect forms (or conjugated and unconjugated) is termed total bilirubin. Routine analytical procedures exist for the determination of total bilirubin and for the measurement of direct bilirubin. The indirect fraction is obtained by subtracting the direct value from the total value. The determination of direct as well as total bilirubin is used in differentiating certain types of jaundice.
Clinical Significance
Any increase in formation or retention of bilirubin by the body may result in jaundice, a condition characterized by an increase in the bilirubin level in the serum and the presence of a yellowish pigmentation in the skin.
Jaundice may be classified as prehepatic, hepatic, or post-hepatic. In prehepatic jaundice, excess bilirubin production (hemolysis) is responsible. Hepatic jaundice occurs when either the removal of bilirubin from the blood or conjugation of bilirubin by the liver is defective. This can have
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 the common bile duct. It is necessary to distinguish between the causes of jaundice early in the disease prior to the onset of complications, as the course of treatment is dependent on the cause of the jaundice.
Hemolytic jaundice is caused by overproduction of bilirubin due to excessive hemolysis and the inability of the liver to adequately remove this pigment from the blood. This condition is usually associated with elevated values of serum indirect bilirubin.
Cirrhosis of the liver and infectious or toxic hepatitis are caused by some type of intrahepatic obstruction, where production of bilirubin is not increased, but accumulates and is discharged back into 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, particularly the common bile duct, results in a reflux of bilirubin into the blood. This condition, when uncomplicated, is associated with elevated serum bilirubin only of the direct type.
Measurement of total bilirubin and determination of the direct and indirect fractions is important in 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
1. Van den Bergh, Malloy and Evelyn Reaction — In an aqueous solution, Ehrlich's diazo reagent reacts with the direct bilirubin in the serum to form a pink to reddish-purple colored compound (azobilirubin). It is read at one minute. In a 50% methyl alcohol solution, Ehrlich's diazo reagent reacts with the total bilirubin in the serum to form a pink to reddish-purple colored compound. (Read at 30 minutes.)
2. Methods of Jendrassik and Grof — Serum or plasma is added to a solution of sodium acetate and caffeine-sodium benzoate. The sodium acetate buffers the pH of the diazo reaction, while the caffeine-sodium benzoate accelerates the coupling of bilirubin with diazotized sulfanilic acid. The azobilirubin color develops within 10 minutes. (An accelerating agent facilitates the coupling of albumin-bound bilirubin with the diazo reagent.) 3. ASTRA — The ASTRA System Direct Bilirubin Chemistry Module employs a modification of
the Jendrassik-Grof rate method.
4. ACA
a. Conjugated Bilirubin – Conjugated bilirubin reacts with DSA under acid conditions to form a red chromophore. The absorbance due to the chromophore is directly proportional to the conjugated bilirubin in the sample and is measured using a two-filter (540-600 nm) end point technique.
Conjugated bilirubin + DSA + H 6 Red chromophore+ (non-absorbing at 540 nm) (absorbs at 540 nm)
b. Total Bilirubin – Total bilirubin reacts with DSA under acid conditions to form a red chromophore. Lithium dodecyl sulfate (LDS) is employed to solubilize the unconjugated bilirubin. The absorbance due to the chromophore is directly proportional to the bilirubin in the sample and is measured using a two-filter (540-600 nm) end point technique.
Bilirubin + DSA + H + LDS Red chromophore (non-absorbing at 540 nm) (absorbs at 540 nm)
c. Neonatal bilirubin (up to 21 days) – The absorbance of the sample, measured using a two-filter (452-540 nm) differential technique is directly proportional to the bilirubin concentration. Absorbance at 452 nm is due to the bilirubin concentration, and, if present, hemoglobin. At 540 nm, bilirubin does not absorb, while hemoglobin exhibits the same absorbance as it does at 452 nm. The use of 540 nm as the blanking wavelength thus eliminates any hemoglobin contribution from the total absorbance at 452 nm.
Bilirubin in newborn babies can be read in this direct spectrophotometric procedure in part 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 children from being suitable.
Procedure
Total and Direct Bilirubin (Sigma #605) Quantitative, Colorimetric Principle of Reaction
Bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. The color of this derivative is pH dependent, occurring as pink in acid or neutral medium and blue under alkaline conditions.
Direct (conjugated) bilirubin couples with diazotized sulfanilic acid (p-diazobenzenesulfonic acid), forming a blue color at alkaline pH.
> blue color azobilirubin Direct bilirubin (conjugated) + diazotized sulfanilic acid alkaline pH
Indirect (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 Direct and Indirect fractions and reflects the Total bilirubin concentration.
Total bilirubin + caffeine-benzoate-acetate mixture + diazotized sulfanilic acid 6 azobilirubin
Supplies and Reagents
1. caffeine reagent (caffeine, sodium benzoate, sodium acetate)
2. alkaline tartrate – CAUTION: Strong base. Avoid contact with skin and clothing.
3. HCl (0.05 N)
4. Diazo Reagent (sulfanilic acid, sodium nitrite). Reconstitute one vial Diazo with 6.0 mL HCl.
Stable five days at 2-6°C.
5. Cysteine Reagent. Prepare by adding 10.5 mL DIH O. Cap, shake. Stable three months2
(room temperature) in the dark.
6. Bilirubin reference. Assayed dry preparation containing bilirubin in a protein base for use as a control or for calibration purposes. The actual bilirubin concentration appears on the vial label.
7. Standard, controls (2), and unknowns.
Specimen Collection and Storage
Fresh serum is recommended, but heparinized plasma is also acceptable. Specimens must be protected from both artificial light and sunlight during processing and storage as bilirubin will undergo auto-oxidation to biliverdin.. The use of a serum blank eliminates interference from hemolysis and lipemia.
Preparation of Calibration Curve
1. Reconstitute bilirubin reference with 3.0 mL water. Let stand for several minutes and swirl or invert to mix.
2. Number three test tubes and pipet solutions as indicated in the chart below
Tube # Bilirubin Reference W ater
Dilution M ultiplication
Factor (F)
Bilirubin (m g/dL) - (F) x listed value of Bilirubin
Reference Absorbance
1 2 3
0.05 mL 0.10 mL 0.20 mL
0.15 mL 0.10 mL
–
0.25 0.50 1.00
3. To each tube add in the sequence shown: (mix after each addition) a. 1.0 mL caffeine reagent
b. 0.5 mL diazo reagent c. 0.1 mL cysteine solution d. 1.5 mL alkaline tartrate
4. Transfer solutions to cuvets and record absorbance of all tubes using water as a reference at 600 nm. (Read within 30 minutes.)
5. Calculate the bilirubin concentrations for each tube by multiplying the listed value for the bilirubin reference by the appropriate dilution factor and record.
6. Plot a calibration curve of the absorbance vs. concentration.
Procedural Notes
1. For screening purposes, the serum blank may be omitted, since the contribution by serum to the final absorbance in this procedure is generally minor.
2. A serum blank should be included primarily when assaying highly turbid sera or control or grossly hemolyzed specimens.
3. Results are not significantly affected by hemoglobin concentrations up to 280 mg/dL.
4. When the serum blank is omitted, the total and direct bilirubin tubes are read versus water as a reference.
Working Procedure
1. Set up Blank tube only on specimens that are hemolyzed or lipemic.
2. To appropriately labeled test tubes add the following:
Blank Total Direct tube tube tube
a. serum 0.2 0.2 0.2
b. hydrochloric acid 0.5 — 1.0
c. caffeine reagent 1.0 1.0 —
d. diazo reagent — 0.5 0.5
Mix well
e. cysteine solution 0.1 0.1 0.1
Mix well
f. alkaline tartrate 1.5 1.5 1.5
Mix well
3. Transfer to cuvets and read absorbance of all tubes, including blank using DI water as a reference at 600 nm.
RESULTS
Use the prepared calibration curve to determine the concentration of your unknown samples.
Determine total and direct bilirubin levels from the curve. The indirect bilirubin is the difference between the total and the direct. Record all results on worksheet.
Normal Values
Total Direct
Adults 0.2-1.2 mg/dL 0.3 mg/dL
Infants 1.0-12.0 mg/dL —
Name Date Bilirubin Worksheets
I. Calibration Curve Wavelength _____________
Linearity _____________ Spectrophotometer Used _____________________
Identification Absorbance Reference Concentration Value * Standard Tube 1 ____________
Standard Tube 2 ____________
Standard Tube 3 ____________
* see Preparation of Calibration Curve
II. Total Bilirubin
Identification AbsorbanceBlank’s AbsorbanceTest’s AbsorbanceCorrected Concentration (units)
Control 1 ____________
Control 2 ____________
Calculation formula(s) and examples
III. Direct Bilirubin
Identification Blank’s Absorbance
Test’s Absorbance
Corrected Absorbance
Concentration (units)
Control 1 ____________
Control 2 ____________
Calculation formula(s) and examples
IV. Indirect Bilirubin
Identification Concentration (units) Control 1 ____________
Control 2 ____________
Calculation formula(s) and examples
NOTES:
1. Show at least one example calculation for indirect bilirubin on the back of this page.
2. The bilirubin curve must accompany the results sheet.
3. See the back of this page to evaluate and report control results.
Total Bilirubin Quality Control
Your Results Controls’ range of expected results.
In control?
Yes / No Level 1 ID______________
Level 2ID_______________
Accepting Patient Results? Reason
Direct Bilirubin Quality Control
Your Results Controls’ range of expected results.
In control?
Yes / No Level 1 ID______________
Level 2ID_______________
Accepting Patient Results? Reason
Name Date Study Questions
Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each question is worth one point. Using lecture notes, reading assignments and information presented in this lab, answer the following questions.
1. Describe the relationship between hemoglobin and bilirubin.
2. Free bilirubin appears in the plasma attached to .
3. List three acceptable adjectives or names for the bilirubin molecule before it is conjugated.
(3 points)
4. The conjugation of bilirubin occurs at what location?
5. During the conjugation process, bilirubin will be combined with what substance?
6. List three acceptable adjectives or names for the bilirubin molecule after it has been conjugated. (3 points)
7. How are bilirubin values used?
8. List reagents needed to perform the direct bilirubin procedure. (½ point each, 2 points total)
9. What different reagent(s) are needed for the total bilirubin procedure? What is (are) their purpose(s)? (2 points)
10. How is urobilinogen related to bilirubin?
11. What special procedures are needed in the handling of bilirubin samples? Why?
12. What components are in Diazo Reagent?
13. Associate the different basic types of jaundice with increased levels of bilirubin by completing the following chart. (3 points)
increased bilirubin levels seen / often associated in this type of jaundice indirect
direct total
