Lab (3): Liver Function profile (LFT) T.A Nouf Alshareef KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012 nf.shareef@hotmil.com

Liver Function profile (LFT) Liver enzymes Execratory function Synthetic function Clinical Lab tests Bilirubin, ALP Proteins TP, Alb, A/G ratio AST, ALT, GGT, ALP

What is Bilirubin ??? is the product of heme degradation (80% hemoglobin, 20% other hemo-protein as cytochrome, myoglobin). Bilirubin is transferred to liver to undergo further metabolic process called (conjugation) to make it more water soluble. Then conjugated bilirubin is excreted in bile to help in food digestion and the excess amount is excreted in urine and stool. Elevated levels of bilirubin in blood and urine indicate certain diseases.

Structure Bilirubin consists of four open chain pyrrols, unlike heme which consists of four rings pyrrols called (porphyrin).

Bilirubin metabolism: Bilirubin is water in-soluble and is carried in plasma bound to albumin (as carrier). When it reaches to the liver, bilirubin is taken by specific carrier meachanism. Pathway of Bilirubin: Liver ……bile…….intestine ……re abosrbe in blood ( back to liver or go to kidney) or go to large intestine

UDP-glucuronyl transferase In liver: Bilirubin is conjugated with Glucouronic acid to produce bilirubin diglucuronides, which is water soluble and readily transported to bile. Blilirubin + Glucouronic acid bilirubin diglucuronides Further metabolic processes are occurred in intestine and kidney (figure) UDP-glucuronyl transferase "water soluble" "Conjugated BIL" "water in soluble" Bile

Types of bilirubin in serum: Indirect bilirubin: is un conjugated or water insoluble, it is called indirect because it reacts slowly, so it indicates indirect reaching to reagent. Direct bilirubin: is conjugated or water soluble it is called direct because it reacts faster, so it indicates direct reaching to reagent. Total bilirubin = D+ ID Knowing the level of each type of bilirubin has diagnostic important.

Jaundice: Pre-hepatic jaundice is a medical term describes the elevation of bilirubin in blood result in yellow color of skin and sclera. Types of Jaundice: according to the cause of jaundice it is classified to three main types: Pre-hepatic jaundice Hepatic jaundice Post-hepatic (most common type)

Post-hepatic jaundice Pre-hepatic jaundice Due to liver cell damage due to cancer or cirrhosis Due to obstruction of hepato-biliary duct. “obstructive jaundice” D.Bil is formed in liver but can’t pass to bile, so it accumulates in liver and leaks to blood “instead of bile” Increase in RBCs breakdown due to hemolytic anemia. Rate of RBCs degradation and Hb production more than ability of liver to convert it to conjugated form Cause High ID.Bil, T.Bil D.Bil high ID.Bil > D.Bil Type of Bil AST, ALT ALP (high) K+ (high) Hematology: CBC, low Hb Conformation test

Physiologic Jaundice of the newborn: High bilirubin levels is common in newborns age (1-3 days). After birth the newborns breaking down the excess RBCs they are born with and, because the newborn’s liver is not fully mature, it is unable to process the extra bilirubin, leads to elevate its level in blood and other body tissues. This situation usually resolves itself within a few days. SO, WHAT TYPE OF JUNDUCE IS THIS ???

Usually newborn is treated by phototherapy which breakdown bilirubin (ID<<<<D) and convert it to the photo isomer form which is more soluble. Very high bilirubin is danger and toxic it may cause brain damage and effect on muscles, eyes and leading to death.

Experiment: Measuring serum bilirubin level Principle: Sulphanalic acid + NaNO3 diazotized sulphanalic acid (DSA) DSA + Bilirubin “D” Azobilirubin “purple” Bilirubin + DSA + accelerator Total bil. (methanol )

Kit components Reagents: Sulfanalic acid reagent Sodium nitrate reagent Methanol reagent Bilirubinequavalent standard (5mg/dl T.bil; 2.5 mg/dl D.bil)

Use this to calculate D.bil Use this to calculate T.bil Procedure: Test Test blank 1.4 ml Sulfanilic acid 25ml - NaNO3 dis. H2O Mix, stand for 1 min 100 ml Sample - Read the Abs after 1 min at 540nm, (Blank dis. H2O) Use this to calculate D.bil 1.5 ml Methanol - Mix by inversion, stand 5 min or more - Read Abs. at 540nm (Blank dis. H2O) Use this to calculate T.bil - Pour Bilirubinequavelant standard in clean cuvette read Abs. at 540nm Read the Abs after 1 min at 540nm, (blank dis. H2O)

Calculations Abs (test) - Abs (test blank) X 2.5 mg/dl Abs of Bilirubinequavelant is 5mg/dl T.bil 2.5 mg/dl D.bil D.Bil: Abs (test) - Abs (test blank) X 2.5 mg/dl T.Bil: Abs (test) - Abs (test blank) X 5 mg/dl To convert to mmol/L multiply by 17.1 Abs of Bilirubinequavelant Abs of Bilirubinequavelant

D.Bil: 0.5 mg/dl or 8.6 mmol/L T.Bil: 1 mg/dl or 17.1 mmol/L Normal range D.Bil: 0.5 mg/dl or 8.6 mmol/L T.Bil: 1 mg/dl or 17.1 mmol/L