Quantitative Determination of Total and Direct Bilirubin in Serum Dept.of Biochemistry

Bilirubin Bilirubin is the yellow breakdown product of normal heme catabolism. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, urine, and the yellow discoloration in jaundice.

Bilirubin Metabolism Bilirubin formation Transport of bilirubin in plasma Hepatic bilirubin transport  Hepatic uptake  Conjugation  Biliary excretion Enterohepatic circulation

BLOOD CELLS LIVER Bilirubin diglucuronide (water-soluble) 2 UDP-glucuronic acid via bile duct to intestines Stercobilin excreted in feces Urobilinogen formed by bacteria KIDNEY Urobilin excreted in urine CO Biliverdin IX  Heme oxygenase O2O2 Bilirubin (water-insoluble) NADP + NADPH Biliverdin reductase Heme Globin Hemoglobin reabsorbed into blood Bilirubin (water-insoluble) via blood to the liver INTESTINE Catabolism of bilirubin (UB ~ Albumin Complex)

Because bilirubin is highly insoluble in water, it must be converted into a soluble conjugate before elimination from the body. In the liver, uridine diphosphate (UDP)-glucuronyl transferase converts bilirubin to a mixture of monoglucuronides and diglucuronides, referred to as conjugated bilirubin, which is then secreted into the bile by an ATP-dependent transporter. This process is highly efficient under normal conditions, so plasma unconjugated bilirubin concentrations remain low.

＊ Conjugation Bilirubin monoglucuronide bilirubin UDP UDP- glucuronyl transferase UDPGA Bilirubin diglucuronide UDPGA UDP UDP- glucuronyl transferase (UDP glucuronic acid)

structure of bilirubin diglucuronide

Serum bilirubin and jaundice Conjugated bilirubin is also called direct reacting bilirubin or hepatobilirubin. ＊ Serum bilirubin 1 ～ 16  mol/l (0.1 ～ 1mg/dl) Free bilirubin is also called indirect reacting bilirubin or hemobilirubin.

free bilirubin conjugated bilirubin Binding with Glucuronic acid no yes Reacting with the diazo reagent Slow and indirect Rapid and direct solubility in watersmalllarge Discharged via kidneynoyes Pass through the membrane of cell yesno Difference of two bilirubins

Hyperbilirubinemia When bilirubin in the blood exceeds 1mg/dl (17  mol/l ), hyperbilirubinemia exists. –prehepatic or haemolytic cause: may be due to the production of more bilirubin than the normal liver can excrete –hepatic cause: may result form the failure of a damaged liver to excrete bilirubin produced in normal amounts –In the absence of hepatic damage, obstruction to the excretory ducts of the liver- by preventing the excretion of bilirubin - will also cause hyperbilirubinemia.

A. Hemolytic anemia excess hemolysis  unconjugated bilirubin (in blood) B. Hepatitis  unconjugated bilirubin (in blood)  conjugated bilirubin (in blood) C. Biliary duct stone  conjugated bilirubin (in blood) Examples of hyperbilirubinemia

Jaundice Definition : Accumulation of bilirubin or its conjugates in body tissues produces jaundice (ie, icterus), which is characterized by high plasma bilirubin levels and deposition of yellow bilirubin pigments in skin, sclera, mucous membranes, and other less visible tissues.

Laboratory results in patients with jaundice normal Hemolytic jaundice Hepatocellular jaundice Obstructive jaundice Serum bilirubin total < 1mg/dl > 1 > 1 > 1 direct 0~ 0.8mg/dl ↑ indirect < 1 Urine bile pigments urobilirubin ––+ urobilinogenA few ↑ uncertainty ↓ urobilinA few ↑ uncertainty ↓ Color of fecesnormaldark Simple or normal Clay color ↑ ↑ ↑

Principle (Colorimetric Method – DMSO) Bilirubin reacts with diazotised sulphanilic acid to form a purple colored azobilirubin complex. Of the two fractions presents in serum, conjugated and free bilirubin loosely bound to albumin –conjugated bilirubin reacts directly in aqueous solution (bilirubin direct) –free bilirubin requires solubilization with dimethylsulphoxide (DMSO) to react (bilirubin indirect). –In the determination of indirect bilirubin, the direct is also determined, the results correspond to total bilirubin. The intensity of the color formed is proportional to the bilirubin concentration in the sample.

Specimens & Materials Specimen: serum Working reagent TB: –Sulfanilic acid, Dimethylsulphoxide (DMSO), Hydrochloric acid (HCl), Sodium nitrite Working reagent DB: –Sulfanilic acid, Hydrochloric acid (HCl), Sodium nitrite C S =20  mol/L Water bath Test tubes Pipettes Spectrophotometer

Method (T-bil) BTBTT STST Working reagent TB 1.5ml - Distilled water 100  l -- Serum -100  l - Standard TB--1.5ml Mix well, incubate for 6 mins at 37  C Mix well, measure the optical density of T T and S T setting zero with B T, λ=578nm.

Method (D-bil) BDBD TDTD SDSD Working reagent DB 1.5ml - Distilled water 100  l -- Serum -100  l - Standard DB--1.5ml Mix well, incubate for 3 mins at 37  C Mix well, measure the optical density of T D and S D setting zero with B D, λ=578nm.

Method (T-bil) BTBTT STST Working reagent TB 1.5ml - Distilled water 100  l -- Serum -100  l - Standard TB--1.5ml Mix well, incubate for 6 mins at 37  C - Mix well, measure the optical density of T T and S T setting zero with B T, λ=578nm.

Method (D-bil) BDBD TDTD SDSD Working reagent DB 1.5ml - Distilled water 100  l -- Serum -100  l - Standard DB--1.5ml Mix well, incubate for 3 mins at 37  C - Mix well, measure the optical density of T D and S D setting zero with B D, λ=578nm.

Calculation C T (  mol/L)=A T /A S x C S Normal value of T-bil: 2~18  mol/L Normal value of D-bil: 2~8  mol/L

Next experiment Glucose tolerance test (p33)

Micropipette Never try to measure a volume that the micropipettor cannot measure. Micropipettes have 3 positions: 1. Rest position 2. First stop 3. Second stop

Operating the Operating the Micropipette Set the volume Attach the Disposable Tip: Fit the tip to the end of the shaft. Press down and twist slightly to ensure an airtight seal. Depress the plunger to the first stop. Draw up the liquid: Immerse the tip 2-3mm into the liquid. Release the plunger back to the rest position. Wait a second for liquid to be sucked up into the tip. Dispense the liquid: Touch the tip end to the side wall of the receiving vessel. Depress the plunger to the first stop, wait one second, press the plunger to the second stop to expel all the liquid Withdraw the Pipette, Release the plunger to the rest position. Discard the Tip: Press ejector button to discard tip.

Spectrophotometry 1 ． Power switch 2 ． Wavelength selection 3 ． “Mode” 4. “100%T ／ 0A” 5 ． “0 ％ T” 6 ． Cuvette holder (sample compartment) 7 ． Pole sample compartment Pole

1.Switch on, allow 20 min for warm up before use. 2.Adjust wave length of maximal absorption. 3.Prepare test, blank and standard sample. sop up liquid with paper, Place them in the cuvette holder. (Notice: put the blank in position 1, Make sure the cuvette is aligned with the light source. ) 4.Mode “A”, press“100%T ／ 0A”, Set A=0 or T= Pull the pole once time. 6.Change mode to “T”, press“0%T ”, Set T =0 7.Change mode to “A”. 8.pull the pole second time, record A1; third time, record A2; forth time,record A3. Operating steps of spectrophotometry

1 、 distinguish transparence and opaque 2 、 control solution at 2/3 volume 3 、 sop up water with paper 4 、 cleanout ， upend it Cuvette