Chapter 67: Enzyme Diagnostics Flashcards
Enzyme leakage into the plasma may result from
- Cellular necrosis
- Membrane damage due to ischemia
- Increased cellular turnover (normal active growth or malignant disease)
- Induction of intracellular enzymes which increases the concentration gradient
Enzyme activity is measured as
- Substrate converted per unit time
- Optimal conditions of [S], Temp, pH, [cofactors]
Advantages of UV light absorption
- Reduced NAD or NADP to absorb UV light at 340 nm
If the reactions do not use NAD or NADP - coupled reactions
- Particular tissues involved cannot usually be pinpointed
- Except Alcohol DH (liver) & Acid Phosphatase (prostate)
Isoenzyme assay allows
- Differential diagnosis
- They can be separated and quantified
The absorption spectra of niacin and flavin coenzymes
- Follows rate of reduction of NAD (increase in absorption at 340nm)
- Follow rate of reduction of FAD (fall in absorbance at 450nm)
Alkaline phosphates (ALP)
- Membrane bound isoenzymes
- Activity varies with both age and sex
Assay of plasma ALP activity is useful for
- Assessing hepatobiliary and bone diseases
Hepatic disease (ALP)
- Obstructed bile flow (cholestasis, pancreatic cancer) = 10x increase in plasma ALP
- Viral hepatitis = 2x increase in plasma ALP
- ALP only increases in small amounts following hepatocellular damage
Bone disease (ALP)
- Paget’s disease of bone remodelling affecting the femur and hip of elderly men
- Pagest’s = plasma ALP increases 25x
- Ricketts = ALP increases 2-4x
Separation of liver and bone ALP isoenzymes by
- Electrophoresis at pH 8.6
Gamma glutamyl transferase
- Membrane bound enzyme
- High concentrations in liver and kidney
- Production is enhanced by drugs and alcohol
- Used to confirm hepatic origin for ALP elevation
Elevated levels of GGT
- (Often more than 10x) indicate cholestasis and cirrhosis
- More sensitive than AP and more liver specific
- Reyes syndrome, MI and acute pancreatitis as well
Some pathological causes of high GGT include
- Hepatitis (often 5x)
- Alcohol abuse (usually less than 5x)
Aspartate transaminase (AST)
- Glutamic oxaloacetic transaminase (GOT)
- Present in most tissues (primarily skeletal and cardiac muscle, liver and kidney)
- Mitochondrial and cytoplasmic isoenzymes
In alcoholic liver disease levels of AST
- AST > ALT
Assay of plasma AST (activity coupled to MDH) measures
- Myocardial infarction
- Acute liver damage
- Muscular dystrophy
Alanine transaminase (ALT)
- Glutamic pyruvic transaminase (GPT): cytoplasmic enzyme
- Generally lower concentrations than AST in tissues
- In the liver the activity of ALT and AST are similar
ALT levels increase > AST early hepatocellular disease
- AST increase is greater in alcohol-related liver disease
Alanine transaminase assay useful for
- Acute liver damage (like viral hepatitis)
- Monitoring effectiveness of new treatments of liver disease
- Cardiac failure and muscle disease
De Ritis Ratio (DRR)
- AST/ALT
- Numerical values may vary
- May reach 6.0 in alcohol-related liver disease
Increased AST/ALT ratio plasma levels are seen in
- Cirrhosis
- Liver tumors
- Obstructive jaundice
- Burns and shock
Creatine kinase catalyzes
- Reversible phosphorylation of creatine by ATP
- Key to phosphagen metabolism
Creatine kinase requires
- Mg++, but is inhibited by excess ADP
Creatine kinase
- Cytosol of striated muscle, brain, heart tissue
- Exists as a dimer of 2 subunits; M and B (mwt 40,000)
Plasma CK levels are proportional to
- Muscle mass
Plasma CK assay importance
- Skeletal muscle and heart disease
- Differentiation between skeletal muscle and heart disease necessitates isoenzyme assay
Creatine kinase isoenzymes must be distinguished by
- Electrophoresis
Elevated plasma creatine kinase levels are seen in
- Muscle disease
- Myocardial infarction (10x increase)
- Surgery, muscular trauma, heavy exercise, muscle dystrophy (5x increase)
Creatine kinase levels of well-trained athletes will be
- Lower than untrained individuals because they secrete smaller amounts
All types of muscular dystrophy demonstrate
- Increase in CK (mainly CK3, but maybe CK2)
- Diseases of CNS and hypothyroidism show
- 5-fold increase in creatine kinase (CK3)
Decrease in plasma CK levels is observed in
- Hyperthyroidism
Plasma Cardiac Troponin T is better than
- CK-MB in diagnosis and prediction of heart failure
Major sources of amylase
- Salivary glands
- Exocrine pancreas
Amylase
- Small protein that filters well
- Measurable in urine
Plasma amylase may be
- Normal in chronic pancreatic disease
- Elevated in renal disease
Acute pancreatitis
- Caused by gallstones/alcohol
- Elevates plasma amylase (10x) and lipase activity
Acute pancreatitis
- Initially managed medically
- Other acute abdomens usually require laparotomy
Lactate dehydrogenase (LDH)
- Cytoplasm of the majority of cell types
- Concentration is 500-fold that in the plasma
Five distinct isoenzymes of LDH exist (mwt 136kD)
- Tetramers of 4 distinct polypeptide chains
- Polypeptide chain may be 1 of 2 types, H or
Five different combinations constitute the LDH isoenzymes
- H4 isoenzyme = LD1
- M4 isoenzyme = LD5
- All 5 found in plasma of healthy individuals
All LDH isoenzymes catalyze
- Reversible oxidation of lactate
- NAD functions as the H acceptor in the reaction
Plasma levels of LDH isoenzymes
- Healthy: LD2 > LD1 > LD3 > LD4> LD5
- Changes to this pattern in disease
LDH 1 and 2 primarily found in
- Myocardium
- Erythrocytes
LDH 3 primarily found in
- Brain
- Kidney
LDH 4 and 5 primarily found in
- Liver
- Skeletal muscle
Classic plasma LDH changes caused by diseases
- Myocardial infarction
- Hepatic, hemolytic jaundice and other liver disorders
Myocardial infarction plasma LDH changes
- LD1 & LD2 are elevated
- 8 - 12 hours after pain begins
- Peak at 24 - 48 hours
- Levels remain elevated for 7-12 days and decline slowly
LD1 and LD2 may also be elevated in
- Anemia
Specimen hemolysis may generate
- LDH profile that mimics that seen in MI
- cTroponin I now often preferred assay for infarction
Plasma LDH in hepatic anoxia, hemolytic jaundice and other liver disorders
- Elevated up to 10x normal
- LD5 most elevated (combine results with AST/ALT assay)
- Patients with malignant disease (Muscular dystrophy) exhibit elevated serum LDH
The most useful enzyme assays are those that can easily be measured
- Spectrophotometrically
