INTRODUCTION TO CLINICAL CHEMISTRY
Outline
1. Definition 2. Cells3. Role of the Clinical Chemistry Laboratory4. Role of the Technologist
Definition
Clinical Chemistry A basic science that utilizes the specialty of
chemistry to study human beings An applied science when analyses are
performed on body fluids or tissues for diagnosis or treatment of disease
Outline
1. Definition 2. Cells3. Role of the Clinical Chemistry Laboratory4. Role of the Technologist
Composition of Cells
Malfunction
1. Trauma or by invasive agents2. Genetic deficiency of a vital enzyme3. Insufficient supply of essential nutrients4. Insufficient blood and oxygen supply5. Malignancy6. Accumulation of waste products7. Defect in the cellular recognition of signals
Outline
1. Definition 2. Cells3. Role of the Clinical Chemistry Laboratory4. Role of the Technologist
Role of CC Laboratory
Measure chemical changes in the body for diagnosis, therapy and prognosis of disease
Role of CC Laboratory
Measure the concentration of a particular constituent (the analyte) in body fluids
Enzymes (ALT, AST, GGT, CK, LD, etc.)Electrolytes (Na, K, Ca, etc.)Trace Elements (Iron, Mn, Zn, Cu, etc.)Blood Buffering System (H2CO3, HCO3)Liver Secretion and Excretions
Outline
1. Definition 2. Cells3. Role of the Clinical Chemistry Laboratory4. Role of the Technologist
Role of the Medical Technologist
Role of the Medical Technologist
Must understand the tools: Equipment Reagents Principle of the testing methods Knowledge of the medical uses of the determinations
Outline
1. Definition 2. Cells3. Role of the Clinical Chemistry Laboratory4. Role of the Technologist
ENZYMES
Objectives
I. Describe the different factors affecting the enzyme reaction
II. Explain what is meant by zero order kinetics.III. Explain the difference between enzyme activity
and enzyme massIV. Describe and select parameters for optimal
measurement of enzyme activity
Objectives
VI. Discuss why the measurement of a serum enzymes is clinically useful.
VII. Discuss which enzymes and/or isoenzymes are useful in the diagnosis of myocardial infarction, liver disease, and acute pancreatitis.
Chapter Outline
I. IntroductionII. General Properties and DefinitionsIII. Enzyme Classification and NomenclatureIV. Enzyme KineticsV. Enzymes of Clinical Significance
Chapter Outline
I. Introduction Enzyme
i. Biologic proteins that catalyze biochemical reactionsii. Not consumed or changed in compositioniii. Found in all body tissue (intracellular) and is ↑ in
serum after cell injury
Chapter Outline
Chapter Outline
I. Introduction Function of Enzymes
i. Hydration of Carbon Dioxide (respiration)ii. Nerve Inductioniii. Muscle Contractioniv. Nutrient Degradation (Digestion)v. Growth and Reproductionvi. Energy Storage and Use
Chapter Outline
I. IntroductionII. General Properties and DefinitionsIII. Enzyme Classification and NomenclatureIV. Enzyme KineticsV. Enzymes of Clinical Significance
Chapter Outline
II. General Properties and DefinitionsA. Components of an EnzymeB. Terms associated with an enzyme
Enzymes
II. General Properties and DefinitionsA. Components of an Enzyme
i. Active Site A cavity of an enzyme where substrates bind
and undergo a chemical reaction. ii. Allosteric Site
A cavity other than the active site that binds regulatory (effector) molecules.
Enzymes
II. General Properties and DefinitionsA. Components of an Enzyme
Allosteric promoter
Allosteric Inhibitor
Chapter Outline
II. General Properties and DefinitionsA. Components of an EnzymeB. Terms associated with an enzyme
Enzymes
II. General Properties and DefinitionsB. Terms associated with enzymes
i. Substratesii. Cofactorsiii. Isoenzymeiv. Apoenzymev. Holoenzymesvi. Proenzyme or Zymogensvii. Allosteric enzymesviii. Inhibitors
Enzymes
II. General Properties and DefinitionsB. Terms associated with enzymes
i. Substrates Substances acted upon enzymes Specific for each of their particular enzyme
Enzymes
II. General Properties and DefinitionsB. Terms associated with enzymes
ii. Cofactors Non protein substances added in the enzyme
substrate complex to manifest the enzyme activitya. Coenzyme or Prosthetic group • An organic cofactor• Nucleotide (E.g. NAD, NADP) and Vitamins
b. Activator • An inorganic cofactor• Metal ion (E.g. Cl-1, Mg++,Cu+)
Enzymes
II. General Properties and DefinitionsB. Terms associated with enzymes
iii. Isoenzyme Similar enzymatic activity but differ in physical,
biochemical and immunologic characteristicsiv. Apoenzyme
The protein portion of the enzyme Subject to denaturation, in which enzyme losses its
activity
Enzymes
II. General Properties and DefinitionsB. Terms associated with enzymes
v. Holoenzyme An active substance formed by combination of a
co-enzyme and an apoenzyme.vi. Proenzyme or Zymogens
An inactive enzyme precursor E.g. Coagulation factors and digestive enzymes
EnzymesII. General Properties and Definitions
B. Terms associated with enzymesvii. Allosteric enzymes Regulator of cellular processes,
but not all enzymes are allosteric. Some can be allosteric provided
that they are composed of quaternary structures with two or more protein chain containing the active sites and regulatory sites (binding sites).
The substances that bind on the regulatory sites are called Regulator
Enzymes
Two kinds of allosteric enzymes: 1. Homoallostery. This is a cooperative substrate
binding and activation wherein substrate is a homotropic effector. Therefore the binding of substrate to one active site alters the substrate binding affinity and/or catalytic activity at other active sites on the multimeric enzyme.
Enzymes
Two kinds of allosteric enzymes: 2. Heteroallostery. This merely involves the
regulation byheterotropic effector molecules, which can be positive (activation) or negative (inhibition). These heterotropic effectors usually bind at a site other than the active site. Furthermore, these effectors can can activate or inhibit the activity of an enzyme..
Enzymes
viii. NHIBITORS OF ENZYMATIC REACTIONS An inhibitor is any compound that reduces the velocity of the enzyme-catalyzed reaction when present in the reaction mixture. Penicillin irreversibly (covalently) inhibits an enzyme involved in bacterial cell wall synthesis.
Ibuprofen and many other nonsteroidal antiinflammatory drugs (NSAIDs) are reversible competitive inhibitors of the cyclooxygenase activity of prostaglandin H2 synthase.
Enzymes
Inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme are said to be active site-directed (or competitive, as they 'compete' with the substrate for the active site).
Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non-active site-directed (or non competitive
KINDS OF INHIBITORS
1. Competitive Inhibition In competitive inhibition, a chemical inhibitor
competes for the active site with the substrates. The question immediately becomes:
Who gets to react with the active site - the inhibitor or the substrate? The answer to this query depends upon the affinity of the enzyme for the substrate and for the inhibitor. Often, the enzyme has a greater affinity for the inhibitor than it does for the substrate.
Examples of competitive inhibitors
In case of Methanol poisoning, it occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness.
Ethanol( an example of competitive inhibitor) is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. It is shown when ethanol is oxidized in preference to methanol.
Consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate.
Inhibitors
2. Uncompetitive Inhibition Occurs when the substrates fit into the active sites of the
enzyme but an inhibitor prevents the release of the product or to stop
enzyme from reacting with substrate to form the product It works well at higher substrate and enzyme concentrations
that substrates are bonded to enzymes. The formation of its binding site only forms when the enzyme and the substrate have interacted amongst themselves. It does not therefore work when additional substrates are trying to be involved.
The enzyme-substrate-inhibitor complex does not produce any product. The binding results in decreasing concentration of substrate binding to
enzyme
Inhibitors
Noncompetitive Inhibition It is rare but there are instances in which it may be encountered. It is a substance that interacts with the enzyme, but usually not at
the active site. It reacts either remote from or very close to the active site. The net effect of a noncompetitive inhibitor is to change the
shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction.
One good example of noncompetitive inhibitor is the nerve gases such as diisopropylfluorophosphate (DFP) that inhibits the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.
Chapter Outline
I. IntroductionII. General Properties and DefinitionsIII. Enzyme Classification and NomenclatureIV. Enzyme KineticsV. Enzymes of Clinical Significance
Chapter Outline
II. General Properties and DefinitionsA. Components of an EnzymeB. Terms associated with an enzyme
Chapter Outline
III. Enzyme Classification and Nomenclature1. Oxidoreductases2. Transferase3. Hydrolases4. Lyases5. Isomerases6. Ligases
Enzyme Classification and Nomenclature
The system for classification of enzymes that also serves as a basis for assigning code numbers to them.
These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning as appearing in example: for
Alcohol:NAD+oxidoreductase as EC number is 1.1.1.1 The first number shows to which of the six main divisions (classes) the
enzyme belongs, The second figure indicates the subclass, The third figure gives the sub-subclass, Tthe fourth figure is the serial number of the enzyme in its sub-subclass.
ENZYME NOMENCLATURE
CLASS RECOMMENDED NAME
ABBREVIATED NAME
E.C CODE NO.
SCIENTIFIC NAME
1. Oxido reductase
Lactate dehydrogenase
LDH 1.1.1.27
L-Lactate NAD+ oxidoreductase
2. Transferase 2.1 Aspartate amino transferase
SGOT ( Serum Glutamate Oxaloacetate transaminase)
2.6.1.1 L-Aspartate ,2-oxaloglutarate Amino transferase
2.2 Alanine aminotransferase
SGPT ( Serum Glutamate Pyruvate transaminase)
2.6.1.2 L-Alanine, 2-oxaloglutarate amino transferase
2.3 Gamma Glutamyl transferase
GGT 2.3.2.2 (5-Glutamyl ) peptide amino acid, 5- glutamyl transferase
2.4 Creatine kinase
CK 2.7.3.2 ATP-creatine, N-Phosphotransferase
ENZYME NOMENCLATURE
CLASS RECOMMENDED NAME
ABBREVIATED NAME
E.C CODE NO.
SCIENTIFIC NAME
3. Hydrolases Alkaline Phosphatase
ALP 3.1.3.1 Ortho-phosphoric, monoester phosphohydrolase (alkaline optimum)
Acid Phosphatase
ACP 3.1.3.2 Ortho-phosphoric, monoester phosphohydrolase (acid optimum)
α-Amylase AMS 3.2.1.1 1,4- D- Glucan, Glucanohydrolase
4. Lyase Aldolase ALD 4.1.2.13 αD –Fructose 1,6 Bis phosphate , D- glyceraldehyde, 3-phosphate lyase
5. Isomerase Triophosphate isomerase
TPP 5.3.1.1 Triose phosphate isomerase
6. Ligase Glutathione Synthetase
GSH-5 6.3.2.3 Gluthione Synthetase
Enzymes
III. Enzyme Classification and Nomenclature1. Oxidoreductases
Catalyze redox reaction between two substrates A- + B → A + B-
E.g: Dehydrogenase (Lactate Dehydrogenase)
Enzymes
III. Enzyme Classification and Nomenclature2. Transferases
Catalyze the transfer of a group (Phosphate, methyl, etc.) between two substrates (A-X + B → A + B-X)
E.g: Transferase (ALT, AST, GGT) and Kinase (CK)
Enzymes
III. Enzyme Classification and Nomenclature2. Transferases
Catalyze the transfer of a group (Phosphate, methyl, etc.) between two substrates (A-X + B → A + B-X)
E.g: Transferase (ALT, AST, GGT) and Kinase (CK)
Enzymes
III. Enzyme Classification and Nomenclature3. Hydrolases
Catalyze hydrolysis of various bonds A–B + H2O → A–OH + B–H E.g: Amylase (AMY), Lipase (LPS), Phosphatase (ALP, ACP)
+
Enzymes
III. Enzyme Classification and Nomenclature4. Lyases
Catalyze the removal of groups from substrates without hydrolysis; the product remain double bonds
ATP → cAMP + PPi Fructose biphosphate aldolase (ALS)
Enzymes
III. Enzyme Classification and Nomenclature5. Isomerases
Catalyze the interconversion of geometric, optical or positional isomers
A → B E.g. Triphosphate isomerase (TPI)
6. Ligases Catalyze the joining of two substrate molecules,
coupled with breaking of pyrophosphate bond in ATP Ab + C → A–C + b Glutathione Synthetase (GSH-S)
Chapter Outline
III. Enzyme Classification and Nomenclature1. Oxidoreductases2. Transferase3. Hydrolases4. Lyases5. Isomerases6. Ligases
Chapter Outline
I. IntroductionII. General Properties and DefinitionsIII. Enzyme Classification and NomenclatureIV. Enzyme KineticsV. Enzymes of Clinical Significance
ENZYME MECHANISMS
1. Lowering the activation energy. It is done by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate—by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).
ENZYME MECHANISMS
Providing an alternative pathway. This mechanism can be illustrated for example, temporarily reacting with the substrate to form an intermediate Enzyme-substrate (ES) complex, which would be impossible in the absence of the enzyme.
ENZYME MECHANISMS
Reducing the reaction entropy change. This can be illustrated by bringing substrates together in the correct orientation to react. Considering enthalpy change (ΔH‡) alone overlooks this effect. It is interesting to note that entropic effect involves destabilization of the ground state, and its contribution to catalysis is relatively small
1.Lock and key hypothesis
MODELS OF ENZYME ACTION
2. Induced Fit Hypothesis
. The change in shape is 'induced' by the approaching substrate molecule. This more sophisticated model relies on the fact that molecules are flexible because single covalent bonds are free to rotate.
CHARACTERISTICS AND PROPERTIES OF ENZYMES
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Enzymes
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymes
Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach
Enzymes
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymes
i. Relationship between Enzyme, Substrate and Product
Enzymes
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymes
i. Relationship between Enzyme, Substrate and Product
Enzymes
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymes
i. Absolute specificity Combines with only one substrate and catalyzes
only one reaction (E.g. CK, LD)ii. Group specificity
Combine with all substrates containing a particular chemical group (E.g. ACP, ALP)
iii. Bond specificity Specific to chemical bonds (E.g. AMY, LPS)
iv. Sterioisometric specificity Combine with one optical isomer (E.g. LDH, G6PD)
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
1. Substrate Concentration2. Enzyme Concentration3. pH4. Temperature5. Cofactors6. Inhibitors
Order of Reaction
the order of the reaction can be specified in terms of the order with respect to each specific reactant or the overall order of the reaction.
Consider the reaction mA + nB<===> C. The rate equation is R = k[A]m[B]n. If the exponent m in the equation is 1, then the reaction is said
tobe First order with respect to A. If m = 2, In then, 2A + 1B <===> 1C) then it is said to be second
order with respect to A and first order with respect to B. Now, if m = 1 and n = 1, since it is first order with respect to A and
B, then the overall order of the reactionis said to be Second order (or m + n).
HALF -LIFE
HALF-LIFE By definition, Half-life (t1/2) is the time required
for half of the original concentration of the limiting reactant tobe used up as the reaction takes place or half-life is equal to 0.69 /K.
Thus, the larger the rate constant (K), the faster will deplete the substrate.
As noted, in a first order reaction, the half-life is inversely proportional to the rate constant (k).
LOWERING ACTIVATION ENERGY
1. Increase the proximity of the reactants, 2. Increase the concentration of the reactants, 3. Increase the surface area of the reactants 4. Increase the temperature of the reactants, 5. Use a catalyst (a substance which speeds up a
chemical reaction but is not used up), 6. Use an enyzme.
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
1. Substrate Concentration First order kinetics (Michaelis- Menten hypothesis)
Reaction rate is proportional to the substrate concentration.
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
2. Enzyme Concentration Zero-order kinetics
Only a fixed number of substrate (in excess) is converted to product per second
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
Substrate and Enzyme Concentration First order and Zero Order kinetics
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
3. pH Common range 7.0-8.0 Controlled by buffers
4. Temperature Within ±0.1°C Enzyme is active
at 25°C, 30°C, 37°C.
Enzymes Sources Substrates Optimum pH
Sucrase Intestine Sucrose 6.2
Ribonuclease Pancreas 3’-5’0-Cytidylyl –adenine
7.0
Α- Glucosidase Yeast Methyl-α-D-glucoside
5.4
Acetylcholinesterase
Erythrocytes Acetylcholine 7.5
Enolase Rabbit Muscle 2-Phospho-D-Glycerate
6.8
Arginase Beef Liver L-Arginine 8.4-9.7
Pepsin Gastric mucosa Acetyl L-Phenylalanine , L-phenylalanine
1.5-2.5
Optimum pH of the Different Enzymes
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
5. Cofactors Activators: Metalic (Ca2+) and Non Metallic (Cl- ) Coenzymes (prosthetic groups): 2nd substrates (NAD)
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
6. Inhibitors
Chapter Outline
IV. Enzyme Kineticsii. Factors that Influence Enzymatic Reactions
1. Substrate Concentration2. Enzyme Concentration3. pH4. Temperature5. Cofactors6. Inhibitors
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
Measurement of catalytic activity1. ↑ in product concentration2. ↓ in substrate concentration3. ↓ or ↑ in coenzyme concentration (NADH)4. ↑ in altered enzyme concentration
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
Measurement of catalytic activity1. ↑ in product concentration2. ↓ in substrate concentration3. ↓ or ↑ in coenzyme concentration (NADH)4. ↑ in altered enzyme concentration
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
Measurement of catalytic activitya. Dependent on enzyme concentrationb. Performed in zero-order kinetics (linear phase)
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay2. Continuous-monitoring or kinetic assays
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay
Reagents are combined and the amount of reaction is measured (AMS, LPS, ACP, ALP)
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay
Reagents are combined and the amount of reaction is measured.
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
General methods of measuring enzymatic reaction2. Continuous-monitoring or kinetic assays
Measurements at specific time intervals Rate of change in substrate, cofactor, product.
Chapter Outline
IV. Enzyme Kineticsiii. Measurement of Enzyme Activity
General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay2. Continuous-monitoring or kinetic assays
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Chapter Outline
IV. Enzyme Kineticsiv. Calculation of Enzyme Activity
1. IU (EC) Amount of enzyme that will catalyze the reaction
of 1 μmol of substrate per minute (μmol /min)
2. Kat (SI) Amount of enzyme that will catalyze the reaction
of 1 mol of substrate per second (mol/s)
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Chapter Outline
IV. Enzyme Kineticsv. Measurement of Enzyme Mass
Immunoassays Electrophoresis
Chapter Outline
IV. Enzyme Kineticsi. Catalytic Mechanism of Enzymesii. Factors that Influence Enzymatic Reactionsiii. Measurement of Enzyme Activityiv. Calculation of Enzyme Activityv. Measurement of Enzyme Mass
Chapter Outline
I. IntroductionII. General Properties and DefinitionsIII. Enzyme Classification and NomenclatureIV. Enzyme KineticsV. Enzymes of Clinical Significance
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. CK2. AST3. LDH
B. Liver Enzymes1. ALT2. ALP3. LDH4. GGT
C. Pancreatic Enzymes1. AMS2. LPS
D. Other Enzymes1. ACP2. G-6-PDH
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LDH)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGT)
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total CKc. Diagnostic Significance of CK Isoenzymed. Methods of Determination of CK Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatine Kinase (CK)a. Function, Tissue Source and Clinical Significance
Storage of high-energy creatine phosphate in muscle cells
Highest activities in skeletal muscle, heart (AMI), and brain tissue
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total CKc. Diagnostic Significance of CK Isoenzymed. Methods of Determination of CK Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
i. Forward Reaction (Tanzer-Givarg)ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
i. Forward Reaction (Tanzer-Givarg)
Auxiliary enzyme
Indicator enzyme
Coupled-enzyme assay
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
i. Forward Reaction (Tanzer-Givarg)• Measure ↓ in absorbance at 340 nm• Optimum pH is 9.0
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
ii. Reverse Reaction (Oliver-Rosalki)• ↑ in absorbance at 340 nm• 6x faster than forward reaction• Optimum pH: 6.8
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination
i. Forward Reaction (Tanzer-Givarg)ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)b. Methods of Determination Source of Error
• Hemolysis cause false ↑ CK due to AK activity• CK is inactivated by light• Physical activity and IM injections cause ↑ CK
Reference Range• Male, 15-160 U/L : Female, 15-130 U/L• CK-MB: <6% of total CK
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total CKc. Diagnostic Significance of CK Isoenzymed. Methods of Determination of CK Isoenzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)c. Diagnostic Significance of CK Isoenzymes
CK-3 / CK-MM / Muscle type
CK- 2 / CK-MB / Hybrid Type
CK-1 / CK-BB / Brain Type
Slowest mobility 2nd fastest Migrate fastestMajor isoenzyme in striated muscle and
normal serum
Significant quantities in heart tissues
Highest concentration in CNS, GI tract and uterus (pregnancy)
Enzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)c. Diagnostic Significance of CK Isoenzymes After MI, CK-MB (>6%) begin to rise within 4-8 hrs,
peak at 12-24 hrs, and return to normal in 48-72 hrs.
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total CKc. Diagnostic Significance of CK Isoenzymed. Methods of Determination of CK Isoenzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)d. Methods of Determination of CK Isoenzymes
Reference Values:94-100% CK-MM0-6% CK-MB
Separation of CK isoenzymes by electrophoresis
Normal Control Myocardial infarction
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK) Other CK Isoenzymes
a. Macro-CK• Migrate midway CK-MM and CK-MB• CK-BB complexed with IgG/IgA• CK-MM with LPP
b. Mitocondrial CK (CK-Mi)• Migrates cathodal to CK-MM• Bound to mitochondrial membranes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total CKc. Diagnostic Significance of CK Isoenzymed. Methods of Determination of CK Isoenzymes
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Lactate Dehydrogenase (LDH)4. Gamma glutamyl transferase (GGT)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total AST
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significance
Serum glutamic-oxaloacetic transaminase (SGOT) Transfer of amino group in aspartate to α-keto. Involved in the synthesis and degradation of AA. Highest activities in cardiac, liver and skeletal
muscle.
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significance
• AST levels begin to rise in 6-8 hours, peak at 24 hours, and return to normal in 5 days.
• Also ↑ in hepatocellular and skeletal muscle dis.
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AST
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)b. Methods of Determination of AST
Karmen Method Uses malate dehydrogenase and monitors ↓ in
absorbance at 340 nm Falsely ↑ in hemolyzed sample Reference Range: 5 – 30 U/L
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AST
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGT)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total LDHc. Diagnostic Significance of LDH Isoenzymed. Methods of Determination of LDH Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significance
Interconversion of lactate and pyruvate Widely distributed, highest activities in heart,
hepatic, skeletal muscle and RBC
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total LDHc. Diagnostic Significance of LDH Isoenzymed. Methods of Determination of LDH Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)b. Methods of Determination of Total LDH
a. Wrobleuski – Cabaud or Wacker method Forward Reaction (Lactate Pyruvate)
b. Wrobleuski – La Due Reverse Reaction (Pyruvate Lactate)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)b. Methods of Determination of Total LDH
a. Wrobleuski – Cabaud and Wacker method Forward Reaction (Lactate Pyruvate) ↑ in absorbance is monitored at 340 nm Optimal pH is 8.3 – 8.9
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)b. Methods of Determination of Total LDH
b. Wrobleuski - La Due Reverse Reaction (Pyruvate Lactate) ↓ in absorbance is monitored at 340 nm Optimal pH is 7.1 to 7.4
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)b. Methods of Determination of Total LDH
c. α-hydroxybutyrate dehydrogenase (α-HBD) Has greater affinity of H subunits Represent LDH-1
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)b. Methods of Determination of Total LDH
LD begin to rise within 10-24 hrs, peak at 48-72 hrs, and remains elevated for 10 days.
Reference Range: 100-225 U/L
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total LDHc. Diagnostic Significance of LDH Isoenzymed. Methods of Determination of LDH Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase Isoenzymes (LDH Isoenzymes)c. Diagnostic Significance of LDH Isoenzyme
Tetramer containing two active sub-unitsIsoenzyme Tissue Disorder (↑)
LDH-1 (HHHH)LDH-2 (HHHM) Heart, RBC MI, Hemolytic anemia
RI, Megaloblastic anemiaLDH-3 (HHMM) Lung, Spleen, Pancreas Pulmonary embolismLDH-4 (HMMM)LDH-5 (MMMM) Liver, Skeletal Muscle
Hepatic injurySkeletal muscle injury
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total LDHc. Diagnostic Significance of LDH Isoenzymed. Methods of Determination for LDH Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)d. Methods of Determination of LDH Isoenzymes
Relative concentration in normal serum: LDH-2>LDH-1>LDH-3>LDH-4>LDH-5
In AMI and Intravascular hemolysis, LDH-1 and LDH-2 demonstrate a Flipped pattern (LDH-1 > LDH-2)
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)d. Methods of Determination of LDH Isoenzymes
Correspondence Between CPK and LDH Isoenzyme Patterns
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
3. Lactate Dehydrogenase (LDH)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total LDHc. Diagnostic Significance of LDH Isoenzymed. Methods of Determination for LDH Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceA. MI Profile
1. CK2. AST3. LD
CK-MB AST LDH
Appearance 4-8 hrs 6-8 hrs 10-24 hrs
Peak 12-24 hrs 24 hrs 48-72 hrs
Stay Elevated 3 days 5 days 10 days
MI Profile
Appearance Peak Stay Elevated
Myoglobin 1-4 hrs 6-9 hrs 18-24 days
Troponin (cTN) 4-10 hrs 12-48 hrs 4-10 days
Troponin T (TnT) 4-10 hrs 2 & 4 days 7-10 days
Troponin I (TnI) 4-6 hrs 12-18 hrs 6 days
CK-MB 4-8 hrs 12-24 hrs 3 days
AST 6-8 hrs 24 hrs 5 days
LDH 10-24 hrs 48-72 hrs 10 days
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGT)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total AST
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significance
Serum glutamic-pyruvic transaminase (SGPT) Transfer of an amino group between alanine
and α-ketoglutarate ↑ in hepatocellular disorders
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total ALT
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)b. Methods of Determination of Total ALT
i. Walker Methodii. Reitmann-Frankel
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)b. Methods of Determination of Total ALT
i. Walker Method Uses LD and monitors ↓ in absorbance (340 nm) Reference Range: 6-37 U/L
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)b. Methods of Determination of Total ALT
ii. Reitmann-Frankel Reagent: 2,4 dinitrophenyl hydrazine (2,4-DNPH) End Color: Brown
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT) De Ritis Ratio
The AST/ALT Ratio Differentiates the cause of hepatic disorder Ratio > 1 Non viral origin (alcohol hepatitis) Ratio < 1 Viral in origin
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
AST/SGOT ALT/SGPTAST/SGOT ALT/SGPTOrgan Affected Heart Liver
AST/SGOT ALT/SGPTOrgan Affected Heart Liver
Substrate Aspartateα-ketoglutarate
Alanineα-ketoglutarate
AST/SGOT ALT/SGPTOrgan Affected Heart Liver
Substrate Aspartateα-ketoglutarate
Alanineα-ketoglutarate
End Products Glutamic acidOxaloacetic acid
Glutamic acidPyruvic acid
AST/SGOT ALT/SGPTOrgan Affected Heart Liver
Substrate Aspartateα-ketoglutarate
Alanineα-ketoglutarate
End Products Glutamic acidOxaloacetic acid
Glutamic acidPyruvic acid
Test KarmenReitman-Frankel
WalkerReitman-Frankel
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total AST
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGT)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significanceb. Methods of Determination for ALPc. Diagnostic Significance of ALP Isoenzymed. Methods of Determination for ALP Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significance
Catalyze the hydrolysis of phosphomonoesters Requires Mg2+ activator Evaluation of hepatobiliary and bone disorders.
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significanceb. Methods of Determination of ALPc. Diagnostic Significance of ALP Isoenzymed. Methods of Determination for ALP Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphataseb. Methods of Determination of ALP
Bowers and McComb Based on molar absorptivity of p-Nitrophenol
Absorbance is measured at 405 nm
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)b. Methods of Determination of ALP
Methods Substrate End Product1-4. Bodansky, Shinowara, Jones, Reinhart β-glycero-phosphate Inorganic PO4
+ Glycerol5. Bessy, Lowry & Brock6. Bowers & McComb p-nitrophenyl phosphate p-nitrophenol
7. King and Armstrong Phenyl phosphate phenol8. Huggins & Talalay Phenolpthalein diphosphate phenol9. Moss α-napthol phosphate α-napthol
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)b. Methods of Determination of ALP
Reference Range 30 – 90 U/L (adult) 70 – 220 U/L (0 – 3 months) 50 – 260 U/L (3 - 10 years) 60 – 295 U/L (10 - puberty)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significanceb. Methods of Determination for ALPc. Diagnostic Significance of ALP Isoenzymed. Methods of Determination for ALP Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)c. Diagnostic Significance of ALP Isoenzyme
1. Liver ALP2. Bone ALP3. Placental ALP4. Intestinal ALP
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)c. Diagnostic Significance of ALP Isoenzyme
1. Liver ALP ↑ in liver diseases Fractions: Major liver and fast liver (α1) band
2. Bone ALP ↑ in bone disease, healing of bone fractures
and physiologic bone growth
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)c. Diagnostic Significance of ALP Isoenzyme
3. Placental ALP ↑ in pregnancy
4. Intestinal ALP Blood groups B or O, ↑ in fatty meal ↑ GIT disorders
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)c. Diagnostic Significance of ALP Isoenzyme
1. Liver ALP2. Bone ALP3. Placental ALP4. Intestinal ALP
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significanceb. Methods of Determination of ALPc. Diagnostic Significance of ALP Isoenzymed. Methods of Determination for ALP Isoenzymes
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
i. Difference by Heat Stabilityii. Chemical Inhibitioniii. Electrophoresis
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
i. Difference by Heat Stability Serum is heated at 56°C for 10 minutes
1. Liver ALP ALP residual activity is ↓ to >20%
2. Bone ALP ALP residual activity is ↓ to <20% Heat labile fraction
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
i. Difference by Heat Stabilityii. Selective Chemical Inhibitioniii. Electrophoresis
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
ii. Selective Chemical Inhibition Placental and Intestinal ALP are inhibited by
phenylalanine (chemical inhibition)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
i. Difference by Heat Stabilityii. Selective Chemical Inhibitioniii. Electrophoresis
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatased. Methods of Determination for ALP Isoenzymes
iii. Electrophoresis
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
Source of Enzyme
LiverBoneIntestinePlacentaRegan (Carcinoma)
Source of Enzyme L-Phenylalanine
Liver 10Bone 10Intestine 75Placenta 80Regan (Carcinoma) 80
Source of Enzyme L-Phenylalanine Heat (56°C for 15 mins or Urea (3M)
Liver 10 60Bone 10 90Intestine 75 60Placenta 80 0Regan (Carcinoma) 80 0
Source of Enzyme L-Phenylalanine Heat (56°C : 15m) Urea (3M)
Order of Migration (Anodal)
Liver 10 60 1Bone 10 90 2Intestine 75 60 4Placenta 80 0 3Regan (Carcinoma) 80 0 3
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes
i. Difference by Heat Stabilityii. Selective Chemical Inhibitioniii. Electrophoresis
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significanceb. Methods of Determination of ALPc. Diagnostic Significance of ALP Isoenzymed. Methods of Determination for ALP Isoenzymes
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGT)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination for GGT
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significance
Catalyze the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids
Diagnosis hepatobiliary disorders (obstructive liver disease) and chronic alcoholism
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significance
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination for GGT
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)b. Methods of Determination for GGT
Szaz Assay Absorbance of p-Nitroaniline is measured at
405-420 nm
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of GGT
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. Creatinine Kinase (CK)2. Aspartate Aminotransferase (AST)3. Lactate Dehydrogenase (LD)
B. Liver Enzymes1. Alanine Aminotransferase (ALT)2. Alkaline Phosphatase (ALP)3. Gamma glutamyl transferase (GGTP)
Chapter Outline
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)2. Lipase (LPS)
D. Prostate Enzymes1. Acid Phosphatase (ACP)2. Glucose-6-phosphate dehydrogenase (G-6-PDH)
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AMS
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)a. Function, Tissue Source and Clinical Significance
Breakdown of starch via α, 1-4 branching linkages
Increased in acute pancreatitis Requires Ca2+ and Cl- for activation Rise at 2-12 h, peak at 24 h and return to
normal within 3-5 d
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)a. Function, Tissue Source and Clinical Significance
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AMS
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination of AMS
Amylase Methodologies1. Amyloclastic2. Chromogenic3. Saccharogenic4. Continuous monitoring
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination of AMS
Amylase Methodologies1. Amyloclastic Measures the disappearance of starch substrate
Starch-iodine comp (dark-blue) ↓color intensity2. Saccharogenic Measures the appearance of the product
Starch reducing sugars
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination of AMS
Amylase Methodologies
3. ChromogenicMeasures the ↑ in colorstarch - dye starch-dye fragments
4. Continuous Monitoring
Coupling of several enzyme systems to monitor amylase activity
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination
Amylase Methodologies4. Continuous Monitoring
Coupling of several enzyme systems to monitor amylase activity
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination
Amylase Methodologies1. Amyloclastic2. Chromogenic3. Saccharogenic4. Continuous monitoring
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination
Amylase Isoenzymesi. Salivary Amylase • ptyalin • fast moving
ii. Pancreatic Amylase • amylopsin • slow moving
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)b. Methods of Determination
Amylase Isoenzymes
http://www.clinchem.org/cgi/reprint/30/3/387.pdf
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Chapter Outline
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)2. Lipase (LPS)
D. Other Enzymes1. Acid Phosphatase (ACP)2. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)a. Function, Tissue Source and Clinical Significance
Hydrolyzes of fats to produce alcohols and FA Earliest marker for acute pancreatitis Larger molecule, remains in circulation (7 days)
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)b. Methods of Determinations
Cherry Crandall TietzSubstrate 50% olive oil 50% olive oil (triolein)Titrating agent 0.4N NaOH 0.4N NaOHIndicator Phenolpthalein Thymolpthalein + VeronalEndpoint FA (Oleic Acid) FA (Oleic Acid)End Color Pink Blue
Enzymes
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
2. Lipase (LPS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Chapter Outline
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)2. Lipase (LPS)
D. Other Enzymes1. Acid Phosphatase (ACP)2. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)a. Function, Tissue Source and Clinical Significance
Catalyze the hydrolysis of phosphomonoesters Evaluation of metastatic carcinoma of prostate. Forensic investigation of rape
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)b. Methods of Determinations
Phosphatase inhibitorsi. L-tartrate ions
inhibits specific prostatic ACP total ACP - ACP after inhibition = prostatic ACP
ii. Formaldehyde and Cupric ions inhibits red cell ACP
Enzymes
V. Enzymes of Clinical SignificanceB. Liver Enzymes
1. Acid Phosphatase (ACP)b. Methods of Determinations
Assay for Enzyme Activity Reference Range: Prostatic ACP: 0 -3.5 ng/ml
Methods Substrate1. Quantitative end point Thymolpthalein monophosphate2. Continuous monitoring α-napthyl phosphate
Chapter Outline
V. Enzymes of Clinical SignificanceC. Pancreatic Enzymes
1. Amylase (AMS)2. Lipase (LPS)
D. Other Enzymes1. Acid Phosphatase (ACP)2. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
Chapter Outline
V. Enzymes of Clinical SignificanceA. MI Profile
1. CK2. AST3. LDH
B. Liver Enzymes1. ALT2. ALP3. LDH4. GGT
C. Pancreatic Enymes1. AMS2. LPS
D. Other Enzymes1. ACP2. G-6-PDH
Thank You!