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137 Terms
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removes H or adds oxygen; redox rxn
examples of oxidoreductases
lactate dehydrogenase (LD)
- transfers a specific group from one substrate to another; other than H - transfers amino acids
examples of transferases
cleaves carbon bonds by the addition of water
examples of hydrolases
AST tissues of origin
skeletal, cardiac muscle/tissue, hepatocellular tissue**
what causes AST to leak into serum?
AST sample requirements
- serum preferred, heparinized plasma okay - no hemolysis (rbc has AST) - stable (3 days fridge)
AST reaction catalyzed
L-aspartate + alpha-oxoglutarate = oxaloacetate + glutamate *malate dehydrogenase catalyzes second rxn* oxaloacetate + NADH + H = malate + NAD+
AST method of analysis
measures decrease in absorbance from the oxidation of NADH to NAD+ at 340nm
clinical significance of AST
- liver enzyme: toxic/viral hepatitis (50-100x), cirrhosis (4x) (normal in obstruction) - cardiac: inc in AMI - skeletal: inc in MD duchenne; muscle inflammation
ALT tissues of origin
skeletal, cardiac, hepatocellular**
ALT sample requirements
- same as AST (serum or hep plasma) - icterus/turbid will dilute - no hemolysis - less stable (measure within 24 hrs) (decreases with time frozen)
ALT reaction catalyzed
alanine + alpha oxaloacetate = pyruvate + glutamate pyruvate + NADH + H = lactate + NAD+
ALT method of analysis
- kinetic coupling with spec analysis at 340 - measure the decrease in absorbance from the oxidation of NADH
which is more liver specific, AST or ALT?
ALT clinical significance
liver: similar to AST but higher in acute infections (longer half life than AST)
de ritis ratio
- to figure out source of liver disease - AST/ALT >2 = alcoholic link <1 = viral hep, acute inflamm disease, obstructive liver disease
ALP tissues of origin
liver* (biliary), bone*, placenta
to figure out if inc ALP is bone or liver?
measure with GGT or 5 nucleotidase (all three are found in biliary tract just outside of liver)
ALP sample requirements
- serum/hep plasma - no hemolysis - store airtight for less than 6 hours (dec with freeze/thawing) - inc activity at 37 C
ALP cofactors
divalent Mg and Zn (pH 10)
ALP reaction catalyzed
4-NPP = 4 nitrophenoxide (yellow) via ALP, Zn, Mg, pH 10.3
ALP method of analysis
kinetic spec of rate of prod of yellow color at 405 nm, 37 C
heat stability test
- test for ALP isoenzymes - 65 C = placental (regan) - heat labile at 56 C = bone source - stable at 56 C = liver (biliary) (for ten minutes) - stable at 56 C = intestinal (10-30 min) - stable at 65 C = placental (regan)
clinical significance of ALP
- liver: stone (obstruction) (3-10x) - skeletal: pagets, rickets, osteomalacia, hyperPTH - misc: bone growth (kids), pregnancy, enteritis, colititis
ACP tissues of origin
liver, breast, prostate**
ACP optimum activity
- pH 5 - divalent Mg cofactor
ACP uses
- historically: prostate marker, PSAs - in rape cases (seminal fluids) - not super clinically significant anymore
GGT tissues of origin
kidney, liver (hepatobiliary**), pancreas, prostate
GGT sample requirements
- serum preferred, hep plasma okay but can cause turbidity - stable, fridge for 3 days - hemolysis will not affect this
liver panel for hepatobiliary?
liver panel for intrahepatic?
GGT reaction catalyzed
glutamyl3carboxy4nitroanilide + glycylglycine = p-nitroaniline** + glutamyl glycylglycine
GGT method of analysis
spec method of kinetic p-nitroaniline at 410nm at 30C
GGT clinical significance
- liver: obstructive (stones), alcoholic ** cirrhosis, (normal in toxic/viral hepatitis) - misc: prostate, renal, hepatic cancer
AMS tissues of origin
pancreas, salivary glands
AMS sample requirments
- serum or hep plasma - stable for days
AMS cofactors
Ca2+ and Cl- pH 6.9-7
measures the disappearance of starch substrate
measures the appearance of the product
measures the increasing color from production of product coupled with chromogenic dye
enzymatic - AMS
coupling of several enzyme systems to monitor amylase activity (maltotetraose)
AMS method of analysis
- measures hydrolysis of maltotetraose at 340 nm - measures production of NADH
which enzyme is exclusively used to diagnosis pancreatitis?
AMS clinical significance
- acute panc - chronic panc (normal due to prolonged damage) - obstructive liver disease, acute alcoholism
- artifactual increase of AMS - caused by AMS binding to IgG/A (form large complex) - urine AMS will be decreased - clinically insignificant
LPS tissue of origin
LPS cofactors
intestine bile acids (act like detergents)
LPS sample requirements
- serum or hep plasma - stable for days
lipase function
makes glycerol and three fatty acids
LPS reaction catalyzed
- very long - final product is quinonemine dye (colored) - LPS does first step - ends in a glycerol and 3 fatty acids
LPS method of analysis
- rate of prod of colored dye is used to monitor reaction (spec) - colored dyes: methylresorufin or 1,2 diglyceride
clinical significance of LPS
- acute panc (stays higher longer than AMS) - chronic panc: normal levels
LD tissues of origin
skeletal, cardiac, liver, rbcs, kidney, lungs, tumor cells
LD function
lactate to pyruvate using NAD and Zn as activator
heart, kidneys, rbc
RES, wbc
lung, spleen, other tissues
kidney, placenta, pancreas
skeletal muscle, liver (parenchymal cells)
what is the order of LDs from highest to lowest conc?
2, 1, 3, 4, 5
isoenzymes of LD
made of four polypeptides forming a tetramer (H and M)
LD sample requirements
- serum preferred (no hep plasma for electrophoresis) - no hemolysis (LD in rbcs) - stable at RT (no fridge) (M poly is unstable) - LD4/5 is heat labile
LD reaction catalyzed
lactate + NAD+ = pyruvate + NADH reverse rxn more favorable but more interferences so forward is measured
LD method of analysis
spec measuring rate of increase in absorbance at 340 nm as NADH is made
clinical significance of LD
- cardiac: AMI or hemolyzed sample (LD1 flipped pattern) - skeletal: MD duchenne - liver: toxic/viral hep (LD5) or obstruction (N to sl inc) - PA, HA, megaloblastic anemias (LD1 flipped pattern) much higher than AMI
CK tissue of origin
wide cellular distribution
CK isoenzymes
3 MM - skeletal (99%) 2 MB - cardiac (2%) 1 BB - brain (0%) dimer of two isomers
CK sample requirements
- serum preferred (no hep plasma for electro) - no hemolysis (mild is okay) (from g6PD) - unstable, affected by light (keep it dark)
CK catalyzed reaction
G6P + NADP = 6-phosphogluconate + NADHP + H requires ATP and Mg2+
CK method of analysis
- kinetic reverse rxn coupled assay with pH 6.4 - spec assay of increased absorbance at 340nm of NADPH at 37C
CK clinical significance
- skeletal: duchennes, inflamm (viral or polymyositis) (normal in neurogenic muscle disorders) (only CK MM) - cardiac: AMI (total inc) - CNS: (ckBB) trauma or pathology - misc: tumors of brain, lung, GI; normal in neonates; hypothyroidism (inc ckMM) nothing super specific
cholinesterase (CHE)
- responsible for nerve transmission - hydrolase - to check for exposure to organophosphates, insecticides, sensitive to anesthesia
acetylcholinesterase (ACHE)
- "true" CHE - liver, heart, pancreas
psuedocholinesterase (PCHE)
- brain (white matter), serum, liver - measured to check for poisoning instead of tissue damage
CHE sample requirements
- serum preferred - no hemolysis (rbc has CHE) - stable for hours
CHE clinical significance
- liver: parenchymal cell damage (dec) - exposure to organophosphates (dec) - dibucaine can determine genetic variants (anesthesia, succinyl choline)
enzymes seen in obstructive (hepatobiliary)
- causes: stones, neoplasms - inc in ALP, GGT
enzymes seen in parenchymal (hepatocellular)
- causes: inflammation from virus, bacteria, toxin - cell death/necrosis (inc AST, ALT, LD4/5) - from loss of cell synthesis function (dec CHE)
enzymes seen in cirrhosis
- combined hepatocellular necrosis and hepatobiliary fibrosis - causes: biliary, wilsons, alcoholic - cell death/necrosis (inc in AST, ALT, ALP, GGT) - loss of cell synthesis function (dec ceruloplasmin in wilsons)
enzymes seen in bone disease
- pagets, ostetitis, rickets, osteomalacia - inc in ALP, ACP
enzymes in pancreatitis
- viral, bacterial, toxic exposure - acute has elevated, chronic has near normal - inc in AMS, LIP in serum - inc in AMS in urine
enzymes in muscular dystrophy
- duchennes or progressive disorder - inc ckMM, AST, LD4/5 - diminish with degeneration of muscle mass
enzymes in prostatic cancer
inc in ACP
what are the four major types of CV disease?
- coronary heart disease - cerebrovascular disease - peripheral arterial disease - aortic atherosclerotic disease
- damage to endo of artery - wbcs go to area - start plaque formation - LDL also enters cells causing cytokine release - as it grows, artery narrows (fibrous cap) - when piece breaks off = emboli
- no specific symptoms - detected during routine exam - increase risk of dying from stroke, MI, heart failure, kidney failure
how to define hypertension
- systolic of 140 - diastolic of 90 - taking antihypertensive medication
- resulting from coronary heart disease - myocardial necrosis due to prolonged ischemia - categorized by size of infarct - cannot repair damaged cells
criteria for MI diagnosis
- hx of chest pain - ecg changes in pattern - serum cardiac markers initially rise and fall - serial samples (baseline, 6-9, 12-14 hrs) of markers
- from coronary heart disease - structural or functional cardiac disorder that impairs the ability of the ventricle to fill or eject blood (edema) - kidneys retain xs fluid (no venous pressure) - 4 stages
what is an ideal cardiac marker?
- released rapidly from heart - specific and sensitive to heart (not in other tissues and rises early) - stay in circulation for days - assays designed to detect low concs
historic cardiac markers
total CK, LD1, AST, ALT (not specific)
current cardiac markers
ckMB and troponins
- leaks from ischemic cardiac muscle cells - return to normal in 2-3 days - ratio of 2/1 >1.5 = AMI - % of total CK activity - good for detecting 2nd MI
how to measure ckMB?
- IA sandwich with monoclonal anti CK-2 Ab - >3% = AMI - can be elevated in trauma
- not cardiac specific - heme containing protein responsible for oxygen deposition in muscle - early marker and cleared at 24 hrs - not useful anymore
troponin complex
- 3 subunits - TnI,TnT, TnC - regulates skeletal and cardiac muscle
TnI function
inhibits ATPase activity of actin and myosin
TnT function
binds tropomyosin
TnC function
Ca2+ receptor (regulates contraction and reverses effects of TnI)
troponin isoforms
cTnI: cardiac specific cTnT: cardiac and skeletal
troponin rise and fall times
- rise within 3-12 hours - peak at 12-24 - stay elevated for up to 3 weeks (T is 3; I is 2) **good for late detection
- only cardiac - used for critically ill or multi organ failure pts - no interferences from muscle injury, exercise, acute/chronic muscle diseases
what are the preferred cardiac biomarkers?
cTnI and cTnT (might also order ckMB)
- very high sensitivity troponin assay with CV <10% - super low detection limit and early - detects in healthy pts
single sample rule out
- using hs-cTn - using very low value with high sensitivity for MI - high NPV so we can rule out AMI - do not use for pts who come in before 2 hr onset of chest pain
- acute phase serum protein - biomarker for atherosclerosis - used to measure risk of AMI/stroke - not for diagnostic or prognostic
hsCRP risk interpretation values
<1 = low CVD risk 1-3 = average risk >3 = high risk >10 = look at other non-CV causes of inflammation
- marker for atherosclerosis/CVD - causes cholesterol to convert to LDL inc risk for clots
- marker for PE and CHF - regulates BP and fluid balance - releases in response to ventricle volume expansion/overload
- longer half life than BNP - inactive form (1:1 with BNP) >20 = high probability of CHF
d dimer
- product of fibrin degradation - inc in PE (normal can rule out PE)
markers for AMI?
ckMB, troponins, and high sensitivity troponins
differentiate heart failure from lung disease?
BNP and NTproBNP
markers to measure risk of PE
cardiac troponins, BNP, NT-proBNP, d dimers
markers for CV risk
troponins, homocysteine, hsCRP
ckMB rise and fall times in AMI
4-8 18-24 1-3 days
protein portion without prosthetic group
prosthetic group
non protein cofactor (bound coenzymes/ion/cofactor)
apoenzyme + prosthetic group (active form)
organic cofactor (NAD/NADPH)
inorganic cofactors (Zn, Mg)
- copper for ceruloplasmin - zinc for carbonic anhydrase
lag phase
- time delayed for activation - time needed for mixing of substrate and enzyme to make ES complex - no abs change
linear phase
- all enzyme saturated (substrate in xs) - zero order kinetics - rate depends on substrate conc - constant abs change over time
substrate depletion phase
- all product - substrate no longer in xs - no abs change
michaelis menten
relates velocity of enzyme to substrate conc
first order kinetics
velocity proportional to substrate conc
zero order kinetics
velocity not dependent on substrate conc
competitive inhibitor
- X graph - Km inc - binds to active side of enzyme
noncompetitive inhibitor
- V graph - Vmax dec - binds to allosteric/active site changing enzyme structure
uncompetitive inhibitor
- parallel graph - Km and Vmax dec - binds to ES complex to inhibit product formation
endpoint analysis
- measures at fixed time/single point - measures amt of product - assumes zero order (not good)
kinetic rate analysis
- multiple point, continuous monitoring - demonstrates linearity of rxn - measuring activity - if enzyme conc too high, will give false low (correct by using less sample)