This document reports on a student's practical experiment to determine bilirubin levels using a colorimetric method. It includes background information on bilirubin, how it is produced and metabolized in the body. The method, reagents, procedure, results and calculations for determining total and direct bilirubin levels are described. Normal bilirubin ranges and potential causes of elevated levels are presented, along with a discussion of bilirubin metabolism and conditions that can affect it like jaundice. Common treatment approaches like phototherapy and drugs that influence bilirubin conjugation are also summarized.

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER:Dr OKUN
HANDING DATE: 11 /4/2017
SUBJECT: PRACTICAL REPPORT ON COLOMETRIC DETERMINATION
OF BILLIRUBIN.
ELVIS L
©

2. Objectives
quantitative,diagnosticdeterminationof bilirubinusingcolometricmethod.
Background
Bilirubinisthe yellowbreakdownproductof normal heme catabolism. Bilirubinisexcretedinbileand
urine,andelevatedlevelsmayindicatecertaindiseases.Itisresponsible forthe yellow colorof bruises,
urine,andthe yellowdiscolorationinjaundice.
The average level of the bilirubinproducedinhumansfromdifferentsourcesrangesbetween250to
300 mg/day,of which85% is derivedfromthe heme moietyof the haemoglobinreleasedfrom
senescenterythrocytesthatare destroyedinthe reticuloendothelialsystem.The remaining15 % is
producedfromerythrocytesdestroyedinthe bone marrow andfromcatabolismof otherheme
containingproteinssuchascytochromesandmyoglobin.
Afterit isproducedinthe peripheral tissues,bilirubinistransportedtothe liverinassociationwith
albumin.Inthe liver,bilirubinis conjugatedwithglucuronicacidforsolubilizationandsubsequent
transportthroughthe bile ductandeliminationviathe digestivetract.Disease orconditionswhich,
throughhemolyticprocesses,produce bilirubinfasterthanthe livercanmetabolizeit, cause the levelsof
unconjugated(indirect) bilirubintoincrease inthe circulation.Bile ductobstructionordamage to
hepatocellularstructure causesincreasesinthe levelsof bothconjugated(direct) andunconjugated
(indirect) bilirubininthe circulation.
The intensityof the colorformedisproportional tothe bilirubinconcentrationinthe sample.
Method
Colorimetricmethod.
Assay Principle
Bilirubinisconvertedtocoloreddiazotizedsulfanilicacidandmeasuredphotometrically.Of the two
fractionspresentsinserum,bilirubinglucuromideandfree bilirubinlooselyboundtoalbumin.onlythe
formerreactsdirectlyinaqueoussolution(bilirubindirect),whilefree bilirubinrequiressolubilization
withdimethylsulfoxide (DMSO) toreact(bilirubinindirect).Inthe determinationof indirectbilirubinthe
directisalso determined,the resultscorrespondtototal bilirubin.
Reagentsand apparatus
-Testsample
-spectrophotometerset at 555Nm
-Roomtemperature
-R2 solution(totalbillirubin estimation)
-R1 solution(directbillirubinestimation)
-Distilledwater.

3. SpecimenPrecautions:
-Serumor plasma
-Avoidhaemolysis
-Avoidlightexposure
-Storage for3days indark and refrigeratorformonthsif freezedat -700
C
-Urine sample collectedwithin24hrs
Procedure
1. Assayconditions.
Wavelength…………555nM
Cuvettete……………1cmlightpack
Temprature…………15 – 250
C
2. Adjustspectrophotometertozerowithdistilledwater.
3. Pipette tocuvette
DirectB Blank Total B Blank
1.5 1.5 - - R1(D)µm
- - 1.5 1.5 R2(I)µm
50 - - - R3µl
100 100 100 100 Sample
Absorbance
4. Mix andincubate exactlyfor5minutesat15 – 250
C
5. Readthe absorbance(A)
Resultsand calculation
DirectB Blank Total B Blank
1.5 1.5 - - R1(D)µm
- - 1.5 1.5 R2(I)µm
50 - - - R3µl
100 100 100 100 Sample
0.060 0.039 0.069 0.051 Absorbance
Calculations
Withfactor:
Absorbance of sample –absorbance of sample blankx factor= mg/dLbillirubininsample
Theoretical factor:Billirubin(T) =19.1 : billirubin(D) =14
Conversionfactor: mg/dl x 17.1 = µmol/L
i.Directbillirubinestimation(D)
=( 0.060 – 0.039) x 14.0

4. =0.294mg/dm
=(0.294mg/dm x 17.1) = 5.0274 µmol/L
ii.Total bilirubinestimation(T)
= ( 0.069 – 0.051) x 19.1
=0.3438mg/dl
=( 0.3438mg/dl x 17.1) µmol/L
=5.879 µmol/L
Normal Ranges
Bilirubinform Normal value
Total (elderly,adult,child)
(newborn)
Critical value (adult)
(newborn)
0.1 to 1.1 mg/dL
1.0 to 12.0 mg/dL
>12 mg/dL
>15 mg/dL
Pre-hepatic,unconjugated,indirect 0.0 to 0.8 mg/dL
Post-hepatic,conjugated,direct 0.0 to 0.25 mg/dL
Fecal urobilinogen 40 to 280 mg/day
Urine 0.0 to 0.02 mg/dL
Interpretation:the billirubinlevel of the sample ( bothtotal anddirectbillirubin)are withinnormal
ranges.
Discussionand conclusion
Billirubinisformedfromheme degradation.Heme isdegradedbyreticuloendothelial cells
(mononuclearphagocytesof the spleen,liver,andbone marrow).

5. Bilirubinisinsoluble inwaterandisresponsible forthe toxiceffects. Thisunconjugated(indirect)
bilirubinistransportedinthe serumboundtoalbumin.
Cause of Jaundice:Excessbilirubin(hyperbilirubinemia) isthe maincause of jaundice.Bilirubin,
whichisresponsibleforthe yellowcolorof jaundice,isanormal part of the pigmentreleasedfrom
the breakdownof "used"redbloodcells.
Procesing:Unconjugated(indirect,pre-hepatic) bilirubinistransportedfromthe surface of the
hepatocyte tothe endoplasmicreticulumthroughthe bindingof ligandin. Ittakesseveral weeksfor
ligandinlevelstoincrease inneonates. Asa result,neonatal jaundice isnotuncommon.
Phototherapytransformsunconjugated(indirect)bilirubinintoawater-solubleform.
Glucuronicacidis addedto bilirubin(catalyzedbyglucuronyltransferase) toproduce the conjugated
(direct) mono- anddiglucuronides. UDP-glucuronyl transferasedeficiencies - milddeficiency
(Glibert’ssyndrome),severe deficiency(Crigler-Najjar)
Conjugatedbilirubins,whichare watersoluble,maybe excretedinthe urine andfeces. The
diglucuronideisprimarilyexcretedinnormal bile. Almostall of the bilirubinproducedisexcretedas
one of the componentsof bile salts.Bilirubinisthe pigmentthatgivesbile itscharacteristicbright
greenishyellow color.
Excretion:Whenthe bile saltsreachthe intestineviathe commonbile duct,the bilirubinisactedon
by bacteriato formchemical compoundscalledurobilinogens.Mostof the urobilinogenisexcreted
inthe feces;some isreabsorbedandgoesthroughthe liveragainanda small amountisexcretedin
the urine.Urobilinogengivesfecestheirdarkcolor.Anabsence of bilirubininthe intestine,suchas
may occur withbile ductobstruction,blocksthe conversionof bilirubintourobilinogen,resultingin
clay-coloredstools.Some of the urobilinogenthatisproducedinthe intestineisreabsorbedand
recycledthroughthe liver.
Commonestmethodsof estimatingbillirubininneonates: Clinical Assessment, Ingram
icterometer:,andTranscutaneousbilirubinometer.
Laboratory valuesand clinical relevance:Elevatedserumbilirubinlevels - increasedproduction,
decreasedconjugation,decreasedsecretionbythe liver,orblockage of the bile ducts. Incasesof
increasedproduction,ordecreasedconjugation,the unconjugatedorindirectformof bilirubinwill
be elevated.
Unconjugatedhyperbilirubinemia - acceleratederythrocytehemolysisinthe newborn
(erythroblastosisfetalis),absence of glucuronyl transferase,orhepatocellulardisease.
Conjugatedhyperbilirubinemia - obstruction of the biliaryducts,aswithgallstonesorhepatocellular
diseasessuchascirrhosisor hepatitis.
Elevatedserumbilirubintestresultsmayalsobe causedbythe effectsof manydifferentdrugs,
includingantibiotics,barbiturates,steroids,ororal contraceptives.
Chronicacquiredliverdiseases,the serumbilirubinconcentrationisusuallynormal until asignificant
amountof liverdamage hasoccurredand cirrhosisispresent.

6. Acute liverdisease,the bilirubin isusuallyincreasedinrelationtothe severityof the acute process.
Associatedsyndromes(Congenital hyperbilirubinemia):
1.Crigler-NajjarSyndrome:Autosomal recessive .Extremelyrare <200 casesworldwide –gene
frequencyis< 1:1000. Highincidence inthe “plainpeople of Pennsylvania”(AmishandMennonites).
Characterizedbya complete absence ormarkedreductioninbilirubinconjugation.Presentwitha
severe unconjugatedhyperbilirubinemiathatusuallypresentsatbirth.Afflictedindividualsare ata high
riskfor kernicterus .Conditionisfatal whenthe enzyme iscompletelyabsent.Treatedbyphototherapy
(10-12 hrs/day) andlivertransplantbyage 5.
2.Dubin-JohnsonandRotor’sSyndromes:Characterizedbyimpairedbiliarysecretionof conjugated
bilirubin.Presentwithaconjugatedhyperbilirubinemiathatisusuallymild
3.Dubin-JohnsonandRotor’sSyndromes:Characterizedbyimpairedbiliarysecretionof conjugated
bilirubin.Presentwithaconjugatedhyperbilirubinemiathatisusuallymild
Treatment & Therapeutic Considerations:
PHOTOTHERAPY
-Throughabsorptionof the wavelengthsatthe blue endof the spectrum(blue,greenandwhite light),
bilirubinisconvertedintowater-soluble photoisomers.Thistransformation enhancesthe molecule’s
excretionintobilewithoutconjugation.
PHENOBARBITAL
-Thisdrug isnot approvedbyFDA for use inneitheradultnorpediatrichyperbilirubinemiapatients,due
to possibilityof significantsystemicside-effects.
-Exactpathwayis notknown,butit isbelievedtoact as an inducingagentonUDP-
glucuronosyltransferase,therebyimprovingconjugationof bilirubinanditsexcretion.
ALBUMIN
-A 25% infusioncanbe usedintreatinghyperbilirubinemia(esp.due tohemolyticdisease).
-Itis usedinconjunctionwithexchangetransfusiontobindbilirubin,enhancingitsremoval.
CLOFIBRATE(ATROMID-S)
-Thisdrug hasbeenshowntoreduce bilirubinlevelsviaanunknownmechanism.
-Clofibrate isalsoassociatedwithincreasedriskof developingcholelithiasis,cholecystitis,aswell as
functional liverabnormalities,whichcanworsenhyperbilirubinemia.
PERCUTANEOUSTRANSHEPATICCHOLANGIOGRAPHY
-Allowsextractionof stonesandthusremoval of the source of obstructionwhenpresent.

7. References
1. Mims, L., Gooden,D.S.Phototherapyforneonatal hyperbilirubinemia:. Phys.Med.Biol.1974;19:
263.
2. With,T. K., Biology of Bile Pigment.s, Ante Frost-Hansen,Copenhagen(1954).
3. Stoner,R. E., and Weisberg,H.F.billirubinmetabolism: Clin. Chem. 3, 22 (1957).
4. Malloy, H. T., andEvelyn,K.A., J. Biol. Chem. 119, 481 (1937).
5. Overbeek,J.T.0., Vink,C.L. J., and Deenstra,H., Eec. Tray. Chim. ı 74, 85
(1955).