1. Khadija Balubaid KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (BIOC 416) 2013 Liver Function profile (LFT) Enzymes

2. 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

5. Liver profile  Profile : is group of tests specific for one organ  Liver function tests (LFTs or LFs), are groups of clinical biochemistry laboratory blood (serum or plasma) or urine assays designed to give information about the state of a patient's liver  These tests can be used to  detect the presence of liver disease  distinguish among different types of liver disorders  Gauge the extent of known liver damage,  follow the response to treatment.

7. SGPT (Serum Glutamate Pyruvate Transaminase) Liver enzymes Produced by liver cells. ALT found primarily in liver. Normal : 7 – 41 U/L Upto 300U/L – nonspecific, any type of liver disorder(CLD…cirrhosis /malignancy) >1000U/L – extensive hepatocellular damage ( viral hepatitis, ischemic liver injury, toxin /drug induced liver injury ) ALT (alanine amino transferase) or

8. 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.

9.  Less sensitive that ALT  Synthesized by : liver, cardiac muscle, skeletal muscles, kidneys, brain, pancreas, lungs, leucocytes and RBC  Normal – 12 – 38U/L  AST – liver, cardiac muscles, skeletal muscle, kidneys, brain, pancreas, lungs, leucocytes, RBC in decreasing order.  2 Iso enzymes- cytoplasmic, mitochondrial  Mild degree of tissue injury – cytoplasmic form in serum  Severe injury – mitochondrial type in serum AST (Aspartate amino transferase) or SGOT (Serum Glutamate Oxaloacetate Transaminase)

10.  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  ALT: < 35U/L, AST: <40U/L  elevated ALT,AST : acute hepatitis (viral or toxic ), chronic hepatitis and cirrhosis,biliary obstruction

11. ALP (alkaline phosphatase) It is related to bile duct. ALP normal level: 30-130 U/L 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.  Hepatocellular carcinoma

12.  Normal physiological elevation :  During pregnancy  During child growth  To assess the etiology of ALP elevation, GGT and bilirubin levels are also measured.

13.  Used in body for syn of glutathione  11 iso enzymes  Normal : 9 – 58 U/L  Produced by liver, kidney and pancreas, intestinal cells,and prostate  Elevated in: toxins, alcoholism,obstructive Jaundice,and neoplasm of liver  Slightly high normally in males prostate  To detect alcohol abuse  Rised even when other LFT are normal in alcohalics.  GGT falls rapidly within few days after abstinence.  Used To confirm hepatic etiology of ALP elevation GGT (Gamma Glutamic Transpeptidase)

14. General indications Conformational procedures IndicationsEnzymes 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, cirohsisGGT In general, every enzyme can gives you specific indication:

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

16. Notes: Samples: Unhemolyzed serum or plasma collected in heparin or EDTA tube.(why?) Stability of AST in serum: 2 days at 20-25 o C or 4 days at 2-8 o C

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

18. Procedure Prepare working reagent: by mixing reagent 1 and 2 together Zero adjust the spectrophotometer with air or dis. H 2 O 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 mlWorking reagent 100  l Sample (serum)

19. Calculations: 25 o C 18 U/LMen 15 U/LWomen AST catalytic conc. U/L = mean A X factor * factor = 1946 Reference value “ normal rang”:

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