I.
II.
III.
IV.
V.
Introduction to Enzymes
General Properties & Definitions
Enzyme Classification and
Nomenclature
Enzyme Kinetics
Enzymes of Clinical Significance
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
i. Forward Reaction (Tanzer-Givarg)
ii. Reverse Reaction (Oliver-Rosalki)
A.
MI Profile
1. Creatine Kinase (CK)
a. Creatine : 20mM
b. ATP/NADH : 2mM
c. PEP : 20mM
d. PK : 3000 U/L
e. LD : 3000 U/L
f. Magnesium : 10mM
b. Total CK Determination
i. Forward Reaction (Tanzer-Givarg)
Coupled-enzyme assay
Auxiliary enzyme
Indicator enzyme
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
i. Forward Reaction (Tanzer-Givarg)
• Measure ↓ in abs. at 340 nm
• pH: 9.0
A.
MI Profile
1. Creatine Kinase (CK)
a. Creatine PO4 : 20mM
b. ADP / NADP : 2mM
c. Glucose : 20mM
d. Hexokinase : 3000 U/L
e. G-6-PD : 3000 U/L
f. Magnesium : 10mM
b. Total CK Determination
ii. Reverse Reaction (Oliver-Rosalki)
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
ii. Reverse Reaction (Oliver-Rosalki)
• Measure ↑ abs. at 340 nm
• 6x faster than forward rxn.
• pH: 6.8
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
i. Forward Reaction (Tanzer-Givarg)
ii. Reverse Reaction (Oliver-Rosalki)
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
§ Source of Error
• Hemolysis: ↑ CK due to ↑ AK activity
• Physical activity & IM injections: ↑ CK
• Light: Inactivates CK
A.
MI Profile
1. Creatine Kinase (CK)
b. Total CK Determination
§ Reference Range
• M: 15-160 U/L
• F: 15-130 U/L
• CK-MB: <6% of total CK
Reference Values:
CK-MM à 94-100%
CK-MB à 0-6%
Reference Values:
CK-MM à 94-100%
CK-MB à 0-6%
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
A.
MI Profile
2. Aspartate Aminotransferase (AST)
b. Total AST Determination
• Karmen Method
•
•
•
Uses malate dehydrogenase
Measure ↓ in absorbance at 340 nm
Falsely ↑ in hemolyzed sample
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
A.
MI Profile
3. Lactate Dehydrogenase (LDH)
b. Total LDH Determination
i.
Wrobleuski – Cabaud or Wacker method
§ Forward Reaction (Lactate à Pyruvate)
ii. Wrobleuski – La Due
§ Reverse Reaction (Pyruvate à Lactate)
A.
MI Profile
3. Lactate Dehydrogenase (LDH)
b. Total LDH Determination
i.
Wrobleuski – Cabaud or Wacker method
• Forward Reaction (Lactate à Pyruvate)
• ↑ in absorbance is monitored at 340 nm
• pH: 8.3 – 8.9
A.
MI Profile
3. Lactate Dehydrogenase (LDH)
b. Total LDH Determination
ii. Wrobleuski – La Due
• Reverse Reaction (Pyruvate à Lactate)
• ↓ in absorbance is monitored at 340 nm
• pH: 7.1–7.4, 3x faster
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
b. Total ALT Determination
i. Walker Method
ii. Reitmann-Frankel
iii. De Ritis Ratio
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
b. Total ALT Determination
i. Walker Method
• Uses Lactate Dehydrogenase
• Monitors ↓ in absorbance (340 nm)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
b. Total ALT Determination
ii. Reitmann-Frankel
• Reagent: 2,4 dinitrophenyl
hydrazine (2,4-DNPH)
• End Color: Brown
˜
Reitmann Frankel
i. Principle
a. Colorimetric, Fixed time
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
b. Total ALT Determination
iii. De Ritis Ratio
• The AST/ALT Ratio
• Ratio > 1 : Non viral origin (alcohol)
• Ratio < 1 : Viral in origin
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
B.
Liver Enzymes
2. Alkaline Phosphatase (ALP)
b. ALP Determination
i. Bowers and McComb
§
§
Based on molar absorptivity of p-Nitrophenol
Absorbance is measured at 405 nm
B.
Liver Enzymes
2. Alkaline Phosphatase (ALP)
ii. Other Methods
Substrate
1-4. Bodansky, Shinowara,
β-glycero-phosphate
Jones, Reinhart
5. Bessy, Lowry & Brock
p-nitrophenyl phosphate
6. Bowers & McComb
7. King and Armstrong
Phenyl phosphate
Phenolpthalein diphosphate
8. Huggins & Talalay
9. Moss
α-napthol phosphate
End Product
Inorganic PO4
+ Glycerol
p-nitrophenol
phenol
phenol
α-napthol
A.
MI Profile
1. Creatine Kinase (CK)
2. Aspartate Aminotransferase (AST)
3. Lactate Dehydrogenase (LDH)
B.
Liver Enzymes
1. Alanine Aminotransferase (ALT)
2. Alkaline Phosphatase (ALP)
3. Gamma Glutamyl Transferase (GGT)
B.
Liver Enzymes
3. Gamma Glutamyl Transferase (GGT)
b. GGT Determination
• Szaz Assay
• Absorbance of p-Nitroaniline is
measured at 405-420 nm
C.
Pancreatic Enzymes
1. Amylase (AMS)
2. Lipase (LPS)
D.
Prostate Enzymes
1. Acid Phosphatase (ACP)
C.
Pancreatic Enzymes
1. Amylase (AMS)
b. AMS Determination
Amylase Methodologies
i. Amyloclastic
ii . Saccharogenic
iii. Chromogenic
iv. Continuous
Monitoring
Starch-iodine comp (dark-blue) à ↓color intensity
Starch à reducing sugars
Starch-dye à Starch-dye fragments (↑ in color)
Coupled enzyme assays
C.
Pancreatic Enzymes
1. Amylase (AMS)
2. Lipase (LPS)
D.
Prostate Enzymes
1. Acid Phosphatase (ACP)
C.
Pancreatic Enzymes
2. Lipase (LPS)
b. LPS Determination
Substrate
Endpoint
Indicator
End Color
Cherry Crandall
Tietz
50% olive oil
FA (Oleic Acid)
Phenolpthalein
Pink
50% olive oil (triolein)
FA (Oleic Acid)
Thymolpthalein + Veronal
Blue
C.
Pancreatic Enzymes
1. Amylase (AMS)
2. Lipase (LPS)
D.
Prostate Enzymes
1. Acid Phosphatase (ACP)
D.
Prostate Enzymes
1. Acid Phosphatase (ACP)
b. ACP Determination
• Assay for Enzyme Activity
Methods
i. Bowers and McComb
ii. Quantitative end point
iii. Continuous monitoring
Substrate
p-nitrophenyl-phosphate
Thymolpthalein monophosphate
α-napthyl phosphate
D.
Prostate Enzymes
1. Acid Phosphatase (ACP)
c. Phosphatase inhibitors
L-tartrate ions
i.
•
•
Inhibits prostatic ACP
Total ACP – ACP after inhibition =
prostatic ACP
Formaldehyde and Cupric ions
ii.
•
Inhibits red cell ACP