clinical chemistry Flashcards
how is glucose measured?
not easy to measure glucose itself, so instead measure reduced product (NADPH)
glucose + ATP –>
glucose-6-phosphate + ADP
enzyme = hexokinase
glucose-6-P + NADP –>
glucono-lactone-6-P + NADPH
enzyme= G6P dehydrogenase
when measuring serum enzymes, describe importance of Km
want [substrate] > km
describe isozymes
closely related enzymes that catalyze the same reaction but have slightly different aa composition and come from different genes. usually have different charges (helps with electrophoresis) and different kinetics (vmax, Km), some are tissue specific
describe the forms of LDH
tetramer, 5 forms Muscle (M) and heart (H) - HHHH, HHHM, HHMM, HMMM, MMMM
elevation of which 2 forms of LDH indicate MI
elevation of LDH 1 (HHHH) and LDH2 (HHHM)
where, besides the heart, is LDH1 and LDH2 found?
in RBCs, consequence that these levels can be increased with hemolysis
which form is high in liver and skeletal mm?
MMMM (LDH5)
how is LDH visualized?
redox mechanism that results in conversion of NBT from oxidized (colorless) to reduced (blue) form
when does LDH increase after MI?
peaks at about 24 hours
3 problems with using LDH for MI detection
- other conditions may also elevate LDH levels
- electrophoresis is labor intensive and slow
- takes 12-24 hrs to appear
how is CPK measured?
couple with glucose reaction (common to both reactions is ATP), then measure NADPH
what can interfere with CPK measurement?
adenylate kinase- which catalyzes 2 ADP –> ATP and AMP and will thus elevate ATP
how can problem with adenylate kinase be avoided?
use a control that is run first without creatinine-P, this will yield the ATP that comes from the adenylate kinase run, then run the reaction with creatinine-P and take the difference
isozymes of CPK
dimeric- Muscle (M), brain (B)
MM, MB, BB
what increased CPK isozyme indicates MI
MB
what are other reasons, aside from MI, that will increase MB?
previously damaged mm and regenerating mm (like that of athletes in training)
describe the rise of CPK-MB vs. rise of LDH1/2 after MI
CPK-MB rises faster than LDH1/2
preferred enzyme for detecting MI
Troponin (T and I forms)
why are TnT and TnI good for detecting MI?
- low serum levels normally
- rise rapidly, can be measured early
- high specificity for cardiac tissue
describe RIA process (4)
- attach antibody to polymer
- add sample, remove excess, protein of interest binds Ab
- add second radio labeled ab to protein of interest
- measure radioactivity present
describe ELISA process (5)
- attach antibody to polymer
- add sample, remove excess, protein of interest binds Ab
- add second labeled Ab (with enzyme attached) to bind protein
- enzyme takes non-flourescent substrate and makes fluorescent product
- fluorescence is measured
