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ClinicalEnzymology
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ENZYMES
Organic catalyst that hasten a chemical reaction without
themselves being consumed or undergoing a chemical change.
Protein in nature with a high degree of specificity for a certain
substrate or class of substrate
SUBSTRATE
Are substances that are acted upon by enzymes
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Enzyme Nomenclature
Enzymes are named acc to the ff:
A. Name of substrate with the addition of the
suffix ase
Ex.
Lipid-lipase
Proteins-protease
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B. Type of reaction they catalyse
Ex.
Transferase
transfer of AMINO GROUP from one substrate to another
Kinase
transfer of PHOSPHATE GROUP from a high energy phosphate
compound to its substrate
Phosphatase effect of hydrolysis on phosphate esters
Dehydrogenase
removal of hydrogen atoms from its substrate
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C. Numerical designation given by the Enzyme
Commission(E.C.)
Ex.
E.C. 1.1.127 Lactate dehydrogenase
E.C. 3.2.1.1 Amylase
1stnumberclass to which the enzyme belongs
2ndand 3rdnumbersub class
4thspecific serial number of the enzyme
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GENERAL CLASSIFICATION OF ENZYMES
Based on the reaction mechanism each enzyme
catalyzes
1. OXIDO-REDUCTASES
removal or addition of electrons
Reduction-oxidation(Redox) reaction
Oxidation-loss of electron
Reduction-gain of electron
Ex.
Removal of hydrogenDehydrogenase
Lactate dehydrogenase
Glucose-6-phosphate dehydrogenase
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Lactate dehydrogenase
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2. TRANSFERASES
Catalyze the transfer of a chemical group from
one substrate to another
Ex.
Aminotransferase -Amino group
Aspartate aminotransferase
Alanine aminotransferase
Kinase/phosphokinase -Phosphate group
Creatine kinase/creatine phosphokinase
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AST-Aspartate Aminotransferase
SGOT-Serum Glutamate Oxaloacetate Transaminase
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ALT-Alanine Aminotransferase
SGPT-Serum Glutamate Pyruvate Transaminase
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3. HYDROLASES
Hydrolyze the splitting of a bond by the addition of
water(hydrolysis reaction)
Ex.: ALP Alkaline phosphatase
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4. Lyases
Removes groups from substrate without hydrolysis
The product contains double bonds
Ex. Aldolase
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5. ISOMERASES
Intramolecular rearrangement of the substrate compound
Same molecular formula but different physical structure
6. LIGASES
Synthethases
Joins two substrate molecules together
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Terms associated with enzymes:
HOLOENZYMES
Active substance formed by the combination of
coenzyme(cofactor) and apoenzyme
HOLOENZYME
APOENZYME COFACTOR
(PROTEIN MOIETY) (NON-PROTEIN MOIETY)
COENZYME
ACTIVATOR
VITAMINS METAL IONS
NAD Zn+2
NADP Ca+2
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APOENZYME
Protein portion of an enzyme
Subject to denaturation, in which enzyme loses its activity
Catalytically inactive protein when cofactor is removed
Heat labile and dialyzable
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COFACTOR
Non-protein subs/compounds needed by an enzyme before
enzymatic activity can be manifested
Thermostable and dialyzable
2 types:
A. Coenzyme
B. Activator
A. Coenzyme
Organic molecule
It hasten enzymatic reaction but undergoes a change or is
consumed to another product
Ex.
NAD-Nicotinamide Adenine Dinucleotide
NADP-Nicotinamide Adenine Dinucleotide Phosphate
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B.Activator
Metal ion
In such, the metal ion may serve as:
A. A bridge to hold the substrate and enzyme together
B. The primary catalytic center
C. As stabilizing agent in the conformation for catalytic activity
Ex.
Amylase Cl-, Br
LDH- Zn+2
Lipase Ca+2
CPK Mg+2
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ISOENZYME
Enzymes present in an individual with similar enzymatic activity
but differ in their physical biochemical and immunological
characteristics
Ex. LDHLactate dehydrogenase
LDH 1
LDH 2
LDH 3
LDH 4
LDH 5
CKCreatine kinase
CK-MB
CK-MM
CK-BB
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METALLOENZYME
Enzyme whose metal ions are intrinsically part of the molecule
such as catalases and cytochrome oxidase
PROENZYME Inactive precursor of enzymes
Also referred to as zymogen
SUBSTRATE Substances acted upon by the enzyme which are specific for ach
of heir particular enzymes
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ENZYME KINETICS
An enzyme(E) catalyses a reaction by combining with its substrate(S)to create an enzyme-substrate complex(ES).
E + S ---------ES
The ES complex according to Michelis and Menten can either:
dissociate back to E + S
breakdown to product(P) and free enzyme(provided that the producthas a low affinity for the enzyme)
K2 E + S ---------ES--------E + S
K3
E + S ---------ES--------P + E
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ENZYME SPECIFICITY
Theories that explain the high degree of specificity of an enzyme for
their particular substrate or class of substrate:
1. Emil Fischers LOCK and KEY THEORY:
Rigid enzyme molecule into which the substrate fits The shape of the key(substrate) must conform into the lock(enzyme)
2. KochlandsINDUCED FIT THEORY
It is based on the attachment of a substrate to the active site of an
enzyme, which then causes conformational changes in the enzyme.
This theory is more acceptable because the protein molecule is
flexible enough to allow conformational changes and also allows
some explanation on the influence of hormones on enzymatic
activity.
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TYPES OF REACTION ORDER:
1. Zero Order Reaction
The rate of reaction is linear with the time,
independent of concentration of the substrate and
directly proportional to enzyme concentration.
2. First Order Reaction
The rate of reaction is determined by the
concentration of substrate as well as of enzymes(therate of reaction changes continuously with time as the
substrate is consumed.
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FACTORS AFFECTING ENZYME REACTIONS:
1. Enzyme concentration
in enzyme concentration,in rate of reaction
2. Substrate concentration
in substrate concentration, in rate of reaction Upon reaching maximal value of conc. of substrate, it does
not result in increased rate of reaction.
3. Temperature
For every 10 C rise in temp, results to 2-3 times increase inthe rate of reaction
37-40 Coptimum temperature for enzyme activity
>40 C-proteins undergo denaturation and loses its
functional ability
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4. Hydrogen ion concentration or pH
some enzymatic reactions proceed at their fastest rate at an
optimum pH.
Extreme pH causes denaturation of enzymes.
Ex.
Pepsin--- active at pH 2.0
Alkaline phosphataseactive at pH 8.6-10
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INHIBITORS:
COMPETITIVEINHIBITOR
Substances that compete with the substrate for enzyme binding
because they are chemically analogous to the substrate and bind
to the active sites of the enzyme.
High substrate concentration will overcome the effect of theinhibitor.
NON-COMPETITIVE INHIBITOR
Substances that does not resemble the substrate and bind to the
enzyme in areas other than the active site.
They do not compete with the substrate.
Increasing the substrate concentration will not overcome the
inhibition.
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Units in measuring enzyme
activity International unit(IU)
Amount of enzyme that catalyzes the conversion of
1 micromole of substrate per minute under controlled condition
Katal unit(KU)
Amount of enzyme that catalyzes the conversion of 1 mole of
substrate per second under controlled condition
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Means in measuring enzyme
activity A. change in coenzyme concentration
B. Increase in product concentration
C. Decrease in substrate concentration
Called an inverse technique
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Pitfall in enzyme assays
Hemolysis causes falsely elevated values due to release ofenzymes from RBCs.
Serum rather than plasma is preferred specimen.
Anticoagulants have adverse effect on enzyme activity.
Lactascent or milky serum causes variable absorpton by thespectrophotometer.
Storage:
Most enzymes are stable at 6 for at least 24 hours.
For a longer period of time, temperature of -20 C or
lower are used to ensure preservation of enzyme activity CK must be kept at -70 C to retain activity.
For the LD4 and LD5, room temperature only, because it isinactivated at refrigerator temperature.
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Principles of diagnostic serum
enzymology CAUSES OF INCREASED SERUM ENZYME LEVELS:
Impaired removal of enzymes from plasma.
In pathologic conditions involving tissue necrosis and
degeneration.
Increased permeability of the cell membrane.
Physiological or pathological increase in the number of cells or
increase in the rate of production of cells.
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CAUSES OF DECREASED ENZYME LEVELS:
Increased removal of enzyme from the plasma.
Decreased synthesis due to organ impairment, injury or removal.
Malnutrition leading to decreased enzyme production.
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oxidoreductases
A. LDH
E.C.: 1.1.1.27
Recommended Name: Lactate dehydrogenase
Systematic name: L-Lactate: NAD+ oxidoreductase
Action:
Conversion of lactic and pyruvic acid
Hydrogen-transfer enzyme
Coenzyme: NAD
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EQUATION:
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Tissue source:
Heart, liver, skeletal muscle, kidney and RBCs
Lungs, smooth muscles, brain
Diagnostic Significance: Cardiac, hepatic, skeletal muscle, renal and hematologic dse.
Highest level is seen in pernicious anemia and Hematologic
disorders
In AMI, LDH rise within 12-24 hrs
peak within 48-72 hrs
remains elevated for 10 days
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Ldh isoenzymes
ISOENZYME TISSUE DISORDERS:
LDH-1
(heat
stable)
HHHH 14-26% Heart
RBC
MI
Hemolytic anemia
LDH-2
(heatstable)
HHHM 29-39% Heart
RBCRenal cortex
Megaloblastic anemia
Acute renal infarctionhemolysis
LDH-3 HHM
M
20-26% Lung
Lymphocytes
SpleenPancreas
Brain
Embolism
Pneumonia
LymphocytosisPancreatitis
CA
LDH-4
(heat labile)
HMM
M
8-16% Liver Hepatic injury
LDH-5
(heat labile)
MMM
M
6-16% Skeletal muscle Skeletal muscle injury
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LDH-6 Alcohol dehydrogenase
Increased in Arterosclerotic cardiovascular failure
Elevated level signifies grave prognosis and impeding death
Techniques in measuring LD isoenzymes: A. Physical
Electrophoresis
Selective absorption on Diethylaminoethyl cellulose(DEAE)Solvent precipitation technique
Heat denaturation at 65 C for 30 mins
B. Chemical Substrate-product relationship
Coenzyme affinty
Differential inhibition of LD activity C. Immunological test
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Sources of error:
Hemolysis
Unstable serum
Spx should be stored at 25 C and analyzed within 48 hrs
LDH-5most labile
Clinical Significance:
LD levels are markedly increased in the ff:
Megaoblastic anemia Pulmunary infarctionGranulocytic leukemia Hodgkins Dse
Hemolytic anemia Infectious mnonucleosis
Progressive Muscular Dystrophy(PMD)
l h h
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Glucose-6-phosphate
dehydrogenase E.C.: 1.1.1.49
Recommended name: Glucose-6-Phosphate Dehydrogenase
Systematic name: D-glucose-6-phosphate: NADP+ 1-
oxidoreductase
Action: oxidation of glucose-6-phosphate to 6-
phosphogluconate
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Tissue Source:
Adrenal cortex, spleen, thymus, lymph nodes, lactating
mammary glands and RBCs
Diagnostic Significance:
RBC
NADPH is converted into glutathione
Glutathione
Anti-oxidant
protects hgb from oxidation
Dec. G6PD=dec. NADPH=dec. Gluthathione
Dec. Gluthathionemay cause hemolysis/damage to cell membrane
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Decreased G6PD
G6PD deficiency
Inherited sex-linked trait
May cause drug-induced hemolytic anemia/exposure to oxidizing
substances
Increased G6PD
Myocardial Infarction and Megaloblastic anemias
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Assays:
Specimen used:
Deficiency of enzymered cell hemolysate
Elevation of enzyme levelserum
Reference Range:
10-15 u/g hgb
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TRANSFERASE
AST
E.C.: 2.6.1.1
Recommended name: Aspartate aminotransferase
Systematic name: L-aspartate:2-oxaloglutarate
aminotransferase
Action:
Transaminase-synthesis and degradation of amino acid
Transfer of amino group between aspartate and a-ketoglutarate
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Tissue source:
Cardiac muscle, liver, skeletal muscle
Kidney, pancreas, RBC
Diagnostic Significance:
Hepatocellular and Skeletal Dse.
AMI
AST rise at 6-8 hrs
Peak at 24 hrs
Return to normal within 5 days
Decreased in
pregnant women
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Assays: Rietmann-Frankel Method
Substrate: aspartic alpha ketoglurate
Color developer: 2,4 dinitrophenyl hydrazine
End Product: glutamic acid and oxaloacetic acid
End color: brown
Karmen Method
Indicator: Malate dehydrogenase
Absorbance: 340 nm
pH: 7.3-7.8
Babson and Read Method
Color developer: diazonium salt
End color: violet
Source of error:
Hemolysis Falsely increased results
Alcohol lowers AST values
Muscle trauma like intramuscular injectiions, exercise, or surgical operation can significantly increase ATS levels
Reference Range: 5-30 u/L
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ALT
E.C.: 2.6.1.2
Recommended name: Alanine aminotransferase
Systematic name: L-alanine: pyruvate aminotransferase
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Tissue source:
Liver
Liver-specific enzyme
Diagnostic significxanc:
Hepatic Disorders
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Assay:
Reitman and Frankel Method
Substrate: alanine alpha ketoglutarate
End Product: glutamate and pyruvic acid
Walker method
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Assay:
Indicator: LDH
Absorbance:340 nm
pH: 7.3-7.8
Source of error:
Stable for 3-4 days at 4 C
Reference
range: 6-37 u/L
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CK
E.C.: 2.7.3.2
Recommended name: Creatine kinase, creatinephosphokinase
Systematic name: ATP: Creatine N-phosphotransferase
Action:
Associated with ATP regeneration in contractile and transportsystems
Physiologic Function:
Muscle cells, creatine phosphate
Production of ATP
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Ck isoenzymes
ISOENZYME LOCATION
CK-1 CK-BB(BRAIN) BRAIN, BLADDER, LUNG,
PROSTATE, UTERUS,
COLON, STOMACH,
THYROID
CK-2 CK-MB(HYBRID) CARDIAC, SKELETALMUSCLE
CK-3 CK-MM(MUSCLE) CARDIAC, SKELETAL
MUSCLE
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CK-BB
Highest concentrations in CNS, GI tract and uterus
Elevated in px with CA
Useful tumor-associated marker
Increased in CNS damage, tumors, child-birth
CK-MB
Major isoenzyme found in striated muscle and normal serum
Elevated in cardiac disorders
In AMI, CK rise at 4-8 hours
Peak in 12-24 hrs
Return to normal within 48-72 hours
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Macro-CK
Midway between CK-MM and CK-MB
CK-Mi(mitochondrial CK)
muscle, brain, liver
Assay:
Electrophoresis
Reference method used for measurement of isoenzymes
Ion-exchange Chromatography
Radio Immunoassay(RIA)
Immuno-inhibition
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GGT
E.C.: 2.3.2.2
Recommended name: Gamma-glutamyl transferase
Systematic name: (5-glutamyl) peptide: amno acid-5-glutamyl
transferase
Action: transfer of gamma-glutamyl residue from gamma-glutamyl peptides to amino acid, H2O and other smallpeptides
Involved in peptide and protein synthesis, regulation of tissuegluthathione levels and transport of amino acid across cellmembrane
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Tissue source:
Kidney, brain, prostate, pancreas and liver
Diagnostic significance
Liver
Biliary ductules
Hepatobiliary disorders
Increased in patients taking drugs such as warfarin, phenobarbitaland phenytoin
Alcoholism
GGT assays:
Useful in monitoring the effects of abstention from alcohol and areused by alcohol treatment centers
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Assay:
Substrate: gamma glutamyl-p-nitroanilide
Absorbance: 405 to 420 nm
Source of error:
Stable for 1 week at 4 C
Reference Range:
Male: 6-45 u/L
Female: 5-30 u/L
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Optimum pH: 9.0-10.0 Activator: Mg 2+
Tissue source:
Intestines, liver, bone, spleen, placenta, kidney, RBC
Diagnostic Significance:
Increased in:
Hepatobiliary and Bone disorders
Obstructive conditions
Pagets dse, Osteomalacia, Ricketts
Pregnancy
Decreased in:
Hypophosphatasia
Malnutrition
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Considerations in ALP assays:
Physiologically elevated in growing children and in pregnant
women in the third trimester
EDTA inactivates ALP owing to chelation of magnesium ions
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Alp isoenzymes
ISOENZYME
LIVER FASTEST
BONE HEAT LABILE
PLACENTA HEAT STABLE
INHIBITED BY PHENYLALANINE
INTESTINES SLOWEST
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Regan-Nagao Isoenzyme
Carcino-placental ALP
Detected in various CA(lung, breast, ovarian and colon)
Assay:
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Assays:
A. Substrate: beta-glycerophosphate
End Product: inorganic phosphate+glycerol
1. Bodansky
2. Shinowara3. Jones
4. Reinhart
B. Substrate: phenylphosphae
End product: phenol
1. King and Armstrong
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C. Substrate: p-nitrophenyl phosphate
Product: p- nitrphenol or yellow phenoxide ion
1. Bessey, Lowry and Brock
2. Bowers and Mc comb
D. Substrate: phenolphthalein diphosphateProduct: phenolphthalein red
1. Huggins and Talalay
E. Substrate: alpha naphthol phosphate
Product: alpha naphtol
F. Substrate: Buffered phenolphthalein phosphate
End product: free phenolphthalein
1. Klein, Babson and Read
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Klein, Babson and Read
Substrate: Buffered phenolphthalein phosphate
End product: free phenolphthalein
Color developer: NaOH
End color: pink
Bessey, Lowry and Brock
Substrate: p-nitrophenyl phosphate
Product: p- nitrphenol or yellow phenoxide ionColor developer: 0.02 N NaOH
End color: yellow
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ACP
E.C. : 3.1.3.2
Recommended name: Acid phosphatase
Systematic name:
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Tissue source:
Prostategland
RBC, Platelet, liver, spleen, milk, bone marrow
To differentiate Prostatic ACP and RBC ACP inhibitors areadded:
L-tartrate ions; Prostatic ACP
Formaldehyde and Cupric ions: RBC and Platelet ACP
Total ACP-ACP after the addition of L-tartrate= Prostatic ACP
Optimum pH: 4.9-5.0
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Assays:
Methods: Substrate End product
1. Bodansky Beta-glycerophosphate Inorganic phosphate
2. Gutman and Gutman Phenyl phosphate phenol
3. Fishman and Lerner Phenyl phosphate phenol
4. King Armstrong PNPP P-nitrophenol
5. Shinowara PNPP
6. Bessy, Lowry annd
Brock
PNPP
7. Babson, Read and
Phillips
Alpha naphthyl
phosphate
Alpha-napthol
8. Roy and Hillman Thymolphthalein
monophosphate
Free thymolphthalein
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Roy and Hillman
Thymolphthalein monohosphate
Most specific substrate for ACP
Color developer: Na OH- Na Carbonate solution
End color: blue
Babson, Read and Phillips
Color developer: tetra azotized orthodianisidine
End color: brown
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Source of error:
Serum ACP is higher than plasma ACP
Hemolysis
Diagnostic significance:
Increased in:
Metastatic prostate carcinoma
A. moderate elevation of TOTAL ACP
Female breast CA
Pagets Dse
Hyper parathyroidism
B. Non-Prostatic ACP elevation
Niemann-Picks dse
Gauchersdse
Myelocytic leukemia
Also present in seminal fluid Used in forensic cases such as in rape
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Reference range:
Prostatic ACP
0-35 IU/L
Total ACP Male: 2.5-117 IU/L
Female: 0.2-3.5 IU/L
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AMS
E.C. : 3.2.1.1
Recommended name: a-amylase
Systematic name: 1,4-D-glucan glcanohydrolase
Action:
Breakdown of starch and glycogen
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2 isoenzymes:
1. Salivary
Ptyalin(fast)
2. Pancreatic
Amylopsin(slow)
Starch/glycogen glucose, maltose, dextrins
Activators:
Ca2+
Cl-
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Tissue source:
Pancreas, salivary glands
Skeletal muscles, small intestine, fallopian tube
Diagnostic Significance:
Acute pancreatitis
Levels reaches 4-6 times the normal value and normalizes
within 3-4 days
Mumps, perforated petic ulcers, appendicitis, ruptured ectopicpregnancy, dissecting aortic aneurysm, biliary tract disease
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Assay for enzyme activity: Amyloclastic
Decrease in substrate concentration/disappearance of starch
Saccharogenic
Amount of reducing sugars produced
Chromogenic
Measures increasing color from production of product coupled withchromogenic dye
Continous monitoring
Coupling of several enzyme system to monitor amylase activity
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Reference Range:
Serum:
25-130IU/L
Urine
1-15 IU/L
Considerations in AMS assays:
Mouth pipetting is not done
Lipemic specimens cause a reduction in amylase activity
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LPS
E.C. : 3.1.1.3
Recommended name: Lipase
Systematic name: Triacyl glycerol acylhydrolase
Action:
Hydrolisis of esters in the alpha-position to yield beta-
monoglycerides and fatty acids
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Tissue Source: Pancreas, stomach and small intestine
Diagnostic Significance:
Acute pancreatitis
Assay for enzyme Activity:
Cherry-Crandal
Substrate: olive oil
Indicator: phenolphthalein
End color: pink
Incubation period: 24 hrs
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Tietz and Templaton Indicator: Thymolphthalein
End coor: blue
Incubation period: 4-6 hrs
Source of error:
Hemolysis
Falsely decreased result
Hgbacts as inhibitor for enzymatic activity
Reference Range:
0-1.0 IU/mL
1.0-1.5 cherry-crandal unit
l
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lyases
ALDOLASE
E.C.: 4.1.2.13
Recommended name: Fructose diphophate
Systematic name: D-D-fructose-1, 6-bidiphosphate, D-
glyceraldehyde phosphate lyase Action:
Spitting of D-fructose di phosphate to D-glyceraldehyde
phosphate and dihydroxy acetone phosphate
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3 isoenzymes:
A. Aldolase A- skeletal muscle
B. Aldolase B- liver, kidney and WBC
C. Aldolase C- brain
Assay:
Pinto, Kaplan and Van Dreal
Sibley and Lehninger
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Diagnostic Significance:
Severe:
Muscle degradation
Viral hepatitis
Moderate:
Gangrene
Megaloblastic anemia
Granulocytic leukemia
Metastatic liver CA
Psychosis
Trichinosis