Download Enzymes of Clinical Significance

I. General Description of Enzymes
A. Chemical composition
1. Enzymes are proteins, compounds of high molecular weight, containing
Carbon (C), hydrogen (H), oxygen (O), sulfur (S), nitrogen (N)
2. Possess primary, secondary, tertiary (sometimes quaternary) structure
3. Same properties as other proteins
a. Charge (isoelectric point, electrophoresis)
b. Solubility
c. Denaturation (extremes in heat, pH, chemical)
d. Non-specific or specific (antigenic) binding
4. Some enzymes have iosenzyme forms:
a. Different form of an enzyme due to differing arrangements of the
polypeptide units in the quaternary structure
b. Maintains specificity for the same chemical reaction
c. Possess unique diagnostic value: show specificity to certain organ
systems
1) Isoenzymes of creatine kinase (CK)
CK-MM to skeletal muscle
CK-MB to cardiac tissue
CK-BB
2) Isoenzymes of lactate dehydrogenase (LD)
LD1 and LD2 to cardiac tissue
LD4 and LD5 to liver and skeletal muscle
B. Biochemical Function
1. Catalyze chemical reactions: accelerates specific
chemical reactions
toward equilibrium
S
P
Substrate (S) + Enzyme
(ES
Product (P) +Enzyme
2. Lowers the energy of activation required for the
reaction to proceed
3. Properties of enzymes
a. Effective in small concentrations
b. Remain unchanged in the reaction, not
consumed in the reaction
c. Effect speed of reaction (not final
concentration of substrate or
products)
d. Show greater degree of specificity than
chemical catalysts
e. Unstable and easily destroyed
4. Cofactors:
substances associated with some enzymes that are needed for
maximal enzyme activity
a. Coenzyme: organic cofactors (NAD, NADH);
serve as second
substrates for enzyme reactions
b. Activators: inorganic cofactors (Ca++, Fe++,
Zn++, K+, Mg++)
C. Importance of Enzymes in Health and Disease
1. Enzymes are intracellular
2. Enzyme concentration differs dependent
upon type of tissue
a. AST: high concentration in liver
b. Amylase: high concentration in pancreas and
salivary glands
Enzymes of Clinical Significance
Creatine Kinase •
1. Specimen collection and handling •
a. Serum is preferred, especially if fractionation/electrophoresis also •
ordered •
b. Heparinized plasma may be acceptable; •
Many anticoagulants (fluoride, EDTA, sodium citrate) inhibit •
enzyme activity and required activators (Ca2+, Mg2+) •
c. No hemolysis: intracellular components interfere with chemical •
reaction in laboratory measurements (NADH, ATP, HK, etc) •
d. Avoid lipemia, especially for spectrophotometric analysis •
e. Relatively unstable: analyze within 4 hours of collection, else •
stable at 2-6oC for up to one week; stability increases when stored •
at lower temps (-20oC) •
Wide tissue distribution: injury, tissue necrosis
will cause elevation of this
enzyme in plasma/serum
a. Skeletal muscle
b. Heart tissue
c. Brain, nerve tissue
Method of Measurment
3. Physiologic reaction: CK is a transferase: ATP storage and generation in
contractile or transport systems, predominantly in muscle cells.
Creatine + ATP
CK􀃆
creatine phosphate + ADP
4. Method of measurement
Most common method: modified Rosalki-Oliver method
creatine phosphate + ADP
CK
creatine + ATP
ATP + glucose
HK
ADP + glucose-6-phosphate
Glucose-6-phosphate + NADP+
(Increased ABS at 340 nm)
G6PD
6-phosphogluconate +NADPH
Isoenzymes: clinical relevance of CK activity often depends more on
isoenzyme fractionation than on total levels:
a. Isoenzymes occur as a dimer:
1) B polypeptide units
2) M polypeptide units
b. Three isoenzyme forms: numbered by their anodal (+)
electrophoresis mobility with ‘fraction 1’ migrating fastest towards
the anode
1) CK1 = CK-BB (brain type) Normal: ~0%
2) CK2 = CK-MB (hybrid, heart) Normal: <5%
3) CK3 = CK-MM (muscle type) Normal: 95-100%
c. Immunologic measurement of CK-MB
1) Immunoinhibition of subunit M (anti-M)
2) Measure CK before and after inhibition
3) Disadvantage: falsely elevated if CK-BB activity present
d. Enzyme immunoassay measurement of CK-MB
(mass)
A ‘sandwich technique’ that uses 2 antibodies:
one directed
against the ‘M’ subunit, the other against the ‘B’
subunit which is
also labeled with a reagent enzyme that
catalyzes the conversion of
substrate to product
6. Reference Ranges: affected by gender,
muscle mass, exercise, age •
Total CK: •
Male: 15-160 IU/L •
Female: 15-130 IU/L •
CK-MB: <5% of total CK •
7. Clinical significance
a. Acute Myocardial Infarction (AMI)
1) Increased total CK, increased CK-MB
2) Total CK increase 7-12 times normal with
rise apparent at 4-6 hours post MI
peaks at 18-24 hours and
returns to normal by 72 hours
3) The rise, peak and fall of CK-MB parallels that of total CK,
but a little sooner
4) Other cardiac events (trauma/inflammation) variable
results
b. Skeletal muscle disease
1) Increased total CK, normal CK-MB (increased CK-MM)
2) Muscular dystrophy, especially Duchenne type: may
see
total CK 50x ULN
3) Inflammation of muscle, especially viral, also malignant
hyperthermia
4) Normal CK activity in neurogenic muscle disorders:
multiple sclerosis, myasthenia gravis, poliomyelitis
c. Central nervous system disorders
1) Increased total CK, normal CK-MB (increased
CK-BB)
2) Trauma or pathology indicated when CK-BB is
present
3) Cerebral vascular disease, encephalopathy,
stroke (CK-BB )may be normal
d. Other
1) Neonates: normally have 1-3% CK-BB
2) Hypothyroidism: increased total CK, increased
CK-MM
3) CK activity is NORMAL in liver disease
4) Effect of hemolysis is procedural and not due
to CK
activity in RBC
B. Lactate dehydrogenase (LD)
1. Specimen collection and handling
a. Serum is preferred, especially if fractionation/electrophoresis also
ordered
b. Heparinized plasma may be acceptable; many anticoagulants
(fluoride, EDTA, sodium citrate) inhibit enzyme activity
c. No hemolysis: LD-1 activity in the RBC is 150x greater than that
in plasma
d. Avoid lipemia, especially for spectrophotometric analysis
e. Relatively unstable: analyze within 4 hours of collection, else
stable at 2-6oC for up to one week; stability increases when stored
at lower temps (-20oC)
f. LD-4 and LD-5 are especially labile: stable when stored at room
temperature, do not store in refrigerator or freezer unless
glutathione is added as preservative
2. Wide cellular/tissue distribution
a. Skeletal muscle
b. Cardiac muscle/tissue
c. Liver
d. RBC
e. Kidney, lung, tumor cells
3. Physiologic reaction:
• LD is an oxidoreductase: catalyzes the
• interconversion of lactic and pyruvic acids
• Lactate + NAD+ LD Pyruvate + NADH +
H+
4. Method of measurement
Forward reaction:
Lactate + NAD+ ---LD (pH 8.3-8.9) Pyruvate + NADH + H+
(Increased ABS at 340 nm)
Reverse reaction:
Pyruvate + NADH ---LD (pH 7.1-7.4) Lactate + NAD+
(Decreased ABS at 340 nm)
Both the forward and reverse reactions have
been used in the clinical lab,
with the rate of the reverse reaction being ~3
times faster allowing
measurement of smaller sample volumes and
shorter reaction times.
However, the forward reaction has better
linearity at this time
5. Isoenzymes: clinical relevance of total LD activity often
depends more on
isoenzyme fractionation correlating to total LD activity
a. Isoenzymes occur as a tetramer
1) H polypeptide units (heart)
2) M polypeptide units (muscle)
b. Five isoenzyme forms: numbered by their anodal
electrophoresis
6. Reference Ranges:
Total LD 100-200 IU/L
LD-1 14-26% of total LD
LD-2 29-39% of total LD
LD-3 20-26% of total LD
LD-4 8-16% of total LD
LD-5 6-16% of total LD
mobility with ‘fraction 1’ migrating fastest towards the anode (+)
1) LD1 = HHHH
Heart, RBC, kidney
2) LD2 = HHHM
3) LD3 = HHMM
4) LD4 = HMMM
Skeletal muscle, liver
5) LD5 = MMMM
6. Reference Ranges:
Total LD 100-200 IU/L
LD-1 14-26% of total LD
LD-2 29-39% of total LD
LD-3 20-26% of total LD
LD-4 8-16% of total LD
LD-5 6-16% of total LD
7. Clinical significance:
a. Hepatocellular disease
1) Increased total LD (7-10 x ULN), increased LD-4, LD-5
2) Toxic and viral hepatitis; infectious mononucleosis
3) Normal or slightly elevated in obstructive liver
disorders
b. Skeletal muscle disease
1) Increased total LD, increased LD-4 and LD-5
2) Muscular dystrophy, especially Duchenne type
Skeletal muscle, liver
c. Acute myocardial infarction (AMI)
1) Increased total LD, increased LD-1 and LD-2 with a
flipped ratio
2) Total LD increases 3-7 times normal with
rise apparent at 8-12 hours post MI
peaks around 72 hours and
returns to normal by 7-12 days
3) Not useful for diagnosis of AMI
4) Other cardiac events (trauma/inflammation) variable
results
d. Hemolytic anemia
1) Increased total LD 20-50 times normal,
increased LD-1 and
LD-2 with a false flipped ratio (not due to MI)
2) Untreated Pernicious Anemia, Folate
Magaloblastic
Anemia
3) Hemolytic anemia
C. Aspartate Transaminase (AST) Historical:
Serum glutamate oxaloacetate transaminase (SGOT)
1. Specimen collection and handling
a. Serum preferred, heparinized plasma acceptable;
other anticoagulants inhibit enzyme activity
b. Avoid hemolysis: RBC contain AST
c. Avoid lipemia, especially for spectrophotometric
analysis
d. Relatively stable: analyze within 8 hours of
collection, else store at
2-4oC for up to 4 days
2. Wide cellular/tissue distribution
a. Skeletal muscle
b. Cardiac muscle/tissue
c. Liver
d. Kidney, pancreas, RBC
3. Physiologic reaction:
AST is a transferase; catalyzes the transfer of an amino
group between aspartate and alpha-keto acids.
Important in the
synthesis and degradation of amino acids; ketoacids
formed are used in the TCA cycle to provide a source of
energy
Aspartate + α-ketoglutarate
AST
Oxaloacetate + Glutamate
4. Method of measurement: Karmen method
Aspartate + alpha-ketoglutarate
AST
Oxaloacetate +
Glutamate
Oxaloacetate + NADH + H+ 􀃆 Malate + NAD+
(decreased ABS at 340 nm)
5. Reference Range: 0-56 IU/L
• a. Liver disease: hepatocellular, parenchymal
disease
• 1) Inflammation: toxic or viral hepatitis, infectious
• mononucleosis; activity may reach up to 50-100 x
normal
• 2) Obstructive conditions (cholestasis, some
stages of
• cirrhosis) generally show normal or slightly
increased AST
6. Clinical significance
• a. Liver disease: hepatocellular, parenchymal
disease
• 1) Inflammation: toxic or viral hepatitis, infectious
• mononucleosis; activity may reach up to 50-100 x
normal
• 2) Obstructive conditions (cholestasis, some
stages of
• cirrhosis) generally show normal or slightly
increased AST
b. Skeletal muscle disease
1) Muscular dystrophy, especially Duchenne
type
2) Inflammation of the muscle, malignant
hyperthermia,
crushing muscle injury
3) Neurogenic muscle disorders such as multiple
sclerosis,
myasthenia gravis, poliomyelitis show normal
AST