Enzymes of Clinical Significance

Importance
Enzymes play an important role in
Metabolism, Diagnosis, and
Therapeutics.
All biochemical reactions are
enzyme catalyzed in the living
organism.
Level of enzyme in blood are of
diagnostic importance e.g. it is a
good indicator in disease such as
myocardial infarction.

Creatine Kinase (CK)

Action
Associated with the regeneration and
storage of ATP
Catalyses the following reaction:

Creatine Kinase (CK)

Purpose
Allows the body to store phosphate energy as
creatine phosphate
Energy can be released/provided to muscles
by reversing the reaction.

Source
Skeletal muscle
Heart
Brain
Other

Creatine Kinase (CK) Structure

Dimer consisting of two subunits
Two subunits are further divided into 3
molecular forms or isoenzymes
CK-BB: (CK-1) brain type
Migrates fast on electrophoresis
Small amount found in tissue (brian, lung, bladder, bowel)

CK-MB: (CK-2) hybrid type

Heart, Skeletal

CK-MM: (CK-3) muscle type

Mostly found in healthy people
Striated muscle and normal serum

Creatine Kinase (CK)

Diagnostic Use
Appearance of CK (MB) very sensitive
indicator of MI
Skeletal muscle disorders such as
muscular dystrophy
CNS Disorders
Cerebrovascular accident (CVA)
Seizures
Nerve degeneration

CK Isoenzymes

CK Half-life

Measurement of CK Activity
Forward and reverse reactions
Change in absorbance at 340 nm is
measured
CK
Creatine + ATP Creatine phosphate
+ ADP

Forward Reaction
Coupled with the pyruvate kinaseLDH-NADH system
PK
ADP + phosphoenolpyruvate pyruvate
+ ATP
LD
Pyruvate + NADH + H lactate + NAD

Reverse Reaction
Oliver and Rosalki method
Most common
Coupled with the hexokinase-glucose-6phosphate dehydrogenase-NADP system
KH
ATP + glucose ADP + G-6-P
G-6-PD
G-6-P + NADPH 6-phosphogluconate +
NADPH

Creatine Kinase Specimen

Collection
Enzyme inactivation is prevented by
addition of sulfhydryl compounds: Nacetylcysteine, mercaptoethanol,
thioglycerol or dithiotreitol
Sources of Error
Hemolysis
Interference of adenylate kinase on CK assays
Results in false elevations

Exposure to light
CK is inactivated by light

Creatine Kinase Reference Range

Affected by:
Age
Physical activity
Race
Bed rest (even overnight can decrease
CK)

Total CK
Men: 46-180 U/L
Female: 15-171 U/L

Creatine Kinase
Isoenzyme Testing
Fractionation of CK
Immunoinhibition
Mass assay
Ion-exchange
chromatography
Electrophoresis

Lactate Dehydrogenase (LDH/LD)

Action
Catalyzes a reversible reaction between
pyruvate and lactate with NAD as a
coenzyme
Reaction:

Lactate Dehydrogenase (LDH/LD)

Source
Skeletal muscle
Cardiac muscle
Kidney
RBCs
Widely distributed in the body

Lactate Dehydrogenase (LDH/LD):

Structure
Tetramer
Four polypeptide chains, two subunits
(heart & muscle)
Five combinations of Isoenzymes

Lactate Dehydrogenase (LDH/LD)

Diagnostic Significance
Nonspecific
Increased
Hematologic and neoplastic disorders
Liver disease
Heart problems

Measurement of LDH
Activity
Forward and reverse reactions
LD
Lactate + NAD pyruvate + NADH + H

Rate of reverse reaction is 3x faster

Forward rxn pH: 8.3 to 8.9
Reverse rxn pH: 7.1 to 7.4

LDH/LD Specimen Collection

Sources of Error
LDH-5 is most labile.
Hemolysis
RBCs contain 100-150 times that found in serum

Analyte stability
Run assay asap or store at room temperature for
at most 48 hours

Prolonged contact of serum and cells

Reference Range
140- 280 U/L

Cardiac Troponins
Isoforms
Proteins
Regulate the calcium-dependent
interactions of myosin heads with
actin filaments
Troponin T (TnT)
Troponin I (TnI)
Troponin C (TnC)

 Troponin T (TnT) binds to troponin

complex to troponin myosin
Troponin I (TnI) inhibits the binding
of actin and myosin
Troponin C (TnC) binds tocalcium to
reverse the inhibitory activity of TnI

Liver Enzymes
Transaminases
AST
ALT

Phosphatases
ALP

Transaminases
Retain amino groups during the
degradation of amino acids
Types
Aspartate transaminase (AST)
Aka: Glutamic Oxalocetic transaminase
(SGOT)

Alanine transaminase (ALT)

AKA: Glutamic pyruvic transaminase (SGPT)

Aspartate Aminotransferase (AST)

Sources
Major
Heart
Liver
Muscle

Minor

RBCs
Kidney
Pancreas
Lung

Aspartate Aminotransferase (AST)

Reaction:

Measurement of AST
Activity
Karmen method: malate dehydrogenase
Change in absorbance at 340 nm is
measured
Optimal pH : 7.3 to 7.8
AST
Aspartate + -ketoglutarate oxaloacetate
+ glutamate
MD
Oxaloacetate + NADH + H malate + NAD

AST Specimen Collection

Sources of Error
Hemolysis
Analyte stability
Stable at room temp for 48 hours or 3-4
days refrigerated

Reference Range
5-30 U/L

Alanine Transaminase (ALT)

Transfers an amino group from
alanine to alpha-ketoglutarate to
form glutamate and pyruvate

Alanine Transaminase (ALT)

Sources
Liver (Majority)
Kidney
Heart
Skeletal muscle

Measurement of ALT Activity

Coupled with LDH as indicator
Change in absorbance at 340 nm is
measured
Optimal pH : 7.3 to 7.8
ALT
Alanine + -ketoglutarate pyruvate +
glutamate
LDH
Pyruvate + NADH + H lactate + NAD

ALT Specimen Collection

Sources of Error
Not affected by hemolysis
Analyte stability
3-4 days refrigerated

Diagnostic Significance: AST & ALT

Many diseases can cause increases since
widely distributed in tissues
Liver
Hepatitis
Cirrhosis
Liver cancer

Myocardial Infarction
AST increases most
ALT normal to slightly increased, unless liver
damage accompanies

Diagnostic Significance: AST & ALT

Other
Pulmonary emboli
Muscle injuries
Gangrene
Hemolytic diseases
Progressive Muscular dystrophy

Phosphatases
Removes phosphates from organic
compounds
Functions to facilitate transfer of
metabolites across cell membranes
Alkaline Phosphatase (ALP)
Acid Phosphatase (ACP)

Phosphatases: Sources
Alkaline
Phosphatase

Bone
Liver
Kidney
Placenta
Intestines

Acid Phosphatase

Prostate gland
Seminal fluid
Liver
Spleen
RBCs
Platelets

Alkaline Phosphatase (ALP)

ALP frees inorganic phosphate from
an organic phosphate monoester,
resulting in the production of an
alcohol at an alkaline pH
Maximum activity at pH of 9.0- 10.0

Alkaline Phosphatase (ALP)

Diagnostic Significance
Elevations seen in
During bone activity
Pagets disease

Liver disease, especially in obstructive

disorders
Pregnancy between 16-20 weeks gestation

Decreased levels occur, but not

diagnostic

Measurement of ALP activity

Bowers and McComb
Increase in absorbance at 405 nm
under alkaline conditions (pH 10.2) is
measured.
(Colorless)
ALP (Yellow)
p-Nitrophenyl phosphate pnitrophenol +
phosphate ion

ALP Specimen Collection

Sources of Error
Hemolysis
Delay in processing, false increases can occur

Reference Range (Adult)

30-90 U/L
Normal increases seen in pregnancy,
childhood, adolescence

Acid Phosphatase (ACP)

Diagnostic Significance
Aids in detection of prostatic carcinoma
Other conditions associated with
prostate
Forensic chemistry: Rape cases
Elevated in bone disease

Measurement of ACP
Activity
Thymolphthalein monophosphate.
One of the most specific substrates for prostatic
ACP

Chemical-inhibition using tartrate as the

inhibitor
Prostatic fraction is inhibited by tartrate.
Serum and substrate are incubated both with and
without the addition of L-tartrate.
Total ACP ACP after tartrate inhibition =
prostatic ACP

Measurement of ACP
Activity
Bowers and McComb
Increase in absorbance at 405 nm
under acidic (pH 5) conditions is
measured.
(Colorless)
ALP (Yellow)
p-Nitrophenyl phosphate pnitrophenol +
phosphate ion

ACP Specimen Collection

Sources of Error
Separate serum from cells asap
Decrease in activity seen at room temp
Hemolysis
Reference Range (prostatic)
0-4.5 ng/mL

Gamma-Glutamyltransferase (GGT)
Possibly involved in peptide and
protein synthesis, transport of amino
acids and regulation of tissue
glutathione levels
Sources
Kidney
Brain
Prostate
Pancreas
Liver

Gamma-Glutamyltransferase (GGT)
Diagnostic Significance
Sensitive indicator of liver damage
Increased in patients taking enzymeinducing drugs such as warfarin,
phenobarbital and phenytoin
Indicator of alcoholism
Elevated in acute pancreatitis, diabetes
mellitus and MI

Measurement of GGT
Activity
The most widely
accepted substrate
for use in GGT
analysis is glutamyl-pnitroanilide.
p-nitroaniline is a
chromogenic product
with a strong
absorbance at 405 to
420 nm.

GGT Specimen Collection

Sources of Error
Very stable
Hemolysis not an issue

Reference Range
Male: 10-34 U/L
Female: 9-22 U/L

Digestive & Pancreatic Enzymes

Amylase
Lipase

Amylase (AMS)
Digestive enzyme that
hydrolzes/catalyzes the breakdown of
starch and glycogen to
carbohydrates
Smallest enzyme
Sources
Acinar cells of pancreas and salivary
glands

Amylase (AMS)
Diagnostic Significance
Acute pancreatitis
AMS levels rise 2-12 hours after onset of
attack, peak at 24 hrs and return to normal
within 3-5 days

Salivary gland lesions

Mumps

Measurement of AMS
Activity

Measurement of AMS
Activity
Amyloclastic method
AMS is allowed to act on a starch substrate to which
iodine has been attached.
Decrease occurs in the initial dark-blue color intensity
of the starchiodine complex.

Saccharogenic method
The amount of reducing sugars produced is
proportional to AMS activity.
Somogyi units are an expression of the number of mg
of glucose released in 30 minutes at 37C under
specific assay conditions.

Measurement of AMS
Activity
Chromogenic methods
Use a starch substrate to which a
chromogenic dye has been attached,
forming an insoluble dyesubstrate
complex.
Smaller water soluble dye-substrate
fragments are produced which is
proportional to AMS activity.

Measurement of AMS
Activity
Continuous-monitoring
Technique in which the change in
absorbance of NAD at 340 nm is
measured.

Amylase
Sources of Error
Presence of opiates increases levels
Stable

Reference Range
Serum: 30-100 U/L
Urine: 1-17 U/h

Lipase (LPS)
Hydrolyzes triglycerides to produce
alcohols and fatty acids

Source
Pancreas

Lipase (LPS)
Diagnostic Significance
Acute pancreatitis
More specific than amylase
LPS persists longer than AMS

Measurement of LPS Activity

Cherry-Crandall
Used an olive oil substrate and
measured the liberated fatty acids by
titration after a 24-hour incubation.

Cherry-Crandall Modification
Triolein is one substrate now used as a
more pure form of triglyceride.

Measurement of LPS Activity

Turbidimetric methods
Simpler and more rapid
As the fats are hydrolyzed by LPS, the
particles disperse, and the rate of clearing
can be measured as an estimation of LPS
activity.

Colorimetric methods
Based on coupled reactions with enzymes
such as peroxidase or glycerol kinase.

Lipase Specimen Collection

Sources of Error
Stable
Hemolysis results in false decreases

Reference Range
13-60 U/L

G6PD

Adrenal cortex
Spleen
Thymus
Lymph nodes
Lactating mammary glands,
erythrocytes

 G-6-PD deficiency- inherited sex-linked

trait
Drug induced-hemolytic anemia
MI
Megaloblastic anemias
Red cell hemolysate; serum
Reference range: 10-15 U/g Hgb

FIN