enzymes - flashcards

oxidoreductase

removes H or adds oxygen; redox rxn

examples of oxidoreductases

lactate dehydrogenase (LD)

transferases

- transfers a specific group from one substrate to another; other than H - transfers amino acids

examples of transferases

GGT, AST, ALT, CK

hydrolase

cleaves carbon bonds by the addition of water

examples of hydrolases

LPS, ALP, ACP, AMS

AST tissues of origin

skeletal, cardiac muscle/tissue, hepatocellular tissue**

what causes AST to leak into serum?

necrosis

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

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?

ALP, GGT

liver panel for intrahepatic?

AST, ALT

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

amyloclastic

measures the disappearance of starch substrate

saccharogenic

measures the appearance of the product

chromogenic

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

AMS clinical significance

- acute panc - chronic panc (normal due to prolonged damage) - obstructive liver disease, acute alcoholism

macroamylasemia

- 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

pancreas

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

LD1 (HHHH)

heart, kidneys, rbc

LD2 (HHHM)

RES, wbc

LD3 (HHMM)

lung, spleen, other tissues

LD4 (HMMM)

kidney, placenta, pancreas

LD5 (MMMM)

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

atherosclerosis

- 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

hypertension

- 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

MI

- 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

CHF

- 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

ckMB

- 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

myoglobin

- 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

cTnI

- 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)

hs-cTn

- 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

hsCRP

- 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

homocysteine

- marker for atherosclerosis/CVD - causes cholesterol to convert to LDL inc risk for clots

BNP

- marker for PE and CHF - regulates BP and fluid balance - releases in response to ventricle volume expansion/overload

NT-proBNP

- 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

apoenzymes

protein portion without prosthetic group

prosthetic group

non protein cofactor (bound coenzymes/ion/cofactor)

holoenzyme

apoenzyme + prosthetic group (active form)

coenzymes

organic cofactor (NAD/NADPH)

activators

inorganic cofactors (Zn, Mg)

metalloenzymes

- 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)