1. MLAB 2401: Clinical Chemistry Keri 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
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
Multiple measurements of absorbance change are made
Advantages
Depletion of substrate is observable
Improved accuracy

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 Source
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. Creatine Kinase (CK): Structure
Dimer consisting 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. Creatine Kinase: Specimen Collection
Sources of Error
Hemolysis
Interference of adenylate kinase on CK assays
Results in false elevations
Exposure to light
CK is inactivated by light

14. Creatine Kinase: Reference Range
Affected by:
Age
Physical activity
Race
Bed rest (even overnight can decrease CK)
Total CK
Men: U/L
Female: 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 times that found in serum
Analyte stability
Run assay asap or store at room temperature
Prolonged contact of serum and cells
Reference Range
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)
Bone
Liver
Kidney
Placenta
Intestines
Prostate gland
Seminal fluid
Liver
Spleen
RBCs
Platelets

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

33. Alkaline Phosphatase (ALP)
Diagnostic Significance
Elevations seen in
During bone activity
Paget’s disease
Liver disease, especially in obstructive disorders
Pregnancy between 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: 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 Reference Range
Presence of opiates increases levels
Stabile
Reference Range
Serum: 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 .