Enzymes and Methods

Alkaline Phosphatase (ALP)

Heat Fractionation:

ALP has varying heat stability, which can help differentiate its sources:

  • Most Stable: Regan (Fetal ALP)
  • Placenta
  • Intestine
  • Liver
  • Least Stable: Bone

ALP Methods:

  • Low Temperature: Increases ALP activity

Acid Phosphatase (ACP)

  • Room Temperature (1-2 hours): Decreases ACP activity

y-Glutamyltransferase (GTT)

  • Methods are used to measure GGT activity.

5'-Nucleotidase (NTP)

  • Marker of hepatobiliary disease and infiltrative lesions of the liver.
  • Methods are used to measure NTP activity.

Lipase (LPS)

  • Methods are used to measure Lipase activity.

Aldolase

  • Catalyzes the splitting of fructose 1,6-diphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, a reaction in the glycolytic breakdown of glucose to lactic acid.
  • Widely distributed in body cells, especially skeletal muscles, heart muscles, liver, and red cells.

Isoenzymes:

  • Aldolase A: Skeletal muscles
  • Aldolase B: WBC, liver, kidney
  • Aldolase C: Brain tissue

Laboratory Method:

  • Based on the rate at which trioses are formed. One method involves measuring the colored DNPH.

Clinical Significance:

Increased in:

  • Progressive muscular dystrophy
  • Inflammatory muscle disease
  • Myocardial infarction
  • Liver disease or necrosis
  • Pulmonary infarction
  • Some cases of malignancy and megaloblastic anemia

Decreased:

  • Not clinically significant

Inhibitors of ALP and ACP Isoenzymes

  • Phenylalanine: Inhibits Regan ALP, Inhibits Placental ALP Inhibits Intestinal ALP
  • L-leucine: Inhibits Nagao ALP
  • Levamisole: Inhibits Liver ALP, Inhibits Bone ALP
  • 3M Urea: Inhibits Bone ALP
  • L-tartrate ions: Inhibits Prostatic ACP
  • Cupric and Formaldehyde ions: Inhibits RBC ACP

y-Glutamyltransferase (GGT) Methods

Szasz Method:
  • Photometric enzyme reaction.
  • Substrate: L-gamma-p-nitroanilide or L-gamma-3-carboxy-nitroanilide
  • Indicator: Production of p-nitroaniline (yellow) or 5-amino-2-nitrobenzoate is monitored at 405405 nm or 420420 nm.
  • Sample collection: Serum, avoid hemolysis, avoid anticoagulant
Rosalki and Tarrow Method:
  • Substrate: Gamma-glutamyl-p-nitroanilide
Orlowski Method:
  • Substrate: Gamma-glutamyl-p-nitroanilide

Lipase Methods:

Dixon and Purdon Method
Campbell, Biefield, and Goldberg Method
Turbidimetric Enzyme Reaction:
  • Substrate: Olive oil or triolein
  • Indicator reaction: Decrease in turbidity
Titration: Cherry-Crandall Method (Reference method, Classic method)
  • Substrate: Olive oil
  • Indicator: Sodium hydroxide (NaOH) and Phenolphthalein
  • Measure liberated fatty acid (hydrolysis) by titration after 24 hr incubation
  • Observation: Pink color after 24 h incubation
Colorimetric method:
  • Based on coupled reactions with enzymes such as peroxidase or glycerol kinase

Leucine Aminopeptidase (LAP)

  • Catalyzes the hydrolysis of N-terminal residues from certain peptides and amides containing free amino groups.
  • No isoenzyme
  • Tissue sources: Urine, serum, and bile
  • Method:
  • Clinical significance:

Increased in:

  • Hepatobiliary disease
  • Obstructive jaundice
  • Cirrhosis
  • Metastatic carcinoma of liver and pancreatitis

Decreased:

  • Not clinically significant

Ornithine Carbamoyl Transferase (OCT)

  • Catalyzes the reversible conversion of ornithine to citrulline (synthesis of urea).
  • No isoenzyme
  • Tissue source: Liver
  • Method:
  • Clinical significance:

Increased in:

  • Acute viral hepatitis
  • Obstructive jaundice
  • Cirrhosis
  • Metastatic carcinoma
  • Heart failure
  • Cholecystitis

Decreased:

  • Not clinically significant

Serum Cholinesterase (CHE)

  • Monitor effects of relaxants (succinylcholine) after surgery.

  • Marker for organophosphate poisoning (Low CHS)

  • Methods:

  • Electrometric method (Michel Method):

    • pH change

  • Manometric Method:

    • Measures the liberation of CO2CO_2 from the formation of acetic acid as ACETYLCHOLINE is hydrolyzed.

  • Photometric Enzyme reaction (Ellman technique):

    • MOST WIDELY USED
    • Substrate: butyrylthiocholine
    • Principle: Colorimetric, thiocholine derivatives
    • Indicator reaction: Hydrolyzed substrate reacts with colorless 5,5’ dithiobis-2- nitrobenzoic acid (DTNB) to colorless 5- mono thio-nitrobenzoic acid (5-MBNA) monitored at 410410 nm.
    • Sensitive, rapid, and recommended method
    • Sample: serum or heparinized plasma

Ceruloplasmin or Copper Oxidase

  • A blue-colored alpha-2-globulin.
  • Used to diagnose myocardial infarction and chronic infection.
  • Substrate: para-phenylenediamine
  • Goldberg & Rutenberg (Fluorometric & Colorimetric method)