Analytical Enzymology Flashcards
In a diseased state, changes in enzymes can infer: (2)
- Possible location
- Pathological changes in cells/tissues (clinical biomarkers)
The rate of an enzyme catalysed rxn is directly proportional to what
The rate of an enzyme catalysed rxn is directly proportional to the amount of active enzyme present
Enzyme activity rxns monitored by what 2 methods
- decreasing substrate concentration (approaching zero, harder to detect)
- increasing product concentration (easier to detect)
When is ALP (alkaline phosphatase) increased
- Increased in liver disease/damage/bilary obstruction
- Increased in pregnancy, growing children
When is ALP decreased
Hypophosphatasia - genetic deficiency of ALP
ALP is what kind of enzyme & how do these function
It’s a metalloenzyme.
These need a metal ion to function effectively as a catalyst.
What cells is gamma-glutamyltransferase primarily in
Liver cells
When is GGT (gamma-glutamyltransferase) increased
- In liver disease/damage
- In pancreatitis
- In cancer patients - increased serum GGT suggests tumour may have metastasised to the liver
Can amylases pass through the kidney
Yes
When is amylase increased
In acute pancreatitis (increased activity in urine specimen)
What is the main source of lipases
Pancreas
When do lipase levels change
- Increaed in pancreatitis
- Increased/decreased levels if tumour in pancreas
What does Cholinesterase do
Hydrolyses acetylcholine released at the nerve endings
When is cholinesterase increased
Increased activity in serum indicates insecticide poisoning (inhibitors of the enzyme)
Principle of kinetic enzyme assay - alkaline phosphatase test
p-Nitrophenol-phosphate + H2O ——–> p-nitrophenol + phophate
(in the presence of ALP)
Steps in ALP kinetic enzyme assay
- Absorbance measured at 405nm
- Absorbance measured at 1 min intervals, up to 4 mins
- Change in absorbance/min is calculated (delta OD/min)
- Convert delta OD/min to enzyme activity units (U)
- Compare to reference value
What factors affect the rate of enzyme reactions
- Conc of substrate
- pH
- Temperature
- Cofactors
- Inhibitors
- Time
What are the 3 isoforms of creatine kinase
- BB (CK-1) - found in brain
- MB (CK-2) - found in muscle
- MM (CK-3) - found in muscle
When is serum CK elevated
In tissue damage related to skeletal & heart muscle, brain injury
What is the most NB application of creatine kinase
Total CK & CK-2 measurement in diagnosis of myocardial infarction
function of lactate dehydrogenase
Catalyses conversion of pyruvate to lactate
Clin significance of increased serum LDH
3 to 10 times increased in myocardial infarction, no change in angina/pericarditis
What does LDH in urine indicate (LDH too large to be in urine) (4)
- Chronic glomerulonephritis
- Systemic lupus erythematosus
- Diabetic nephrosclerosis
- Bladder & kidney malignancies
Clinical significance of ALP in terms of bones
Bone disease - can have increased osteoblastic activity
Analytical enzymology methodologies
- Electrophoresis
- Immunoassays
Clearance rate & meaurement
Enzymes usually excreted from the body via receptor-mediated endocytosis (liver,spleen, etc) - associated with specific clearance rates
Examples of pre analytical variables
Activity/enzyme content influenced by sample collection & processing - e.g: time, haemolysis
Enzymes as analytical reagents
- eNzymes make excellent analytical reagents due to specificity, selectivity & efficiency
- Used to measure several metabolites & substrates & in development of labelled immunological assays
Enzymes in measurement of metabolites
- High specificity - removes need for preliminary separation/purification
- Analysis carried out directly on complex mixtures
- Coupled rxns often used
Principle of glucose assay - hexokinase method
Hexokinase transfers a phophate group from ATP to glucose to form glucose-6-phosphate (G-6-P) and ADP
Glucose + ATP ———> G-6-P + ADP (in presence of hexokinase)
G-6-P + NADP+ ———-> 6-phosphogluconate + NADPH + H+ (in presence of G-6-PD)
What does the urea method-using urease measure
Kidney damage
Principle of the coupled rxn used in urea method
- Rxn 1: urea + 2H2O ——> 2NH4+ + HCO3- (in presence of urease)
- Rxn 2: 2NH4 + 2aketoglutarate + 2NADH —-> 2Glutamate + 2NAD+ + 2H20
