Introduction

Measurement is an important tool

Enzymology
Janelle
M. Chiasera, PhD, MT(ASCP)
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Clinical diagnoses
Assessing prognosis
Evaluating effective treatment

New enzymes continue to be developed

LD, CK-MB

Thousands identified; 15 measured

Biochemistry
Integral components
Location
Mitochondria
Cytosolic
C t
li
Membranes

Diagnostic utility
Differences in amount of enzyme in tissues
Intracellular (1000 xs greater)
Presence in normal individuals
Apoptosis, efflux, and clearance

Composition and Structure

Composition
Proteins
Isoenzymes

Structure

Enzyme

Major tissue source

Alkaline phosphatase (ALP)

Placenta, intestines, kidney, bone, and

liver

Alanine aminotransferase (ALT)

Liver, kidney

Amylase (AMY)

Pancreas, salivary glands, fallopian tubes

Aspartate aminotranferase (AST)

Heart, liver, skeletal muscle, kidney,

pancreas

Creatine kinase (CK)

Skeletal muscle, myocardium, brain,

colon, stomach, urinary bladder

Gamma glutamyltransferase (GGT)

Kidney, biliary tract of liver

Lactate dehydrogenase (LD)

Brain, heart, erythrocytes, kidney, lung,

skeletal muscle, liver, pancreas, stomach

Lipase (LPS)

Pancreas

Cholinesterase

Plasma, liver

Factors that affect activity

Denaturation
By changing environment
p
pH and Temperature
p

Active site
Confers specificity

Cofactors and Coenzymes

Small compounds, loosely associated
Prosthetic groups and holoenzymes

Factors that affect activity

pH
Activity is pH dependent
y
activity
y should be measured here
Enzyme

Temperature
Activity is temperature dependent
Doubles for every 10o rise in temp
As temp increases, activity increases and
plateaus
Most inactive above 55-60o

Enzyme kinetics

Kinetics

Biological catalysts
They accelerate the rate of a reaction without
being consumed

Michaelis-Menten theory states

E+S
ES
E+P
Rates of forward and reverse = k1, k2
By design substrate is much greater

Enzyme kinetics
Substrate saturation
First-order kinetics
Zero-order kinetics

Michaelis-Menten
Constant Km
(moles/liter)
Reaction rate is
the maximal
velocity
Km is different for
enzymes that can
use the same
substrate
Glucose vs.
Fructose

Enzyme inhibition
Inhibitors
Competitive, noncompetivie, and
uncompetitive
Competitive

Enzyme inhibition
Noncompetitive
Inhibitor combines with free enzyme or
enzyme substrate complex (not in active site)
E+S+I
ES + EI + ESI
E+P

Substance binds free enzyme at active site

Inhibitor directly competes with substrate

E+S+I

ES + EI

E+P

Overcome increase substrate concentration

Enzyme inhibition
Uncompetitive inhibition
Inhibitor binds to the ES complex only
E+S+I
ES + ESI
E+P
What happens if we increase substrate
concentration?

Nomenclature
Source of Confusion
Classification scheme
1st gives name of substrate acted on
2nd (ending in ase)
ase ) indicates type of reaction

6 classes

Oxidases
Transferases
Hydrolases
Lyases
Isomerases
Ligases

Clinical overview
Aminotransferases

AST

Sensitive indicators of necrosis

No metabolic function in serum

AST (30%
L-aspartate + ketoglutarate

Clinical utility

Oxaloacetate + L-glutamate

*Coenzyme required (P-5-P)

2 forms
Cytoplasmic (30%)
Mitochondrial (70%)
Patients with liver disease - in mitochondrial

Liver and biliary tract

Viral hepatitis
Liver hypoxia
Autoimmune hepatitis
Alcoholic liver disease

Skeletal disorders
Duchenne muscular dystrophy
Strenuous physical acticity

Measurement
AST (AKA SGOT)

S, P, U
Heparin, EDTA, oxalate and citrate
St bl refrigerated
Stable
fi
t d
Urine = renal function after transplant
Increased in infants and children
Reference range
8-20 U/L
10-30 U/L

hepatocellular

Aminotransferases
ALT (more liver specific)
Present in only the cytosol of hepatocytes
Requires coenzyme (P-5-P)
Measure hepatocellular diseases
Ratio
ALT/AST (De Ritis Ratio)
Mild liver damage = ALT:AST <1.0
Severe liver damage = ALT:AST is decreased
AST > conc in liver tissue than ALT

Measurement
ALT (AKA SGPT)

S preferred or P
Heparinized or EDTA
RT stable for 3 days
Reference range
10-40 U/L with P-5-P

Phosphohydrolases
ALP
Requires Zn2+ and Mg2+ for activity
Present in most body tissues
Isoenzymes
Distinguished by electrophoresis

ALP present = bone and liver origin

High values dont always relate to disease
Elevations
Bone diseases ( osteoblastic activity)
Hepatobiliary disease
Extrahepatic obstruction (more )
Intrahepatic obstruction
Liver disorders without cholestasis

12x the normal

hepatobiliary

Phosphohydrolases
ALP
Total ALP sensitive but nonspecific
Reference range
38-94 U/L
28-78
28 78 U/L
Measurement
S, P (heparin)
Bowers and McComb
ALP
p-nitrophenol and phosphate
P-nitrophenyl phosphate
(Colorless)
(Yellow)

40-125 u/l

Lactate Dehydrogenase
Cytoplasmic enzyme
LD
Pyruvate + NADH + H
L-lactate + NAD
Highest concentrations in many organs
Increased in a wide range of abnormalities
Molecule
Tetramer with 2 subunits
5 possible isoenzymes
LD-1, LD-2, LD-3, LD-4, LD-5

Measure change in absorbance over time

LD isoenzymes
LD-1 (HHHH), LD-2 (HHHM), LD-3
(HHMM), LD-4 (HMMM, LD-5 (MMMM)
Distinct biochemical properties
LD-1 and LD-2
LD-3 and LD-4
LD-5
Isoenzymes aid in identifying tissue source

LD

Normal = from erythrocytes and platelets

No known function in serum
Metabolized & eliminated by biliary tract
Measurement
S, P (H)
NO HEMOLYSIS
Separate ASAP

Storage
Complex
Analyze ASAP

LD

Creatine kinase

Measurement
Lactate + NAD LD
Pyruvate + NADH + H
Reverse 3xs faster (good and bad)
know pH and
Optimal pH forward = 8.3-8.9
Optimal pH for reverse = 7.1-7.4 absorbance of
Reference Ranges
NAD and NADH
100-225 U/L
80-280 U/L

Creatine + ATP
H

CK

Creatine phosphate + ADP +

Mg
g2+ required
q
Found
Clinical utility
Myocardial & skeletal damage
Other increases seen

100-200 U/L

Dimer with two subunits (M) and (B)

Form 3 isoenzymes (MM, MB, BB)

NAD = 265nm
NADH = 340nm

CK Isoenzymes
Majority in N serum
CK-MB

MM

Highest in myocardium; low in skeletal

CK-BB
C
Historically electrophoresis
Rapid assay

Two macromolecular forms

Macro CK-1
Macro CK-2

CK measurement

S, P(H), CSF, amniotic fluid

NO EDTA, citrate or oxalate
No hemolysis
Adenylate kinase
Unstable at RT
Keep in dark
Reference range

CK

Most commonly used method

CK
Reverse reaction by Oliver and Rosalki
Creatine phosphate + ADP CK
creatine +ATP
ATP + glu HK
ADP + Glu
Glu-6-phosphate
6 phosphate
Glu-6-phosphate + NADP G6PDH 6-phosphogluconate
NAD
+ NADPH

NADH

Reference ranges
Total CK
15-160 U/L male
15-130 U/L female

40-170U/L
25-150U/L

CK-MB
< 6% TCK

Reaction proceeds 2-6 xs faster than forward reaction

Less interferences from side effects
Optimal pH = 6.8

requires Mg for reaction to occur

Amylase
Digestive enzyme
Requires Ca as a cofactor
2 isoenzymes

ACCR
U AMS (U/L) X S Creat (mg/L)
S AMS X U Creat (mg/L)

X 100

Useful for detecting pancreatitis

Increased activity 2-24 hours

Eliminated by renal excretion

Macroamylasemia (benign)
Amylase bound to immunoglobulin
Increased amylase without pancreatitis
Use amylase:creatinine clearance ratio (ACCR)

Amylase
Measurement
Extremely stable in serum
S, P, and U (not acidified)
No EDTA, citrate, oxalate

Reference ranges

N = 2-5%
U AMS ACCR = Macroamylasemia
ACCR = Possible Pancreatitis

Amylase measurement
Substrate method
Starch -amylase
maltose + maltotriose +
dextrins
Maltose + maltotriose -glucosidase glucose
Glucose + O2 Glu Ox H2O2 + glucolactone

Lipase
Digestive enzyme
Catalyses TG hydrolysis
Concentration in pancreas is 100 xs
Diagnose pancreatitis

Three isoforms
In circulation = removed
Amylase v. lipase in kidney
LPS:AMS ratio ( seen in pancreatitis due to alcohol)

Clinical utility
Pancreatitis/renal insufficiency
Measurement
S
Storage
Measurement
Ability of Lipase to hydrolyze FAs from an
emulsion
Measure Turbidity

GGT

Amino acid transferase

Membrane-bound enzyme
Highest activity in kidney
5 isoenzymes
Clinical utility

GGT
Measurement
S,P (H)
No hemolysis

Hepatobiliary diseases/cholestasis
Helpful with regard to extent
Aid in interpreting increased ALP
Marker of alcohol consumption

Cholinesterase
Two of clinical interest
AChE acetlycholinesterase
SChE pseudocholinesterase (syn hepatic
cells)

SChE measured
Marker of poisoning
Detection of variants

Measurement
S, P (H)

Acid phosphatase
Belongs to group of enzymes with ALP
Major difference pH = 5.0
Tissue activity
Prostate (richest), bone, liver, spleen, kidney, RBCs and Plts

Clinical significance
Prostatic cancer

Elevations
Bone diseases (osteoclasts). Also useful in rape
cases

ACP assay
Same as ALP; pH 5.0
Error
Separate immediately (RBC leakage)
RT = ACP
No hemolysis

Reference range
TACP
2.5-11.7 U/L; 0.3-9.2 U/L (females)

Prostatic ACP
0.2-5.0 U/L

Test procedures
Measure product formation
Exception AST and ALT

Product formation

Test procedures
Activity assays or mass assays
MA = protein mass

Determination
From catalytic activity
Criteria

Requirements for cofactors

Optimal concentration
pH and temp
Mechanism for measuring

Test methods
Kinetic methods
Standardized reporting schemes
International Unit
Amo
Amount
nt of en
enzyme
me catal
catalyzes
es the transformation of 1
1.0
0 mol
mol
of substrate /minute

Directly
coupled

Three phases (enz + substrate)

Lag
Linear
Substrate depletion

Isoenzyme patterns
Pattern 1 CK-MB > 6% TCK; LD-1>LD-2
most reliable diagnostic criteria for AMI
confirms AMI has occurred

Pattern 2 CK-MB > 6% TCK; LD-1<LD-2

reflects some degree of myocardial damage
damage, but not
necessarily AMI

Pattern 3 CK-MB < 6% TCK

no AMI has occurred regardless of LD pattern

Pattern 4 CK-MB < 6% TCK; LD-1>LD-2

found in a variety of noncardiac disorders
frequently seen with intravascular hemolysis and
megaloblastic anemia
Lack of MB elevation rules out myocardial damage