1 Bilirubin CC2 L Handout PDF

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Summary

Bilirubin is derived from two main sources. Roughly, 80% of bilirubin is made from the breakdown of hemoglobin in senescent red blood cells, and prematurely destroyed erythroid cells in the bone marrow. The remainder originates from the turnover of various heme-containing proteins found in other tis...

Description

Activity No. 1

Metabolism of Bilirubin

Bilirubin

RBC destruction

-end product of hemoglobin metabolism

-hemoglobin is release

-orange-yellow pigment derive from bilirubin degradation

-hemoglobin in the plasma is carried by transporter protein called haptoglobin

-Transported by albumin Forms: B1 (Unconjugated bilirubin) Non polar bilirubin

B2 (Conjugated bilirubin) Polar bilirubin

Water insoluble bilirubin Indirect bilirubin Hemobilirubin Slow reacting bilirubin

Water soluble bilirubin

Pre-hepatic bilirubin

Post-hepatic, hepatic, obstructive, regurgitative bilirubin

Direct bilirubin Cholebilirubin Prompt bilirubin

o Biliary atresia  The failure of common bile duct to form an opening o Cholecystitis  Inflation of the gallbladder o Cholelithiasis  Formation of the gallstones o Choledochithiasis  Presences of gallstone in the biliary tree Urobilinogen in Urine and feces: Ehrlich, 1901   

Specimen  

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Serum or plasma; urine Avoid hemolyzed samples- may cause interference and may lead to falsely decrease result when Jendrassik method is use Stored in the dark- bilirubin may photo oxidize when expose to light

Temperature Room temperature 4oC -20oC Bilirubin Analysis

Stability 2 days 1 week indefinite

Year

Methods

Sample

Accelerator

1901

Ehrlich

urine

p-dimethyl aminobenzaldehyde

1913

Van den Bergh

serum

Sodium nitrate and HCL

1937

Malloy and Evelyn

serum

50% methanol solution

1938

Jendrassik and Grof

serum

caffeine-sodium Benzoate-acetate

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Quantitative method Basis: p-dimethyl amonobenzaldehyde Steps:  to measure urobilinogen it must be treated with ascorbic acid (reducing agent)  -next add saturated sodium acetate to stop reaction  -add Ehrlich’s reagent to have end color of red End color: red If End color: green- means measuring other compounds like porphobilinogen, sulfonamides, procaine, 5-hyroxyindoleactic acid

Preferred Specimen   

Fresh 2 hr. urine Should be keep cool Protect from light

Detection of fecal urobilinogen    

Reference range 0.1-1.0 Ehrlich unit every 2 hours 0.5-4.0 Ehrlich unit per day Steps:  Aqueous extract of fresh feces  Alkaline ferrous hydroxide as reducing agent  Then add Ehrlich’s reagent

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75-275 Ehrlich unit per 100g of fresh feces 75-400 units per 24 hours’ specimen

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 Total: 0.2-1.0mg/dL (3-17 umol/L) Method for total and conjugated bilirubin

Terwen, 1925  

Alkaline ferrous hydroxide Sodium acetate- eliminate interference such as indole

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Watson, 1936 

petroleum ether- to remove interfering substances

Malloy and Evelyn, 1937   

Specimen: serum End Product: azobilirubin Steps:  Serum is treated by diazo reagent - diazo-A is 0.1 % of diazotized sulfanilic acid and diazo-B is 0.5% sodium nitrite and diazo blank (1.5% HCL)  Accelerator is 50% methanol  End color: red-purple color with wavelength 560 nm  Positive result: pink to pink red to purple azobilirubin

Specimen: serum -neither hemolyzed/ lipemic- cause decrease serum bilirubin -stored in the dark - refrigerated sample is up to 1 week or freezered for 3 months

Direct Spectrophotometry (Bilirubinometry) -

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measurement of total bilirubin in serum analysis of two-component system by measuring absorbance at two wavelengths that provide a numerical index applicable to sera from neonates not applicable to adults: carotenoid compound

Jendrassik-Grof, 1938 Specimen: serum/plasma Steps for conjugated bilirubin:  Treat with diazo reagent  End Color: pink-purple color  Do not need accelerator  Yield conjugated bilirubin only Steps for total bilirubin  Treat with diazo reagent  Use caffeine benzoate as acceleratoraccelerate the coupling of bilirubin with diazo reagent  Terminated by ascorbic acid- destroy the excess concentration of diazo reagent  Add strong alkaline tartrate solution to convert to pink-blue color (600nm)  Result: pink to purple to blue azobilirubin Reference Ranges for bilirubin  Conjugated: 0-0.2 mg/dL (0-3 umol/L)  Unconjugated: 0.2-0.8 mg/dL (3-14 umol/L)

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Formula of delta bilirubin: Total bilirubin - direct bilirubin + indirect bilirubin Reference value:...