1. Course of Advanced Diagnostics Integrated laboratory evaluation of liver Prof. Giuseppe Castaldo, a.y. 2015-16

6. Acute Cytolysis release in blood of liver enzymes Chronic Progressive destruction and regeneration of the liver parenchyma leading to fibrosis Reduced synthesis of proteins -Viral (A, B, C, …) -Ethanol -Drugs -Autoimmune Liver disease

10. Biochemical markers of liver disease Conjugation –Conjugated and unconjugated Bilirubin –Urine urobilin Cytolysis –Alanine Aminotransferase (ALT, GPT) –Aspartate Aminotransferase (AST, GOT) –Lactate Dehydrogenase (LD) Cholestasis –Alkaline phosphatase (AP) –  -Glutamyltransferase (  -GT) Protein biosynthesis –Albumin –Prothrombin time

16. Congenital icterus -Pre-hepatic:Gilbert -Hepatic:Crjgler Najjar, Rotor -Post-hepatic:Dubin Johnson

18. Gilbert Enhanced unconjugated bilirubin ( values between 1,5 and 6 mg/dL, usually < 3 mg/dL) Altered liver uptake and conjugation of bilirubin Frequent alterations of drugs metabolism (i.e., paracetamol) It appears between 20 and 30 years and thus, frequently disappear Normal levels of cytolytic and cholestetic enzymes 8% of adult population, benign Autosomal recessive Reduced activity of bilirubin-uridin-glucuronosil transferase (UGT-1) UGT-1A1 gene (mutation within the TATA box, TA repeat)

19. G A T C 1 2 3 4 5 6 7 8 wt mut (TA) 6 (TA) 7 Gilbert: Two copies of the (TA) 7 repeat

20. Autosomal recessive Altered excretion of conjugated bilirubin in the bile canaliculus Altered MRP2 (multidrug resistence-associated protein 2) Dubin-Johnson Total bilirubin: 2 - 5 mg/dL; conjugated bilirubin: 60-70 % It appears during the II- III rd decade Triggered by pregnancy, surgery and estrogenic therapy Good prognosis

21. Phototherapy Crigler-Najjar rare and severe neonatal onset neurological complications Eye protection

22. type I complete deficiency of the UDP glucuronyl transferase bilirubin levels < 20 mg/dL no conjugated bilirubin in the bile type II partial deficiency of the enzyme bilirubin levels < 20 mg/dL phenobarbital helps to reduce bilirubin

24. Aminotransferases Aspartate aminotransferase (AST) Reference values Male: 10-45 UI/L Female: 5-30 UI/L Alanine aminotransferase (ALT) Reference values Males: 10-40 UI/L Females: 5-35 UI/L

25. ALT AST AST and ALT localization

26. Why AST and ALT are mainly expressed by liver and by muscle?

28. ALT Cytoplasmatic AST Cytoplasmatic Mitochondrial

29. Age and gender effect on reference values of AST and ALT Males  Females

30. Lactate dehydrogenase Catalyzes the reversible pyruvate-lactate conversion Reference values: 340-450 UI/L 12 3 5 5 isoenzymes 4 Tetrameric enzymes H (heart) e M (muscle) subunits

31. Expression of Lactate dehydrogenase isoenzymes 1 2 1 2 1 2 3 1 2 3 4 4 5 1 2 4 5 4 5

32. Why LD catalyzes the reaction in a different direction in liver and in muscle?

33. Biochemical markers of cytolysis (AST, ALT, LDH) ↑ ↑ ↑Acute viral hepatisis ↑ ↑ ↑Acute drugs or ethanol hepatitis ↑ ↑Autoimmune hepatitis ↑ ↑Active chronic hepatitis ↑Hearth failure ↑Cholestasis False positives Acute myocardial infarction Muscle cytolysis

34. Alkaline phosphatase Reference values Adult: 50-190 UI/L Newborn: 110-700 UI/L Child (1-10 years): 110-550 UI/L Puberty: 130-700 UI/L -Physiological: infancy, puberty, pregnancy -Bone disease (osteoblasts, bone osteogenesis): hyperparathyroidism, osteomalacia, osteomyelitis -Hepatobiliary disease: (cholestasis): cirrhosis neoplasia, gallstones -Intestine: acute intestinal diseases

35. Alkaline phosphatase Isoforms

37. Gamma-glutamyltransferase Serum increase in cholestasis May be induced (drugs, ethanol) Reference values Male: 6-28 UI/l Female: 4-18 UI/l Newborn: 10-100 UI/l

38. Localization of  -GT

39. Age and gender effect on the reference values of AP and  -GT Males  Females

40. Biochemical markers of cholestasis (AP and  -GT) Biliary lithiasis Liver neoplasia Biliary neoplasia Pancreatic cancer Biliary cirrhosis False positives -  -GT Ethanol Drugs - AP Pregnancy Bone diseases Ovarian cancer

55. Biochemical markers of liver fibrosis Fibro scan: GGT, ALT, Bilirubin, aptoglobin, apolipoprotein AI, alpha2-macroglobulin

56. Congenital liver diseases -Hemochromatosis -Alpha 1 antitrypsin deficiency -Cystic fibrosis -Wilson’s disease

65. Serum alpha1 antitrypsin (nephelometric) Genetic analysis ?

67. Ridotta cupremia Ridotta ceruloplasmina

69. Pre-hepaticHepaticPost-hepatic S total Bilirubin S con Bilirubin S uncon Bilirubin S AST A AP U urobilinogen U bilirubin Stool

70. How can you discriminate between pre- hepatic icterus and Gilbert ?