This presentation is about Medical importance of Enzymes and their role in clinical medicine. The types of Enzymes are mentioned along with their normal roles and in pathologies. The measurements of enzyme levels are given as well as fluctuations in their levels in the presence of pathology. Hope this will help you.

1. CLINICAL ENZYMOLOGY
1. What are enzymes that are present in blood plasma?
Enzymes are found in all tissues, as well as in plasma Enzy
mes in plasma can be separated into two groups:
Functional plasma e
nzymes
Non-functional plasma en
zymes
Enzymes that are synthesized b
y the liver predominantly, prese
nt in plasma and have function
al role in plasma
E.g. Lipoprotein lipase Clotting
factors
Substrates for these enzymes are NOT pre
sent in tissues (cellular enzymes)
E.g. Pancreatic amylase
Creatine phosphokinase (muscles)
2. What cause intracellular enzymes to be release into plasma?
3. In which clinical ways are enzymes used?
Enzymes are used clinically in three principal ways:
• in diagnosis and prognosis of various disease
• as analytical reagents in the measurement of activity of other enzyme or non-enzyme subs
tances
• as therapeutic agents
4. How does enzyme activity assay?
Enzyme assays usually depend on the measurement of the catalytic activity of the enzyme (m
ore sensitive test), rather than it concentration.
Conditions of the assay are optimized and standardized E.g.
specific color
AMYLASE
REDUCING
SURGARS
reagent
* COLOUR
optimal condition: time,
buffer, t0C
(maltose, glucose)
COMPLEX
CALCULATE
1
^_ READ OPTICAL
USING THE
FORMULA
DENSITY
International Unit of Enzyme activity: (IU/L or m-IU/L)
• One IU is defined as the activity of the enzyme which transforms one micromole of substr
ate per minute under optimal conditions.
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2. 5. What are isoenzymes?
Definition: Are the multiple forms of enzyme which catalyze the SAME REACTIONS and d
iffer from each other by:
-structurally
- electrophorecically
- Synthesis coded by different gene
-localized predominantly in the different organs/tissue -differ from each other by kinetic pro
perties (Km, Vmax or both)
6. What are common cellular enzymes and isoenzymes used for clinical diagnosis?
• AST/ALT (transaminases/aminotransferases)
• ALP (alkaline phosphatase)
• CK (creatine kinase) or CPK (creatine phosphokinase)
• LDH (lactate dehydrogenase)
• GGT (gamma- glytamyl transferase)
• ACP (acidic phosphatase)
• Amylase (AMS)
• Lipase (LPS)
Intracellular Distribution of
Diagnostic Enzymes
Liver Heart Pancreas Salivary
Glands
Bone Muscle Biliary
Tract
Prostate
LD5
ALT
AST
LD1
AST
CK
LPS
AMS
AMS ALP CK ALP
GGT
ACP
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SOME ENZYMES OF CLINICAL INTEREST
1. SERUM CARDIAC MARKERS
Acute myocardial infarction (AMI).
• AMI generally occurs when blood flow increases abruptly after a
thrombotic occlusion of a coronary artery, previously narrowed by at
herosclerosis.
• AMI occurs when a coronary artery thrombus develops rapidly at
a site of vascular injury. This injury is produced by factors such as: ci
garette smoking, hypertension and lipid accumulation.
Cardiac markers
• Certain proteins, called serum cardiac markers, are released into the blood in large quantities
from necrotic muscles after AMI.
1. CK (Creatine phosphokinase).
• Total serum CK rise within 4 to 8 hours, reaches a peak level at 24 to 30 hours and generally
returns to normal by 48 to 72 hours.
• But total CK elevation may be due to:
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3. (i) Intramuscular injection of a narcotic
(ii) hypothyroidism
(iii) cardiac surgery
(iv) electric shock
(v) acute psyhosis
(vi) CNS trauma, extensive brain infarction
(vii) Progressive muscular dystrophy (levels may reach 300-400 times normal).
(viii) Chronic alcoholism
• The determination of CK isoenzymes may be helpful in a differential diagnosis.
• Structure: Dimer. Subunits: M (muscle) and B (brain) in different variation
• CK1 (CK-BB) Brain (trace)
• CK2 (CK-MB) Heart (0-4%)
• CK3(CK -MM) Skeletal muscles (96-100%)
• The elevation CK-MB (CK2), the cardiac isoenzymes, provides a more definitive indication o
f myocardial cell damage than total CK.
• Appearance of this isoenzyme in plasma (> 20% of total CK) is considerably more specific in
AMI.
• But also cardiac surgery, myocarditis often results in elevated serum levels of the CK-MB.
• Diagnosis: Determination of relative index CK-MB/CK-BB.
• If CK-MB/CK-BB ratio > 1.5 is highly sensitive for diagnosis of AMI, particularly 4 to 6 hou
rs after the coronary occlusion.
2. Lactate dehydrogenase □ LDH
• In AMI total serum LDH rises within 12 to 24 hours, reaches peak level at 48 hours and then r
eturn to normal 8th to 14th day.
• But total serum LDH non specific for myocardial tissue.
• Elevated levels of total serum LDH are observed in various other conditions, such as
(i) Liver diseases (cirrhosis, alcoholism, acute viral hepatitis)
(ii) Cancer
(iii) Lung diseases (pulmonary infarction)
(iv) Skeletal muscles diseases
(v) (muscular dystrophy)
(vi) Megaloblastic and pernicious anemia
(vii) Sickle cell disease
Isoenzymes of LDH
Structure: Tetramer. Subunits: H (heart) and M (muscles) in different variation
• LDH1 (H4) cardiac, RBCs (17-27%)
• LDH2 (H3M) cardiac, brain, RBCs (29-39%)
• LDH3 (H2M2) lung, spleen, pancreas, placenta, brain, kidney (19-27%)
• LDH4 (HM3) - liver, skin, kidney (8-10%)
• LDH5 (M4) - liver, skeletal muscles (6-16%)
• The appearance of LDH flip (when LDHi is more than LDH2) is extremely helpful in diagnos
is of AMI.
• The presence of LDH flip a day following or with the detection of CK-MB is essentially diag
nostic of MI.
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4. 3. AST: aspartate transaminases (aminotransferase) or sGOT □ serum glutamic-oxaloacetic trans
aminase.
❖ AST is an enzyme present in tissues of high metabolic activity
heart, liver, skeletal muscle, kidney, brain, pancreas, spleen, lungs
decreasing concentration of enzyme
• The enzyme is released into the circulation following the injury or death of cells
.
• The amount of AST in the blood is directly related to the number of damaged ce
lls and the amount of time that passes between injury to the tissue and the test.
• Following severe cell damage (AMI) the blood AST level rises sharply within th
e first 12 hours with a peak level at 24 hours or over and returns to normal withi
n 3 to 5 days.
• AST is catalyzed the conversion of Aspartic acid to oxaloacetic acid
COOH COOH
COOH
I COOH |
H2N —CH
o
II
-o-
AST ^ = Q H2N-CH
CH2 CH2 + CH2
COOH CH2 CH2 f2
COOH COOH COOH
aspartate alpha-keto-
glutarate
oxaloacetate glutamate
3. Cardiac-specific troponins
cTnT and cTnl are regulatory proteins involved in myocardial contractility.
They are not normally detectable in the blood of healthy individuals, but may increase aft
er AMI to levels over 20 times higher than the normal.
Levels of cTnl may remain elevated for 7 to 10 days after AMI, and cTnT levels may rem
ain elevated for up to 10 to 14 days.
f Aspartate tr
ansaminase
Creatine kinase MB fraction
10-
Creatine kinase
Lactate dehydrogenase
Time course of elevation (days)
Time course of myocardial enzymes appearing in the blood after myocardial inf
arction
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5. 2. SERUM ENZYMES IN LIVER DISEASES
• The liver contains thousands of enzymes, some of which are also present in the serum in v
ery low concentration.
• Liver serum enzyme tests can be classified into 2 groups:
(A) Enzyme whose elevation in serum reflects damage to hepatocytes (Hepatocellular disea
ses). In hepatocellular diseases (such as viral hepatitis or alcoholic liver disease), features
of liver injury, inflammation, and necrosis predominant.)
(B) Enzymes whose elevation in serum reflects cholestasis (bile duct obstruction).
# 1 ENZYMES THAT REFLECT DAMAGE TO HEPATOCYTES
1. Serum transferases (transaminases) : AST, ALT
2. Y-GT
Serum aminoptransf erases (aminotransaminases)
• Aminotransferases (aminotransaminases) are sensitive markers of liver cell injury.
• They include the AST and ALT or another name: serum glutamic-pyruvic transaminases (
s-GPT)
• Alanine aminotransferase (ALT) occurs in high concentration in the liver, and relatively
low concentrations are found in the heart, muscle and kidney.
• This test is primarily used to diagnosis liver disease and to monitor the course of treatment
for hepatitis, and the effects of drug therapy.
ALP is catalyzed the following reaction:
COOH
1 COOH
O
II
C=O
1
1
CH-NH2
1 2
ALT
H,N—CHC—OH 2 1
CH2 + W
CH2
1 2
CH2 1 2 COOH
alpha-keto-
CH3 CH2
alanine
C=O
1
OH
glutamate
glutarate
COOH
I
C=O
I
CH3
pyruvate
+
Normal values:
N Age ALT (U/L) AST (U/L)
1 Adults 7-56 5-40
2 Children 10-35 10-50
3 Newborn 6-50 35-140
• Elevation of serum Dtotal aminotransferases > 1000 U/L occurs exclusively in disorders ass
ociated with extensive hepatocellular injury such as:
(i) viral hepatitis
(ii) toxin or drug-induced liver injury
(iii) Ischemic liver injury (acute heart failure).
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6. Determination of AST and ALT elevation can be helpful diagnostically
N Liver diseases Total
aminotransferases
AST ALT
1.
Acute hepatocellular necrosis
(hepatitis)
Elevated, often > 500
IU
ALT >>> AST
2. Alcoholic liver disease AST/ALT ratio > 2.0-
3.0
AST > 300 IU/L ALT - normal
or decreased due to alcohol
induced deficiency of Vit. B6,
requires for the activity of liver
ALT
3. Chronic hepatocellular disease Elevated,
But usually < 300
elevated elevated
4. Intra- end extrahepatic cholestasis
(obstructive jaundice)
Normal to moderate
elevation
Normal to
mild
elevation
Mild to
moderate
elevation
Serum y-glytamyl transferase (transpeptidase) -GGT or y-GT
GGT is present mainly in: liver, kidney, prostate, spleen.
GGT catalyses the transfer of the Y-glytamyl group from one peptide to another p
eptide or an amino acid.
A B
GGT
B A
+
'ySSs
CH2
CH2
COOQ
Normal values:
Men: 5-85 U/L
Women: 5-55 U/L
CH2
CH2
CoO^
• The Y-GT activity is elevated in all forms of liver disease
• The estimation of Y-GT used to determine liver cell dysfunction and the detect alcohol ind
uced liver disease (chronic alcoholism) and infiltrative diseases (metastasis, tumor).
# 2 ENZYMES THAT REFLECT CHOLESTASIS
The activities of three enzymes - ALP and 5'nucleotidase, Y-GT are usually elevated in ch
olestasis.
5'nucleotidase hydrolyzes nucleotides with a phosphate group on carbon atom 5' of the rib
ose:
H2O
NH
5'nucleotidase
PI
N
V'A
? n—
n
HO
OH OH
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7. • ALP and 5'nucleotidase are found in or near the bile canalicular membrane of hepatocytes,
while Y-GT is located in the endoplasmatic reticulum and in bile duct epithelial cells.
• 5'nucleotidase and Y-GT are rarely elevated in conditions other than liver disease. 5'nucle
otidase:
Normal: 0 to 5 IU/L
N Liver diseases ALP total ALP-1 5'nucleotidase Y-GT
1. Acute hepatocellular
necrosis
(hepatitis)
Normal elevated Normal to slight
elevated
Elevated
2. Alcoholic liver disease Normal to < 3
times normal
elevation
elevated normal Elevated
(often > 4
times
normal)
3. Chronic hepatocellular
disease
Normal to < 3
times normal
elevation
elevated Normal to slight
elevated
elevated
4. Intra- end extrahepatic
cholestasis (obstructive
jaundice)
Elevated, often >4
times normal
elevation
elevated elevated elevated
5 Infiltrative disease (tumor,
metastasis)
Elevated, often >4
times normal
elevation
elevated elevated elevated
3. SERUM ENZYMES IN BONE AND MUSCLES DISEASES
MYOPATHIES
• Skeletal muscles disorders or myopathies are defined as disorders with structural changes
or functional impairment of muscles.
• CK-MM is the muscle enzyme and its serum concentration is elevated in different types of
myopathies.
E.g. Hereditary myopathies - progressive muscular dystrophies (progressive weakness, progressiv
e kyphoscoliosis, impaired pulmonary function, acute gastric dilation, etc). Different types of mu
scular dystrophies are exist: Duchenne, Emery-Dreifuss, Becker etc. with different types of mutat
ion of proteins, play important role in the muscular contractile, stability, etc.
In all these types serum CK-MM greatly elevated ( > 20 -100 times normal).
Different types of muscular dystrophies may be identified by new method: analysis of mutation o
f DNA.
BONE DISEASES
ALP (alkaline phosphatase)
• The enzyme is a monomer and the isoenzymes are due to the difference in the carbohydrate c
ontent:
• ALP 1 (Alpha1-ALP) liver, blood vessels
• ALP 2 (Beta 1- ALP) bone (osteoblasts)
• ALP 3 (Beta 2- ALP) intestinal epithelium, kidney
• ALP 4 (Gamma-ALP) placenta
Diagnostic importance of ALP:
• Increased ALP-2:
(i) Bone diseases: Paget' s disease, Osteoblastic bone tumors, Osteomalacia, Rickets, Skeletal dis
ease
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8. (ii) Obstruction of bile duct, diseases of bile duct
ALP-2 osteosarcoma
• ALP-2 of osteosarcoma cells binds with specific substrate (ELF - end
ogenous light fluorescent substrate) forms a fluorescent precipitate at t
he site of enzymatic activity (blue). For contrasting of these sites uses
green fluorescent precipitation of ELF with alcohol.
TUMOR MARKERS
Tumors markers- enzymes Cancer Non-Neoplastic condition
Acid phosphatase (pH=5.0) Prostate cancer
Prostatitis, prostatic hypertrophy
Total LDH Lymphoma Hepatitis, many other
Neuron-specific enolase Small cell cancer of the lung
afetoprotein Hepatocellular carcinoma Cirrhosis, hepatitis
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