32 - Clinical Assays of Enzymes Flashcards
Immunoassays
Quick & Specific
But only one protein @ a time
no Parallel assays
Detected by:
Color Change / Fluorescence / Radiolabel
Electrophoresis & Chromatography
CHEAP but slower vs immunoasays
Parallel Assay
(more than 1 at a time)
Detection:
Stains / IV absorbance / Fluorescence
Electrophoretic Separations
and it is dependent on what?
SIZE
Larger Size of Particle –> larger drag force –> LESS IT MOVES
CHARGE = Z
Greater the E-Force on the particle –> MORE IT MOVES
Detecting Proteins with ELECTROPHORESIS
what is used?
To REDUCE diffusion & convection –> to IMPROVE resolution
proteins are often electrophoresed in a
GEL MEDIUM
Polyacrylamide / Agrose / Cellulose acetate
this is then treated with a dye or
STAIN
Bromcresol Green / Coomassie Blue
Intensity of the stain : amount of protein present
- *Capillary Electrophoresis**
- *CE**
AUTOMATED & NO GEL MEDIUM
uses a cool capillary tube that supresses the convection
Detected by:
UV Absorbance / MS
To detect:
DRUGS / Proteins
we can use micelles for un-charge analytes
Blood level of enzyme/protein is UNUSUALLY HIGH,
indicates what?
Diagnostic Enzymology
MAY indicate Pathology
different tissues have:
different levels of key ezymes / proteins
and might even have different forms (isozymes / isoforms)
If enzyme / protein levels are well BELOW the normal levels….
Diagnostic Enzymology
There MAY be some damage to the
SECRETING ORGAN
Assays help us do what for
Diagnostic Enzymology?
IDENTIFY the affected tissues
find the EXTENT of damage
and possibly the CAUSE of the damage
Examples of Enzymes and Proteins Assayed for
LIVER DAMAGE
Alkaline Phosphatase
AST / ALT
LDH
5’ nucleotidase / GGT / albumin / complement factors
Examples of Enzymes and Proteins Assayed for
HEART MUSCLE DAMAGE
MYOGLOBIN
TROPONIN I / T
isoforms of creatine kinase + LDH
AST / ALT
MULTPLE/Continous Enzyme Assay
How do we determine the AMOUNT OF ENZYME PRESENT?
Initially, Product increases LINEARLY w/ time
we are Following PRODUCT RELEASE at a rate of time
Δ[P] is proportional to the Amount of Enzyme
so we can COMPARE:
Δ[P]to the standard calibration curve
in order to get the amount of enzyme present
End-Point Assay
one of 2 main ACTIVITY ASSAY types
Start the rxn and incubate for a FIXED TIME @ constant T
then STOP reaction & measure amount of PRODUCT
simple but PRONE TO ERROR
can be automated and done quickly
also must be done in LINEAR REGION of the kinetic curve
Continuous / Multiple Point Assay
one of 2 main ACTIVITY ASSAY types
Start RX & follow it CONTINUOUSLY
with MANY time points
More accurate but MORE Complicated
can be automated and done quickly
also must be done in LINEAR REGION of the kinetic curve
Steps / Precautions AFTER drawing blood sample
before doing the Assay
Centrifugation
seperates Serum from RBC / leukocytes
serum may then be directly assayed or STORED
proteins denature / degrade upon storage
we can try to fight that by reducing temperature to help maintin stability & activity
BUT NOT ALWAYS
Enzyme Stability
<10% change in activity
ALT
can NOT be FROZEN
What does ONE Conventional UNIT ( U ) of enzyme activity
do?
CONVERT
1 MICROmole of substrate
to product in ONE MINUTE
- *Alkaline Phosphatase**
- *ALP** or AP
Function & Catalytic Activity
HIGH ALP:
from Rapid Bone Growth @ puberty
from Placental Release @ 3rd trimester of pregnancy
group of enzymes that
Hydrolyze monoPhosphate esters @ ALKALINE pH
Highest activity in
LIVER / BONE / intestine / kidney / placenta
but is found in most tissues
GGT
Gamma-Glutamyl Transferase
Function & Catalytic Activity
Induced by DRUGS & ALCOHOL
HIGH Serum GGT = Liver Disease
NORMAL in bone disease, ALP might be elevated
- *Microsomal** enzyme that is major in:
- *Glutathione Metabolism**
- *Resorption** of AA’s in kidney
Activity is HIGHEST in KIDNEY
5’-Ntase
5’-Nucleotidase
Function & Catalytic Activity
Serum levels Increased in LIVER DISEASE
but NOT in bone disease
Hydrolyzes Nucleoside 5’-phosphate Esters
microsomal enzyme
Aminotransferases
ALT (Ala) / AST (Asp)
Function & Catalytic Activity
Converts:
AA’s –> corresponding a-Keto acids
ALT = highest in Liver
AST = roughly EQUAL levels in Heart / SkelMuscle / Liver
may be elevated in:
liver disease / MI / Renal infarction
LDH
Lactate Dehydrogenase
Function & Catalytic Activity
Interconverts:
Lactate <–> Pyruvate
using NAD/NADH
cytoplasmic enzyme
HIGHEST levels found in kidney & heart
also found in other tissues
Diagnosing a AMI (Acute Myocardial Infarction)
Presence of at least 2 of the following:
H/O Chest Pain
Evolutionary Changes in ECG
EKG / ECG = electrocardiogram
Elevation in serum of Cardiac Enzymes / Proteins
LDH / Creatine Kinase
Myoglobin / Troponin I & T
Cardiac Markers
ENZYMES
and how do they differ?
LDH & Creatine Kinase isoforms
differ in:
location in cell
how quickly they are released after cell damage
how quickly they are cleared from serum
Cardiac Markers
PROTEINS
Myoglobin** & **Troponins I & T
differ in:
location in cell
how quickly they are released after cell damage
how quickly they are cleared from serum
AMI Markers
1st to Peak = MYOGLOBIN
returns in 1 day
2nd = CK
- *Troponin I** = 1st peak
- *Troponin T** = 2nd peak
Last to Peak = LD
LDH Isoforms
- *Tetrameric enzyme** w/ 2 main subunit types:
- *M & H**
with 5 principle isoforms that are MORE Tissue-specific:
Mixed Isoforms = LD2/3/4
found in a WIDE variety of tissues
Isoforms can be seperated with
ELECTROPHORESIS & QUANTIFIED
LD-1 is found in highest concentrations where?
Lactate Dehydrogenase Isoform
H4
1 of 5 LDH isoforms, highest in
HEART muscle / KIDNEY / RBC
Think LD1 = #1 is heart muscle
LD-5 is found highest concentrations where?
Lactate Dehydrogenase Isoform
M4
one of 5 LDH isoforms found highest in:
LIVER & SKELETAL MUSCLE
What can cause a RISE in TOTAL Serum LDH?
STROKE / HEART ATTACK
LD-1 (H4)
blunt-force TRAUMA
LD-1 RBC
LIVER DAMAGE
LD-5 (M4)
heavy EXERCISE
LD-5 skeletal muscle
Which indicates an ACUTE MI?
LDH Isoform Levels
B & C
LD1 = Heart / Kidney / RBC
HIGH LD1 indicates ACUTE MI
Creatine Kinase
CK
Function & Catalytic Activity
Dimeric enzyme that catalyzes:
REVERSIBLE Phosphorylation of CREATINE
using ATP
Creatine Phosphate = resevoir for “High Potential” Phosphate
for re-phosporylation of ADP during EXERTION
Greatest activity in:
MUSCLE / Brain / Heart
CK Isoenzymes
2 Subunit polypetides:
- *B = Brain (comes FIRST)**
- *M = Muscle**
Three main isoenzymes:
CK-1 = BB
CK-2 = MB
CK-3 = MM
CK-4 = mitochondrial form
Isoforms can be seperated by ELECTROPHORESIS
Through electrophoresis if there is a SHIFT
in CK isoform concentrationfrom
CK-3 -> CK-2
WHAT DOES THAT INDICATE?
MYOCARDIAL INFARCTION
CK-3 = MM (Muscle concentration)
leaks down to:
CK-2 = MB (Mix of Muscle + Brain)
shows that there is a LEAK from MUSCLE -> BRAIN
CK as a DISEASE MARKER
Duschenne’s Muscular Dystrophy
rise in CK-3 & 2 = skel muscle
HEART = MI / other heart conditions
leak from CK-3 –> CK-2 MB
CNS in cerebral ischemia / cerebrovascular disease
CK-3 RISES ALONE, comes likely from VASCULAR cells
TUMORS
neuronal damage –> raise in CK 1
