Lab 3 Liver Function profile LFT Lecturer Nouf

Lab (3): Liver Function profile (LFT) Lecturer Nouf Alshareef KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012 noalshareef@kau. edu. sa

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

What is Bilirubin ? ? ? • Is water insoluble compound produced in spleen from heme degradation. [Heme: iron + organic compound “porphyrin”]. • Heme source in body: 80% from hemoglobin, 20% other hemo-protein: cytochrome, myoglobin)

Structure • Heme four pyrrols rings connected together to form (porphyrin). • Bilirubin consists of open chain of four pyrrols-like rings

Hemoglobin degrading and bilirubin formation Spleen Plasma Hemoglobin Protein and a. a pool Heme iron Iron pool Bilirubin Binds with albumin TO L Liver IVER Conjugation process

• In liver: Bilirubin is conjugated with Glucouronic acid to produce bilirubin diglucuronides, which is water soluble and readily transported to bile. Blilirubin + Glucouronic acid "water in soluble" UDP-glucuronyl transferase bilirubin diglucuronides "water soluble" "Conjugated BIL" Bile • Then conjugated bilirubin is excreted in bile through bile duct to help in food digestion. • The excess amount transferred to intestine to be excreted in urine and stool.

• Elevated levels of bilirubin in blood and urine indicate certain diseases.

Types of bilirubin in serum: ü Direct bilirubin: is conjugated (water soluble bilirubin) in aqueous solution it reacts rapidly with reagent (direct reacting). ü Indirect bilirubin: is unconjugated (water insoluble bilirubin) because it is less soluble in it reacts more slowly with reagent (reaction carried out in methanol). - in this case both conjugated and unconjugated bilirubin are measured given total bilirubin. Unconjugated will calculated by subtracting direct from total and so called indirect. ü Total bilirubin = D+ ID • Knowing the level of each type of bilirubin has diagnostic important.

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: v Pre-hepatic jaundice v Hepatic jaundice v Post-hepatic (most common type)

Cause haemolytic jaundice hepato-cellular jaundice obstructive jaundice Pre-hepatic jaundice Hepatic jaundice Post-hepatic jaundice Due to increase in Due to liver cell damage Due to obstruction of bile duct which RBCs breakdown due (cancer, cirrhosis or prevents passage of to hemolytic anemia. hepatitis) bilirubin into Conjugation of bilirubin intestine. The rate of RBCs decreased (ID. Bil. ). lysis and bilirubin D. Bil will back to production more than Blilirubin that is liver and then to ability of liver to conjugated is not circulation elevating convert it to the efficiently secreted into its level in blood and conjugated form. bile but leaks to blood urine. (D. Bil. ) Type of Bil. ID. Bil > D. Bil, ID. Bil, T. Bil all (High) D. Bil (High) Conformational test K+ ( High) Hematology: CBC (low Hb) ALT, AST (High) ALP ( High)

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, (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 + Na. NO 3 DSA + Bilirubin “D” Bilirubin “ID”+ DSA + accelerator (methanol ) diazotized sulphanalic acid (DSA) Azobilirubin “purple” Total bil.

Procedure: Test blank Test Sulfanilic acid 1. 4 ml Na. NO 3 - 25 ml dis. H 2 O 25 ml - Mix, stand for 1 min Sample ü 100 ml - Read the Abs after 1 min at 540 nm, (Blank dis. H 2 O) Use this to calculate D. bil Methanol 1. 5 ml - Mix by inversion, stand 5 min or more - Read Abs. at 540 nm (Blank dis. H 2 O) Use this to calculate T. bil - Pour Bilirubinequavelant standard in clean cuvette read Abs. at 540 nm, (blank dis. H 2 O)

Calculations • Conc. of Bilirubinequavelant is 5 mg/dl for T. bil, and 2. 5 mg/dl for D. bil D. Bil: Abs (test) Abs (test blank) X 2. 5 mg/dl Abs of Bilirubinequavelant T. Bil: Abs (test) - Abs (test blank) X 5 mg/dl Abs of Bilirubinequavelant • To convert to mmol/L multiply by 17. 1

Normal range D. Bil: 0. 5 mg/dl or 8. 6 mmol/L T. Bil: 1 mg/dl or 17. 1 mmol/L