Principles

Accurate enzyme testing is vital for diagnosing and monitoring a wide array of diseases. Understanding the principles behind these tests is crucial for accurate lab work

Types of Enzyme Assays
- Kinetic Assays (Continuous Monitoring)
- Fixed-Time Assays (Endpoint Assays)
- Enzyme Activity vs. Enzyme Concentration

Kinetic Assays (Continuous Monitoring)

Principle: In kinetic assays, the rate of the enzyme-catalyzed reaction is measured continuously over time. The change in absorbance (or other measurable parameter) is monitored as the reaction proceeds
Process
1. Initiation: The reaction is initiated by adding the sample containing the enzyme to a solution containing the substrate and any necessary cofactors
2. Monitoring: The change in absorbance (or other parameter) is monitored continuously over a fixed period (e.g., 1-5 minutes) using a spectrophotometer or other appropriate instrument
3. Calculation: The rate of the reaction (ΔA/min) is calculated from the slope of the absorbance curve. The enzyme activity is proportional to the reaction rate
Reactions
- Enzyme + Substrate → Product(s)
- The rate of product formation (or substrate consumption) is measured directly
Advantages
- More accurate than fixed-time assays, as they account for non-linearity and lag phases
- Can detect interferences and substrate depletion
Disadvantages
- More complex instrumentation and data analysis
- May be more susceptible to interferences
Examples
- Lactate Dehydrogenase (LDH): Measures the rate of NADH formation or consumption
- Creatine Kinase (CK): Measures the rate of NADPH formation
- Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST): Measure the rate of NADH formation or consumption
- Gamma-Glutamyl Transferase (GGT): Measures the rate of p-nitroaniline formation

Fixed-Time Assays (Endpoint Assays)

Principle: In fixed-time assays, the reaction is allowed to proceed for a fixed period, and the amount of product formed (or substrate consumed) is measured at a single endpoint
Process
1. Incubation: The sample containing the enzyme is incubated with the substrate and any necessary cofactors for a fixed period (e.g., 5 minutes, 10 minutes, 30 minutes)
2. Stopping the Reaction: After the fixed incubation period, the reaction is stopped by adding a reagent that denatures the enzyme or inactivates it
3. Measurement: The amount of product formed (or substrate consumed) is measured at the endpoint using a spectrophotometer or other appropriate instrument
4. Calculation: The enzyme activity is proportional to the amount of product formed (or substrate consumed) during the fixed incubation period
Reactions
- Enzyme + Substrate → Product(s)
- The amount of product formed is measured at a single time point
Advantages
- Simpler and less expensive than kinetic assays
- Easier to automate
Disadvantages
- Less accurate than kinetic assays, as they do not account for non-linearity or lag phases
- More susceptible to interferences
- Can underestimate enzyme activity if the reaction is not linear over the entire incubation period
Examples
- Amylase: Measures the amount of reducing sugars formed from starch hydrolysis
- Lipase: Measures the amount of fatty acids released from triglyceride hydrolysis
- Alkaline Phosphatase (ALP): Measures the amount of p-nitrophenol formed from p-nitrophenyl phosphate

Enzyme Activity vs. Enzyme Concentration

Enzyme Activity Assays: Measure the catalytic activity of the enzyme (i.e., the rate at which it catalyzes a reaction). These assays are used to assess enzyme function and to diagnose conditions involving altered enzyme activity
Enzyme Concentration Assays: Measure the amount of enzyme protein present in the sample, regardless of its activity. These assays are used to assess enzyme levels and to diagnose conditions involving altered enzyme expression or synthesis
Methods for Measuring Enzyme Concentration
- Immunoassays: Use antibodies specific to the enzyme to capture and quantify the enzyme protein. Examples include ELISA, Western blot, and immunoturbidimetry
- Mass Spectrometry: Used to identify and quantify enzyme proteins based on their mass-to-charge ratio

Specific Enzyme Assays

Lactate Dehydrogenase (LDH)
- Principle: Measures the rate of NADH formation or consumption as lactate is converted to pyruvate or vice versa
- Method: Kinetic assay
- Reactions:
  - Lactate + NAD+ ↔︎ Pyruvate + NADH + H+
- Detection: Increase in absorbance at 340 nm due to NADH formation
Creatine Kinase (CK)
- Principle: Measures the rate of ATP formation from creatine phosphate and ADP, or the reverse reaction
- Method: Kinetic assay
- Reactions:
  - Creatine Phosphate + ADP ↔︎ Creatine + ATP
  - ATP + Glucose –(Hexokinase)–> Glucose-6-Phosphate + ADP
  - Glucose-6-Phosphate + NAD+ –(G6PD)–> 6-Phosphogluconate + NADH + H+
- Detection: Increase in absorbance at 340 nm due to NADH formation
Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT)
- Principle: Measure the rate of formation of oxaloacetate and glutamate (AST) or pyruvate and glutamate (ALT)
- Method: Kinetic assay
- Reactions:
  - AST: Aspartate + α-Ketoglutarate ↔︎ Oxaloacetate + Glutamate
  - ALT: Alanine + α-Ketoglutarate ↔︎ Pyruvate + Glutamate
  - Oxaloacetate + NADH + H+ –(Malate Dehydrogenase)–> Malate + NAD+
  - Pyruvate + NADH + H+ –(Lactate Dehydrogenase)–> Lactate + NAD+
- Detection: Decrease in absorbance at 340 nm due to NADH consumption
Gamma-Glutamyl Transferase (GGT)
- Principle: Measures the rate of p-nitroaniline formation from γ-glutamyl-p-nitroanilide
- Method: Kinetic assay
- Reactions:
  - γ-Glutamyl-p-nitroanilide + Amino Acid ↔︎ γ-Glutamyl-Amino Acid + p-Nitroaniline
- Detection: Increase in absorbance at 405 nm due to p-nitroaniline formation
Amylase
- Principle: Measures the hydrolysis of starch to reducing sugars (e.g., maltose)
- Method: Fixed-time assay
- Reactions:
  - Starch + H2O –(Amylase)–> Reducing Sugars
  - Reducing Sugars + Reagent → Colored Product
- Detection: The intensity of the colored product is directly proportional to the amylase activity
Lipase
- Principle: Measures the hydrolysis of triglycerides to glycerol and fatty acids
- Method: Fixed-time assay
- Reactions:
  - Triglycerides + H2O –(Lipase)–> Glycerol + Fatty Acids
  - Glycerol + ATP –(Glycerol Kinase)–> Glycerol-3-Phosphate + ADP
  - Glycerol-3-Phosphate + O2 –(Glycerol-3-Phosphate Oxidase)–> Dihydroxyacetone Phosphate + H2O2
  - H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + H2O
- Detection: The intensity of the colored product is directly proportional to the lipase activity
Alkaline Phosphatase (ALP)
- Principle: Measures the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol
- Method: Kinetic assay
- Reactions:
  - p-Nitrophenyl Phosphate + H2O –(Alkaline Phosphatase)–> p-Nitrophenol + Phosphate
- Detection: Increase in absorbance at 405 nm due to p-nitrophenol formation
Angiotensin-Converting Enzyme (ACE)
- Principle: Measures the conversion of angiotensin I to angiotensin II or the hydrolysis of a synthetic substrate
- Method: Kinetic assay
- Reactions:
  - Angiotensin I –(ACE)–> Angiotensin II
  - Or
  - Synthetic Substrate –(ACE)–> Products
- Detection: The products are measured spectrophotometrically or fluorometrically

Key Terms

Kinetic Assay: An assay in which the rate of the reaction is measured continuously
Fixed-Time Assay: An assay in which the amount of product formed is measured at a single time point
Enzyme Activity: The rate at which an enzyme catalyzes a reaction
Substrate: The molecule acted upon by an enzyme
Product: The molecule produced by an enzyme-catalyzed reaction
Cofactor: A non-protein molecule required for enzyme activity
Spectrophotometry: A method to measure the absorbance of light by a solution
Chromogen: A substance that produces a colored product
ELISA (Enzyme-Linked Immunosorbent Assay): An immunoassay that uses enzyme-labeled antibodies
Chemiluminescence Immunoassay (CLIA): An immunoassay that uses chemiluminescent labels
HPLC (High-Performance Liquid Chromatography): A method to separate and quantify compounds

Procedures

Special Precautions