enzymes - flashcards

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oxidoreductase

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