360 - Serum Enzymes Flashcards
the amount of energy needed to raise all the molecules in 1 mol of a compound at a certain temperature to the transitional state at the peak of the energy barrier
activation energy
- this corresponds to the formation of an activated enzyme-substrate complex
group of enzymes that catalyze the same reaction but are encoded by different genes
isoenzymes
- each has different molecular structure and varying physical, immunological, and biochemical properties
non-protein molecules needed for enzyme activity.
cofactor
ex: activators, coenzymes
inorganic cofactor, that when bound to an enzyme increases the enzyme’s activity
activator
ex: Cl-, Mg2+
organic, low molecular weight, substances which combines with an inactive protein
coenzyme
what is a holoenzyme?
a coenzyme and apoenzyme forms holoenzyme or a complete enzyme
the active form of an apoenzyme formed by the combination of the apoenzyme with its coenzyme (prosthetic group), e.g. alanine transferase and pyridoxal phosphate
T or F. Coenzymes are more complex than activators, e.g. NAD+ and NADP+, vitamins
T!
an inactive form of an enzyme that requires a coenzyme to be converted into an active holoenzyme
apoenzyme
what is a prosthetic group?
a coenzyme bound to an apoenzyme, e.g. pyridoxal phosphate bound to an apoenzyme
denaturation
- change in the structure of a protein accompanied by a loss of activity
- can be caused by extreme pH, elevated temperature, changes in ionic strength and chemical modifiers
- can be reversible or irreversible.
how do enzymes work?
proteins that function as catalysts by lowering the activation energy
they are not consumed or destroyed in the process
Most physiological enzymes perform optimally in the pH range
7.0 to 8.0
Shifts in pH can change the ionization of enzymes active site or the substrate and can cause conformational changes in the structure of the enzyme
Effect of extreme pH shifts
it can irreversibly denature an enzyme
buffers control the pH of enzyme rxns
Most physiological enzymes have optimal activity at this temperature
37C
- As temperature increases interactions between enzymes and substrates become more common
- 10°C increase in temperature doubles the reaction rate
- temperature rises too high = enzyme is inactivated and irreversibly denatured
The rate of the enzymatic reaction is dependent on the concentration of enzyme
zero order kinetics
Describe zero order kinetics
- dependent on enzyme concentration
- occurs at high substrate concentration
- complete saturation of enzyme by excess substrate
- rxn reaches Vmax
rate reaction is directly proportional to the concentration of the substrate
first order kinetics
describe first order kinetics
- velocity of the enzymatic reaction will increase as more substrate is added until Vmax is reached
- as the amount of substrate decreases, the reaction rate decreases
- reaction rate reflects the amount of enzyme-substrate complex formed
describe competitive inhibition
inhibitor = structural analog of substrate and competes w the substrate for the active site of the enzyme
- rxn can reach same Vmax, just slower
- enzyme Km is constant bc more substrate required to overcome inhibition; there is appearance of increased Km
- reverisble
T or F. Competitive inhibition can be overcome by adding more substrate
T! This is so that the substrate concentration is greater than the inhibitor
when an inhibitor, often a metallic ion, binds to the enzyme at an allosteric site causing conformational changes in the enzyme structure
non-competitive inhibition
- not reversible
T or F. In non-competitive inhibition, the inhibitor does not alter the affinity of the enzyme for the substrate
T!
- The substrate concentration does not modify the interaction between the inhibitor and the enzyme, therefore increasing the substrate concentration will not overcome inhibition, and thus Km is unaltered
T or F. In non-competitive inhibition, Vmax is reached
F! binding of the inhibitor to the enzyme slows down the reaction rate, and Vmax cannot be reached
when the inhibitor binds to the enzyme-substrate complex preventing the creation of the product
uncompetitive inhibition
describe uncompetitive inhibition
- adding more substrate increases the amount of enzyme-substrate complex, which worsening the inhibition and free enzyme concentration is reduced = decreased Vmax and decreased in Km.
reversible.
The concentration of an enzyme in a specimen is _____ proportional to the measurable catalytic activity of the enzyme.
directly
how do we measure enzyme activity in the lab?
enzyme activity is measured by monitoring a decrease in substrate concentration or an increase in product concentration by spectrophotometry
describe the fixed point method
- started, incubated for a specified time at a set temperature
- reaction is stopped, and the change in absorbance is measured
- run over a few seconds to a few minutes
- assumed the reaction is constant and linear over time and follows zero-order kinetics
describe continuous monitoring kinetic methods
- started, incubated at a set temperature for a set time
- change in absorbance (or amount of product formed) is measured at multiple time points or continuously until the reaction is stopped
multiple readings verifies a constant linear reaction rate
Why is continuous monitoring preferred to fixed point methodologies?
continuous monitoring is preferred to fixed point methodologies because of the shorter reaction time and the ability to verify zero-order kinetics
tissue distribution of alkaline phosphatase (ALP)
- high in the intestine, liver, bone, and placenta.
- on the surface of red blood cells
clinical significance of ALP
- osteoblasts and hepatocytes
- active bone growth, children = higher levels of ALP than adults
- placental forms of ALP, enter the circulation and result in elevated plasma ALP concentrations during the last trimester of pregnancy
ALP levels in different bone diseases
Paget’s disease, 10 - 25X the normal upper limit
Osteomalacia, rickets, 2 - 4X the normal upper limit
Hyperparathyroidism (slight to mod increase)
In osteoporosis, levels are usually normal or slightly increased
how does the liver respond to any type of hepatobiliary blockage (ALP)?
produce more ALP 3-10X normal upper limit
- similar can be seen in hepatic cancer; 10-20X normal upper limit
- liver diseases such as hepatitis and cirrhosis that affect the parenchymal cells may demonstrate a slight rise in ALP
other causes of increased ALP
- individuals undergoing hemodialysis
- individuals with cancer (due to presence of variant carcinoplacental forms of ALP - Regan + Nagoa)
- these forms of ALP similar to placental form and can cause increased ALP concentrations
Activators for ALP
Zn2+, Mg2+
Inhibitors or ALP
phosphate and anticoagulants: oxalates, citrate, EDTA
limitations for ALP reaction
Serum or heparin plasma should be measured within 4 hours
ALP increases on storage at 4°C and room temperature
DO NOT USE HEMOLYZED SPECIMENS
Bilirubin and lipemia are not causes of significant interference
tissue distribution of lactate dehydrogenase (LD)
- found throughout the body
- highest concentrations of LD are in the heart, liver, skeletal muscles, red blood cells, platelets & lymph nodes
clinical significance of LD
LD is a non-specific indicator of disease
There are primarily three clinical uses for LD levels: anemia, liver disease and heart disease
LD and anemia
- an increase in serum LD is observed in hemolytic anemias and megaloblastic anemias
- LD is also useful for predictive monitoring in leukemia and lymphoma
LD and liver disease
LD is increased in viral hepatitis, cirrhosis, and liver cancer
It is not as specific as GGT or ALT for liver diseases
LD and heart disease
Lactate dehydrogenase is also increased in myocardial infarction but is not as specific as troponins
LD reaction coenzyme
NAD+
LD reaction limitations
serum sample is preferred; platelets in plasma can increase LD levels
Store serum samples at room temperature
Some LD isoenzymes are cold labile
DO NOT USE HEMOLYZED SPECIMENS
Bilirubin and lipemia are not causes of significant interference
ALT (alanine aminotransferase) tissue distribution
ALT is found primarily in the liver and kidneys
clinical significance of ALT
ALT is increased in hepatic diseases
In viral hepatitis and acute hepatic, necrosis ALT can increase 10 - 40X the normal upper limit
Acetaminophen toxicity ALT >85X the normal upper limit
Elevated concentrations of ALT may also be observed in non-alcoholic fatty liver disease, metabolic syndrome
ALT reaction coenzyme
pyridoxal 5’ phosphate (vitamin B6)
ALT rxn limitations
ALT is unstable and should be measured on the same day
ALT is stable at -70°C
Hemolysis may falsely elevate results due to the release of endogenous LD enzyme
Bilirubin and lipemia are not causes of significant interference
GGT (gamma-glutamyl transferase) tissue distribution
GGT is expressed in the kidney, the bile ducts of the liver, pancreas and intestine
GGT clinical significance
- an indicator of hepatobiliary disease, especially biliary obstruction
- elevated in alcoholic hepatitis, heavy alcohol drinkers and liver cancer
- can help in determining whether observed ALP elevations are due to hepatobiliary disease (cholestasis) or skeletal disease as GGT is normal in skeletal disorders and pregnancy
GGT rxn activator
Mg2+
GGT rxn inhibitor
citrate, oxalate, fluoride
GGT rxn limitations
Serum and plasma samples are stable at 4°C
A non-hemolyzed sample is preferred
Bilirubin and lipemia are not causes of significant interference
GGT levels are higher levels in newborns
The following drugs can cause a false increase up to 4X the normal upper limit: ethanol, warfarin, phenobarbital, and phenytoin
tissue distribution of creatine kinase (CK)
CK is expressed in skeletal muscle, heart, brain
clinical significance of CK
CK measurements are used in assessing skeletal muscle diseases and heart disease
CK and muscle
CK is increased in all forms of muscular dystrophy
CK is increased in viral and polymyositis, rhabdomyolysis, and muscle trauma
It is NOT increased in neurogenic diseases, e.g. multiple sclerosis, Parkinsonism
CK and heart
CK is increased in cardiac disorders, including myocardial infarct; however, cardiac troponins are more specific
CK rxn activator
Mg 2+
CK rxn coenzyme
ATP
CK inhibitors
All anticoagulants other than heparin
Mn2+, Ca2+, Zn2+, Cu2+ and excess Mg2+
CK rxn limitations
Serum and heparin plasma specimens are stable for up to 48h at 4°C
Gross hemolysis will interfere with the second hexokinase reaction due to the presence of enzymes and reaction intermediates
Bilirubin and lipemia do not significantly interfere
tissue distribution of amylase
Amylase is found in the salivary glands, pancreas, ovaries, fallopian tubes, and lungs
amylase clinical significance
Amylase activity is increased in salivary gland inflammation, e.g. mumps virus
Amylase activity is increased in acute pancreatitis and other intra-abdominal disorders, e.g. biliary tract disease, intestinal obstruction, appendicitis, and ectopic pregnancy
this enzyme is a more specific enzyme for acute pancreatitis than amylase
lipase
Urine and plasma levels should correlate as the kidney freely filters amylase
amylase activator
Ca2+
absolutely required fo activity
amylase rxn inhibitor
all anticoags except heparin
limitations of amylase rxn
Serum and urine amylase is stable at room temperature
In vitro, plasma triglycerides inhibit amylase activity
Morphine and opiates elevate amylase
Hemolysis, bilirubin, and lipemia do not cause significant interference
distribution of lipase AKA triacylglycerol lipase
Lipase is found in the pancreas, stomach, and small intestine
lipase clinical significance
- used to diagnose acute pancreatitis
> In acute pancreatitis, serum lipase rises 4-8 hours after onset of symptoms, peaks at 24h, and diminishes to normal within 7-14 days - Lipase >3X the normal upper limit is indicative of acute pancreatitis
- also increased in gastric or duodenal ulcers and intestinal obstruction
methodologies to measure lipase
colourimetric, turbidimetric, titrimetric rxns
cofactors of lipase
colipase and bile salts
limitations of lipase
Lipase is stable in serum at RT for one week
Hemolysis should be avoided as hemoglobin inhibits lipase activity
Bilirubin and lipemia interference is method dependent
