CC2 - Enzymes

• Enzymes are biologic proteins that catalyze biochemical reactions, affecting the reaction of organic matter
• During the process of catalyzing reactions, enzymes are not consumed or altered, remaining unchanged before and after the reaction
• Enzymes are found in all body tissues and are increased in serum in conditions like cell injury, degradation, and increased membrane permeability
• Functions of enzymes include promoting hydration of CO2 during respiration, nerve induction for faster nerve impulse transmission, muscle contraction for locomotion, nutrient degradation for faster absorption in the small intestine, growth & reproduction, and energy storage and use
• Liver enzymes like Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) are of clinical significance, with AST involved in the synthesis and degradation of amino acids
• The De Ritis Ratio differentiates the cause of hepatic disorders based on AST and ALT measurements, with a ratio > 1 indicating non-viral origin and < 1 indicating viral origin
• Gamma-Glutamyltransferase (GGT) is a transferase enzyme that catalyzes the transfer of the γ-glutamyl residue, commonly found in the canaliculi of hepatitic cells
• In the study of enzymes, the wavelength requirement can be used as a clue to the possible appearance of the product in a measurement:

  • If between 400-700nm (visible light region), expect a colored product
  • If <400 (UV region) & >700 (IR region), non-visible region = colorless
• Alkaline Phosphatase (ALP) is also known as Alkaline Orthophosphoric Monoester Phosphohydrolase:

  • It is an example of a hydrolase that catalyzes the hydrolysis of various phosphomonoesters at an alkaline pH
  • At high pH, ALP can cleave the monoester bond present in the substrates
  • It liberates inorganic phosphate from an organic phosphate ester with the production of alcohol
  • ALP activity enhancer: requires Mg2+ activator
• ALP activator:

  • An example of a co-factor that enhances enzyme activity by altering the spatial configuration of the active site of the enzyme or enhancing substrate finding
  • Can be metallic (Magnesium, Iron, Zinc, Calcium) or non-metallic (Chloride, Bromide)
  • ALP is not liver-specific; it can be found in other organs
• ALP diagnostic significance:

  • Used for the evaluation of hepatobiliary (obstructive types) and bone disorders like an increase in Paget’s disease (Osteitis deformans)
  • In healthy human serum, ALP is predominantly derived from the liver and bone
• Assay for ALP enzyme activity:

  • Bowers and McComb method is based on the molar absorptivity of p-Nitrophenol
  • Absorbance is measured colorimetrically at 405 nm (visible light region) with a pH of 10.2
• ACP diagnostic significance:

  • Used for the evaluation of metastatic carcinoma of the prostate
  • ACP is not specific for the prostate; PSA is a more specific tumor marker
• ACP assay for enzyme activity:

  • Reference range for Prostatic ACP: 0 - 3.5 ng/ml
  • Adult reference range: 30 - 90 U/L
  • ACP is normally present in seminal fluid
• Differentiation of ALP isoenzymes:

  • Liver ALP is the fastest and increases in liver disease
  • Bone ALP is the most heat-labile fraction and increases in bone diseases, healing of bone fractures, and physiologic bone growth
  • Placental ALP is the most heat-stable and increases during pregnancy
• Creatine Kinase (CK) is also known as Creatine Phosphokinase (CPK):

  • It is a transferase enzyme that catalyzes the transfer of a phosphate group between substrates
  • CK is involved in the storage of high-energy creatine phosphate in muscle cells
• CK sources include skeletal muscle with high activities, heart, and brain:

  • CK is not heart muscle-specific
• Creatine originates in the liver from the amino acids arginine, glycine, and methionine
• From the liver, creatine is transferred to muscle, where it is converted into creatine phosphate through the action of the enzyme CK
• Creatine phosphate serves as a high-energy depot in the muscle, used during muscle metabolism for intense physical activities that require more energy
• Creatine phosphate utilization forms the waste product creatinine, excreted in the kidneys at a constant rate
• Methods for Determination of Creatine:

  • Forward Reaction: Tanzer-Gilvarg
  • Determines ↓ in absorbance at 340 nm
  • Optimum pH is 9.0
  • Indicator: Lactate dehydrogenase
  • Product: Oxidized NAD
  • Reverse Reaction: Oliver-Rosalki
  • Determines ↑ in absorbance at 340 nm
  • Optimum pH: 6.8
  • Product: Reduced NADP
• Source of Error in Creatine Kinase (CK) Testing:

  1. Hemolysis causes false ↑ CK activity due to Adenylate kinase (AK)

  • Abundant enzyme in red cells
  • Released into serum/plasma when rbcs are lysed, ↑ CK activity

  2. CK is inactivated by light (photosensitive)

  • Serum/plasma must be protected immediately after processing
  • Delayed testing: protect with carbon paper or store in an area without light

  3. Physical activity and IM injections cause ↑ CK

  • Normal: predominant CK comes from muscle
  • Higher in males with physical activity & muscle mass
  • Bedridden patients show decreased activity = decrease in CK
• Reference Range for CK:

  • Male: 15 - 160 U/L
  • Female: 15 - 130 U/L
  • CK-MB: <6% of total CK
• CK Isoenzymes:

  • CK-3 / CK-MM: muscle type, composed of 2 M monomers, major isoenzyme in healthy individuals
  • CK-2 / CK-MB: muscle & brain hybrid type, cardiac muscle specific, 1st to elevate in AMI
  • CK-1 / CK-BB: muscle type, least anodal, major isoenzyme in striated muscle in normal serum
• Pancreatic enzymes are secreted specifically by acinar cells of the pancreas and are useful during the digestion process
• AMYLASE:

  • Example of hydrolase that catalyzes the breakdown of starch and glycogen via α, 1-6 branching linkages
  • Smallest enzyme in terms of molecular weight, normally filtered in glomerulus and present in urine
  • Increased in acute pancreatitis, with onset at 2-12 hrs, peak at 24 hrs, and staying elevated for 3-5 days
  • Major tissue source: pancreas; minor tissue sources: adipose tissues, small intestines, salivary gland, fallopian tubes, skeletal muscle
• Assay for Amylase Activity:

  • Amyloclastic: measures the disappearance of starch substrate, with activity inversely proportional to absorbance
  • Saccharogenic: measures the appearance of the product, directly proportional to amylase activity with reducing sugar formed
  • Chromogenic: measures the increasing color from the production of product, directly proportional to amylase with starch dye fragment formed
  • Continuous monitoring: coupling of several enzyme systems to monitor amylase activity
• LIPASE:

  • Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids, specific to pancreatitis and not increased in other disorders
  • Only major source: Acinar cells of pancreas
  • Diagnostic significance: earliest and specific marker for acute pancreatitis, increased after onset in 6 hrs, peak after 24 hrs, and stays elevated for 7 days
• MISCELLANEOUS ENZYMES:

  • Nucleotidase (5'N): a phosphoric monoester hydrolase predominantly secreted in the liver, marker for hepatobiliary disease and infiltrative lesions of the liver
  • Cholinesterase / Pseudocholinesterase: index for parenchymal function, used to monitor the effect of muscle relaxants after surgery, marker for insecticide/pesticide poisoning
  • Angiotensin Converting Enzyme (ACE): primary enzyme in RAAS, converts angiotensin I to II within the lungs, possible indicator of neuronal dysfunction
  • Ceruloplasmin: copper-carrying protein acting as an enzyme, marker and increased in Wilson’s disease
  • Ornithine Carbamoyl Transferase: for hepatobiliary disease, reference values 9 - 20 mU/mL
  • G6PD: function to maintain NADPH in the reduced form in erythrocytes, decreased levels lead to Drug-Induced hemolytic anemia, increased levels seen in acute MI and Megaloblastic anemia