Lab (2): Liver Function profile (LFT) Khadija Balubaid KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012

Profile : is group of tests specific for one organ

Liver Liver is an important organ in human body Synthesis of proteins, glycogen storage, drug metabolism and detoxification process Many diseases can affect liver functions as: Viruses (heptites A,B,C,D,G) Cirrhosis Inflammation Jaundice Fatty liver Albumin, clotting factos

Liver diseases

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

SGPT (Serum Glutamate Pyruvate Transaminase) Liver enzymes ALT (alanine amino transferase) or i v e r SGPT (Serum Glutamate Pyruvate Transaminase) Produced by liver cells. High serum ALT due to: Liver cells damage due to inflammation, virus infection or cell death (why?) when liver cells damaged ALT enzyme leaks to blood stream leads to rise its level in serum. Some medication may also elevate serum ALT, because some drugs cause liver damage leads to rise ALT level. ALT is the most sensitive marker for liver cell damage; since it is only synthesized by liver cells other enzymes may be also synthesized by other organs. or SGPT

AST (Aspartate amino transferase) or SGOT (Serum Glutamate Oxaloacetate Transaminase) Synthesized by : liver, cardiac muscle and skeletal muscles Less sensitive that ALT High serum AST due to: Muscle damage, myocardial infarction (heart attack) and in chronic liver disease. To confirm that high AST is due to heart or muscle injury; other enzyme (creatinine kinase CK) which is specific for heart, is also tested. Because it is less sensitive the ration ALT/AST is calculated

ALP (alkaline phosphatase) It is related to bile duct. It is not specific for bile because it is synthesized also by bone and placenta (isoenzymes) High serum ALP may be due to: bile duct damage (inflammation, cirrhosis or obstruction) In alcohol hepatitis. Normal physiological elevation : During pregnancy During child growth To assess the etiology of ALP elevation, GGT and bilirubin levels are also measured.

GGT (Gamma Glutamic Transpeptidase) Produced by liver, kidney and pancreas Elevated in: toxins, alcohol To confirm hepatic etiology of ALP elevation

Conformational procedures General indications In general, every enzyme can gives you specific indication: Conformational procedures Indications Enzymes Serology (for virus) Biopsy, ultrasound (liver size) Hepatitis. may be due to ( virus, medication, toxin) ALT AST/ALT ratio, CK to confirm heart disease Not specific. May be due to ( muscle disease, heart disease, liver disease) AST GGT to confirm liver Gallbladder ultrasound Bile problems (stone or bilary duct obstruction) Liver disease Normal physiological elevation ( child, pregnancy) ALP Liver toxin, alcohol, cirohsis GGT

Experiment: Measuring serum AST level Principle: The rate of NADH oxidation is directly related to AST activity which measured photometrically. L-aspartate + a oxoglutarate L-glutamate + oxaloacetate Oxaloacetate + NADH + H+ L-malate + NAD + AST MDH

Notes: Samples: Unhemolyzed serum or plasma collected in heparin or EDTA tube. Stability of AST in serum: 2 days at 20-25oC or 4 days at 2-8oC

Kit components Reagent 1: mixture of: buffer (pH 7.5) + substrate (L-aspartate) Reagent 2: mixture of: enzyme (MDH) + coenzyme (NADH)

Procedure Prepare working reagent: by mixing reagent 1 and 2 together Zero adjust the spectrophotometer with air or dis. H2O Prepare the reaction as the following: Mix After 30 sec. read the absorbance at 340nm. (R1) Repeat the reading after 1min and 2 min (R2, R3) Calculate the mean absorbance mean = R1+R2+R3/ 3 or (R2-R1)+(R3-R2)/2 Sample tube 1 ml Working reagent 100 ml Sample (serum)

AST catalytic conc. U/L = mean A X factor Calculations: AST catalytic conc. U/L = mean A X factor * factor = 1946 Reference value “ normal rang”: 25oC 18 U/L Men 15 U/L Women

Interfering factors Therapeutic heparin increase AST Hemolysed blood increase AST Many drugs falsely increase or decrease AST