MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Measurement of Enzymes & Their Clinical Significance

2. Measurement of Enzyme Activity • Often measured by catalytic activity then related to concentration • Enzyme concentration is best measured by its activity or its rate of activity by observing: • Substrate depletion • Product production • Increase/decrease in cofactor/coenzyme • Usually performed in zero-order kinetics

3. Measurement of Enzyme Activity Fixed time Continuous Monitoring/Kinetic Multiple measurements of absorbance change are made Advantages Depletion of substrate is observable Improved accuracy • Measurement of the amount of substrate utilized over a fixed amount of time or by a fixed amount of serum • Problems • Long incubation times • Possible enzyme denaturation • Often a lag phase • Possible substrate depletion

4. Reporting Enzyme Activity • Originally reported as activity units • IUB standardized these as international units (IU) • IU: the amount of enzyme that will convert one micromole of substrate per minute in an assay system • Expressed as units per liter or U/L • Conditions: pH, temperature, substrate,activators • Katal units(SI): express as moles/second

5. Other Methods to Measure Enzymes • Using Enzyme Mass • Measure protein mass NOT catalytic activity • Electrophoresis • Used to differentiate isoenzymes • Time-consuming

6. Enzymes of Clinical Significance

7. Creatine Kinase (CK) • Action • Associated with the regeneration and storage of ATP • Catalyses the following reaction:

8. Creatine Kinase (CK) • Purpose • Allows the body to store phosphate energy as creatine phosphate • Energy can be released/ provided to muscles by reversing the reaction • Source • Skeletal muscle • Heart • Brain • Other

9. CreatineKinase (CK):Structure • Dimerconsisting of two subunits • Two subunits are further divided into 3 molecular forms or isoenzymes • CK-BB: (CK-1)brain type • Migrates fast on electrophoresis • Small amount found in tissue (brian, lung, bladder, bowel) • CK-MB: (CK-2)hybrid type • Heart, Skeletal • CK-MM: (CK-3)Muscle type • Mostly found in healthy people • Striated muscle and normal serum

10. Creatine Kinase (CK) • Diagnostic Use • Appearance of CK (MB) very sensitive indicator of MI • Skeletal muscle disorders such as muscular dystrophy • CNS Disorders • Cerebrovascular accident(CVA) • Seizures • Nerve degeneration

11. CK Isoenzymes

12. What’s in a Number?

13. CreatineKinase: Specimen Collection • Sources of Error • Hemolysis • Interference of adenylatekinase on CK assays • Results in false elevations • Exposure to light • CK is inactivated by light

14. CreatineKinase: Reference Range • Affected by: • Age • Physical activity • Race • Bed rest (even overnight can decrease CK) • Total CK • Men: 46-180 U/L • Female: 15-171 U/L

15. Creatine Kinase • Isoenzyme Testing • Fractionation of CK • Immunoinhibition • Mass Assay • Electrophoresis

16. Lactate Dehydrogenase (LDH/LD) • Action • Catalyzes a reversible reaction between pyruvate and lactate with NAD as a coenzyme • Reaction:

17. Lactate Dehydrogenase (LDH/LD) • Source • Skeletal muscle • Cardiac muscle • Kidney • RBCs • Widely distributed in the body

18. Lactate Dehydrogenase (LDH/LD):Structure • Tetramer • Four polypeptide chains, two subunits (heart & muscle) • Five combinations of Isoenzymes

19. Lactate Dehydrogenase (LDH/LD) • Diagnostic Significance • Nonspecific • Increased • Hematologic and neoplastic disorders • Liver disease • Heart problems

20. Lactate Dehydrogenase(LDH/ LD):Specimen Collection • Sources of Error • Hemolysis • RBCs contain 100-150 times that found in serum • Analyte stability • Run assay asap or store at room temperature • Prolonged contact of serum and cells • Reference Range • 140- 280 U/L

21. Liver Enzymes • Transaminases • AST • ALT • Phosphatases • ALP

22. Transaminases • Retain amino groups during the degradation of amino acids • Types • Aspartate transaminase (AST) • Aka: Glutamic Oxalocetic transaminase (SGOT) • Alanine transaminase (ALT) • AKA: Glutamic pyruvic transaminase (SGPT)

23. Aspartate Aminotransferase( AST) • Sources • Major • Heart • Liver • Muscle • Minor • RBCs • Kidney • Pancreas • Lung

24. Aspartate Aminotransferase( AST) • Reaction: AST

25. AST:Specimen Collection • Sources of Error • Hemolysis • Analyte stability • Stable at room temp for 48 hours or 3-4 days refrigerated • Reference Range • 5-30 U/L

26. Alanine Transaminase (ALT) • Transfers an amino group from alanine to alpha-ketoglutarate to form glutamate and pyruvate ALT

27. Alanine Transaminase (ALT) • Sources • Liver (Majority) • Kidney • Heart • Skeletal muscle

28. ALT:Specimen Collection • Sources of Error • Hemolysis • Analyte stability • 3-4 days refrigerated • Reference Range • 6-37 U/L

29. Diagnostic Significance: AST & ALT • Many diseases can cause increases since widely distributed in tissues • Liver • Hepatitis • Cirrhosis • Liver cancer • Myocardial Infarction • AST increases most • ALT normal to slightly increased, unless liver damage accompanies • Other • Pulmonary emboli • Muscle injuries • Gangrene • Hemolytic diseases • Progressive Muscular dystrophy

30. Phosphatases • Removes phosphates from organic compounds • Functions to facilitate transfer of metabolites across cell membranes • Alkaline Phosphatase (ALP) • Acid Phosphatase (ACP)

31. Phosphatases: Sources Alkaline Phosphatase (ALP) Acid Phosphatase (ACP) Prostate gland Seminal fluid Liver Spleen RBCs Platelets • Bone • Liver • Kidney • Placenta • Intestines

32. Alkaline Phosphatase (ALP) • ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an alkalinepH • Maximum activity at pH of 9.0- 10.0

33. Alkaline Phosphatase (ALP) • Diagnostic Significance • Elevations seen in • During bone activity • Paget’s disease • Liver disease, especially in obstructive disorders • Pregnancy between 16-20 weeks gestation • Decreased levels occur, but not diagnostic

34. Alkaline Phosphatase (ALP):Specimen Collection • Sources of Error • Hemolysis • Delay in processing, false increases can occur • Reference Range (Adult) • 30-90 U/L • NOTE: Normal increases seen in pregnancy, childhood, adolescence

35. Acid Phosphatase (ACP) • Diagnostic Significance • Aids in detection of prostatic carcinoma • Other conditions associated with prostate • Forensic chemistry: Rape cases • Elevated in bone disease

36. Acid Phosphatase (ACP):Specimen Collection • Sources of Error • Separate serum from cells asap • Decrease in activity seen at room temp • Hemolysis • Reference Range (prostatic) • 0-4.5 ng/mL

37. Gamma-Glutamyltransferase (GGT) • Possibly involved in peptide and protein synthesis, transport of amino acids and regulation of tissue glutathione levels • Sources • Kidney • Brain • Prostate • Pancreas • Liver

38. Gamma-Glutamyltransferase (GGT) • Diagnostic Significance • Sensitive indicator of liver damage • Increased in patients taking enzyme-inducing drugs such as warfarin, phenobarbital and phenytoin • Indicator of alcoholism • Elevated in acute pancreatitis, diabetes mellitus and MI

39. GGT:Specimen Collection • Sources of Error • Very stable • Hemolysis not an issue • Reference Range • Male: 10-34 U/L • Female: 9-22 U/L

40. Digestive & Pancreatic Enzymes • Amylase • Lipase

41. Amylase (AMS) • Digestive enzyme that hydrolzes/catalyzes the breakdown of starch and glycogen to carbohydrates • Smallest enzyme • Sources • Acinar cells of pancreas and salivary glands

42. Amylase (AMS) • Diagnostic Significance • Acute pancreatitis • AMS levels rise 2-12 hours after onset of attack, peak at 24 hrs and return to normal within 3-5 days • Salivary gland lesions • Mumps

43. Amylase • Sources of Error • Presence of opiates increases levels • Stabile • Reference Range • Serum: 30-100 U/L • Urine: 1-17 U/h

44. Lipase (LPS) • Hydrolyzes triglycerides to produce alcohols and fatty acids • Source • Pancreas

45. Lipase (LPS) • Diagnostic Significance • Acute pancreatitis • More specific than amylase • LPS persists longer than AMS

46. Lipase:Specimen Collection • Sources of Error • Stabile • Hemolysis results in false decreases • Reference Range • 13-60 U/L

47. References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .