2. • Diagnostic indicators – the activities of many enzymes are
routinely determined in plasma ( rarely in tissue biopsies) for
diagnostic purposes in diseases of the heart, liver, skeletal
muscle, pancreas and other tissues - enzyme diagnostics
• Therapeutic agents – several enzymes are used as drugs;
new approach - enzymotherapy
•
Diagnostic tools – use as chemicals in clinical laboratory
assays
3. ENZYMES IN CLINICAL DIAGNOSIS
plasma –
secretory - produced by tissues (namely
choline
Enzymes
use in
liver), acting in
prothrombin, plasminogen, cerruloplasmin,
esterase; lipoprotein lipase
intracellular – function intracellulary, have no physiological
plasma
- membrane bound – ALP, GMT
- cytosolic –
ALT, AST, LD, MDH
- mitochondrial –
AST, GMDH
- lysosomal ACP
- tissue specific – glucose-6-phosphatase – liver
amylase – pancrease
LD1 – heart
4. • Healthy individuals - levels of intracellular enzymes fairly constant, low
–
the rate of enzyme release from damaged cells into plasma balanced by
the rate of removal of enzyme protein from plasma
⇓
Physiological enzyme levels ⇒ reference values of the enzyme
activities
lab
(determined in clinical laboratory – each
has its own reference values)
• Elevated enzyme activity in the plasma – reflect tissue damage
accompanied by increased release of intracellular enzyme
Skeletal muscle during exertion – physiologically elevated levels of
muscle enzymes in plasma
• Many diagnostically important enzymes = isoenzymes – pattern of
isoenzymes in plasma (determined electroforetically)
– a means of identifying the damaged tissue
5. ALTERATION OF ENZYME PLASMA LEVELS
Increased values – increased cell membrane permeability
anoxia, disturbances of energy metabolism ⇒
cytosolic enzymes – ALT, LD, CK
- cell necrosis ⇒ membrane-bound enzymes – ALP, GMT
mitochondrial enzymes – AST, GMDH
- induction of the enzyme synthesis ← drugs – ALP, GMT
Decreased values – inhibition of the activity ← drugs
- inhibition of the synthesis ← cell damage, drugs
6. Examples of enzymes commonly assayed for diagnostic
purposes
Enzyme
level
Location
Cause of elevated plasma
Acid phosphatase - ACP
Prostate
Prostatic cancer
Alkaline phosphatase – ALP
hypoparathyroidism,
Bone, liver
Rickets,
osteomalacia, obstructive
jaundice, cancer of
bone/liver
Alanine aminotransferase – ALT
circulatory
Liver (muscle,
heart, kidney)
Aspartate aminotransferase – AST Heart, muscle,
muscle
red cells, liver
liver
Amylase - AM
ulcer
γ-Glutamyl transferase – GMT
Hepatitis, jaundice,
faillure with liver congestion
Myocardial infarction,
damage, anemia, hepatitis,
circulatory faillure with
congestion
Pancres
Liver, kidney,
pancreas
Acute pancreatitis, peptic
Hepatitis, alcoholic liver
damage, cholestasis
8. ENZYMES IN THERAPY
• Substitution of missing production of digestive enzymes –
digestive enzymes – pepsin trypsin…
• Removal of deposits of death tissue or fibrin (e.g. in lungs, eyes),
treatment of skin defects – proteinases, nucleases, collagenase
• Acceleration of fibrinolysis in lungs embolization (activation of
plasmin
and plasminogen) – streptokinase, urokinase
9. ENZYMOTHERAPY
Orally administered enzymes – treatment of a variety disorders
- digestive, gastrointestinal, pancreatic
- inflammatory diseases, edema
- immune and autoimmune diseases
(arthritis, multiple sclerosis)
- viral diseases (herpes, AIDS)
- cancer
Mixtures of enzymes of plant and/or animal origin - proteinases,
amylase, lipase - administered as acidoresistent tablets
• Pancreatin – trypsin, chymotrypsin, lipase, amylase
• Wobenzym – pancreatic and plant proteolytic enzymes – trypsin,
=
optimum,
chymotrypsin, papain (Carica papaya), bromelain (ananas)
combination of enzymes with different specificity, pH
stability, interaction with inhibitors and antiproteinases
⇒ multiple action
10. • Mechanism of resorption (transport of large macromolecules
across the intestinal barrier) – paracellular transport, receptor
mediated endocytosis and transcytosis
• Mechanism of action – interaction with plasma antiproteinases –
→complexes
?
factor
α1-antitrypsin, α2-macroglobulin
direct proteolytic action, degradation of adhesive
molecules, secretion of cytokins (tranforming growth
TGF-β), modulation of receptor function
not fully clarified
11. ENZYMES - USE IN LABORATORY ASSAYS
Enzymes isolated from different sources - used for determination of various
substances in the blood, plasma/serum and urine ← enzyme methods
much more specific than chemical methods, the presence of relative
substances with similar chemical properties does not hinder
Components of commercial kits or diagnostic strips
- determination of glucose - glucose oxidase, peroxidase
cholesterol - cholesterol esterase, cholesterol oxidase
peroxidase,
urea – urease, …….
in blood, plasma, serum
- proof of glucose (glucose oxidase), ……..
in blood or urine (strips)
Markes in the immunochemical analysis
- ELISA (=enzyme-linked immunoadsorbent assay) – peroxidase, alkaline
phosphatase
15. THE NITROGENOUS BASES
Purine bases
NH 2
|
C
N
O
||
C
adenine
adenine
N
C
HN
CH
HC
C
N
C
CH
C
N
H
N
guanine
guanine
H 2N
C
N
H
N
Pyrimidine bases
O
||
C
NH 2
|
C
N
O
C
CH
N
H
cytosine
cytosine
CH 3
N
H
O
C
C
CH
O
||
C
CH
N
H
thymine
thymine
HN
CH
C
C
H
O
N
H
uracil
uracil
16. NUCLEOSIDE
• A sugar - base
combination.
Base
Base
N
O
HOCH 2
H
Sugar
Sugar
In this case
In this case
deoxyribose
deoxyribose
H
H
OH
H
H
β-N-glycosidic
β-N-glycosidic
linkage
linkage
19. NUCLEOTIDES
5’-OH on the sugar of a nucleoside is converted into a
phosphate ester.
NH 2
|
C
deoxyadenosine monophosphate
deoxyadenosine monophosphate
(dAMP)
(dAMP)
N
Each is named based on
sugar and base name
and then the number of
phosphates is indicated.
C
N
CH
HC
O
||
-
O-P-O-CH 2
|
O- - H
C
N
N
O
H
H
OH
H
H
20. ATP - adenosine triphosphate
adenine
phosphate chain
O
-
O
P
O-
O
O
P
O-
NH2
O
O
N
P
O
O-
CH2
N
O
OH
ADP
ATP
N
AMP
ribose
OH
N
21. NUCLEOTIDE FUNCTION
• Precursors of DNA, RNA - NTPs
• Energy transport
-
ATP
• Allosteric effectors of enzymes – ATP, ADP, AMP
• Covalent modification of enzymes – ATP
• Intracellular mediators (= second messengers) – cAMP,
cGMP
• Coenzymes – NAD+, NADP+, FAD, CoA-SH
• Activated precursors of polysaccharaides,
glycoproteins, proteoglycans, phospholipids,
glycolipids – UDPG, UDPGA, UDPGal…, CDP-choline,
CDP-diacylglycerol…
• Active groups (group transport) – SAM, PAPS
28. NH 2
|
C
N
C
N
CH
HC
C
N
O
CH 2
-
O
O
P
H
H
O
N
H
H
O H
O-
cAMP – cyclic adenosine monophosphate
-intracellular mediator, second messenger of hormonal signal tranduction
via adenylate cyclase cascade
- mechanism of action: allosteric effector