basic enzymes, CPK, LDH, ALT, AST

isoforms

similar proteins originating from a single gene/family that perform the same/similar roles (via alt splicing)

• catalyze same rxn

• have different structure

  • have varying physical, biochem, immunologic properties

enzymes do not

• change eqlbrm of rxn

• consumed during rxn

  • appear as rxn products

endpoint measurement

measures amount of enzyme by qty of substrate consumed or product formed at end of rxn

kinetic assay/rate analysis

measurements are taken at short intervals & continuously monitored throughout rxn

rate of rxn is proportional to enzyme conc

zero order kinetics

rxn rate is independent of substrate conc

• enzyme is only limiting factor to rxn rate

=> most useful in the lab

first order kinetics

rxn rate is proportional to substrate conc

• limiting factor can be enzyme or substrate conc

Vmax = all free enzyme bound to substrate (max velocity)

Km = Michaelis-Menten constant = substrate conc req for enzyme to reach ½ max

inhibitors

reversible vs irreversible

competitive- binds at active site

• Vmax same

• Km inc → more substrate is req for rxn to reach ½ Vmax

noncompetitive- binds enzyme in site other than active site

• Vmax dec

• Km same

factoring affecting rxn rate

temp

pH

ionic strength

activators/cofactors

inhibitors

auxiliary enzymes

what can cause abnormal serum enzyme activity?

genetic dz

organ dz or damage

inflammation

vit or nutritional defic

creatine kinase (CK) fxn

transfer P group from creatine-P + ADP → ATP + creatine formation for muscle mvmt

inc in MI & muscle dz

inc in trauma, exercise, males > females

CK isoenzymes

what is the nm used if NAD(P) is used in indicator rxn?

340 nm

what is the gold standard for isoenzyme differentiation?

electrophoresis

CK is unstable at RT > 8hr → RFG

CK-MB/CK relative index (RI)

% of CK-MB to total CK

if RI > 2.5 → MI, renal, pulm, or hepatic dz, or normal → need to retest for MI 4-8 hr later

lactate dehyrdrogenase (LDH)

in all cells

L-lactate + NAD → (LDH)→ pyruvate + NADH + H

used to dx

• MI

• liver dz

• lung/kidney tumor

LDH is present in rbc, so _

hemolyzed samples will be falsely inc → reject

5 LDH isoenzymes

normal distribution: LD2 > 1 > 3 > 4 > 5

LD flip: when LD1 > 2>… → MI !

pulmonary embolism: 3>2>…

liver dz if inc LD 4 & 5

LDH spec req

no hemolysis

store at RT

don’t freeze LD4/5 sensi to cold

LDH EL

must be RT

frzn → loss of fractions

LD1 migrates fastest toward anode

Aspartate aminotransferase (AST/SGOT)

liver, brain, pancreas, heart, lungs, sk muscle, rbc (non-specific)

L-Asp + a-ketoglutarate -(AST)→ oxaloacetate + L-Glu

dx of liver & heart dz

no hemolysis!!

Alanine aminotransferase (ALT/SPGT)

m/c in liver

L-Ala + a-ketoglutarate -(ALT)→ pyruvate + L-Glu

liver & heart dz dx

AST/ALT ratios are usually meas’d as biomarkers of liver health

AST vs ALT

AST/ALT ratio

indicates dz, if acute or chronic

most liver dz ALT > AST