Enzymes

INTRODUCTION TO CLINICAL CHEMISTRY
Outline
1.
2.
3. 4.
Definition Cells Role of the Clinical Chemistry Laboratory 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.
2.
3. 4.
Composition of Cells
Malfunction
1.
2.
3. 4. 5. 6. 7.
Trauma or by invasive agents Genetic deficiency of a vital enzyme Insufficient supply of essential nutrients Insufficient blood and oxygen supply Malignancy Accumulation of waste products Defect in the cellular recognition of signals
Outline
1.
2.
3. 4.
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.
2.
3. 4.
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.
2.
3. 4.
ENZYMES
Objectives
I.
II. III.
IV.
Describe the different factors affecting the enzyme reaction Explain what is meant by zero order kinetics. Explain the difference between enzyme activity and enzyme mass Describe and select parameters for optimal measurement of enzyme activity
Objectives
VI.
VII.
Discuss why the measurement of a serum enzymes is clinically useful. Discuss which enzymes and/or isoenzymes are useful in the diagnosis of myocardial infarction, liver disease, and acute pancreatitis.
Chapter Outline
I.
II.
III. IV. V.
Introduction General Properties and Definitions Enzyme Classification and Nomenclature Enzyme Kinetics Enzymes of Clinical Significance
Chapter Outline
I.
Introduction
i. ii.
Enzyme
Biologic proteins that catalyze biochemical reactions Not consumed or changed in composition Found in all body tissue (intracellular) and is in serum after cell injury
iii.
Chapter Outline
Chapter Outline
I.
Introduction
i. ii.
Function of Enzymes
Hydration of Carbon Dioxide (respiration) Nerve Induction Muscle Contraction Nutrient Degradation (Digestion) Growth and Reproduction Energy Storage and Use
iii.
iv. v. vi.
Chapter Outline
I.
II.
III. IV. V.
Chapter Outline
II.
General Properties and Definitions A. Components of an Enzyme B. Terms associated with an enzyme
Enzymes
II.
General Properties and Definitions A. Components of an Enzyme

i.
ii.
Active Site A cavity of an enzyme where substrates bind and undergo a chemical reaction. Allosteric Site A cavity other than the active site that binds regulatory (effector) molecules.
Enzymes
II.
General Properties and Definitions A. Components of an Enzyme

Allosteric Inhibitor
Allosteric promoter
Chapter Outline
II.
Enzymes
II.
General Properties and Definitions B. Terms associated with enzymes

i. ii.
iii.
iv. v.
vi.
vii. viii.
Substrates Cofactors Isoenzyme Apoenzyme Holoenzymes Proenzyme or Zymogens Allosteric enzymes Inhibitors
Enzymes
II.

i.
Substrates Substances acted upon enzymes Specific for each of their particular enzyme
Enzymes
II.

ii.
Cofactors Non protein substances added in the enzyme substrate complex to manifest the enzyme activity a. 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.

iii.
iv.
Isoenzyme Similar enzymatic activity but differ in physical, biochemical and immunologic characteristics Apoenzyme The protein portion of the enzyme Subject to denaturation, in which enzyme losses its activity
Enzymes
II.

v.
vi.
Holoenzyme An active substance formed by combination of a co-enzyme and an apoenzyme. Proenzyme or Zymogens An inactive enzyme precursor E.g. Coagulation factors and digestive enzymes
Enzymes
II. General Properties and Definitions
B. Terms associated with enzymes vii. 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 veloci 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.
II.
III. IV. V.
Chapter Outline
II.
Chapter Outline
III.
Enzyme Classification and Nomenclature

1.
2. 3.
4.
5. 6.
Oxidoreductases Transferase Hydrolases Lyases Isomerases Ligases
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 Lactate dehydrogenase ABBREVIATED NAME LDH E.C CODE NO. 1.1.1.2 7 2.6.1.1 SCIENTIFIC NAME
1. Oxido reductase 2. Transferase
L-Lactate NAD+ oxidoreductase L-Aspartate ,2oxaloglutarate Amino transferase L-Alanine, 2oxaloglutarate amino transferase (5-Glutamyl ) peptide amino acid, 5- glutamyl transferase
2.1 Aspartate SGOT ( Serum amino transferase Glutamate Oxaloacetate transaminase) 2.2 Alanine amino SGPT ( Serum transferase Glutamate Pyruvate transaminase) 2.3 Gamma Glutamyl transferase GGT
2.6.1.2
2.3.2.2
ENZYME NOMENCLATURE
CLASS RECOMMENDE ABBREVIATED D NAME NAME Alkaline Phosphatase ALP E.C CODE NO. 3.1.3.1 SCIENTIFIC NAME
3. Hydrolases
Ortho-phosphoric, monoester phosphohydrolase (alkaline optimum) Ortho-phosphoric, monoester phosphohydrolase (acid optimum) 1,4- D- Glucan, Glucanohydrolase D Fructose 1,6 Bis phosphate , Dglyceraldehyde, 3-
Acid Phosphatase
ACP
3.1.3.2
-Amylase 4. Lyase Aldolase
AMS ALD
3.2.1.1 4.1.2.13
Enzymes
III.

1.

Oxidoreductases
Catalyze redox reaction between two substrates A- + B A + BE.g: Dehydrogenase (Lactate Dehydrogenase)
Enzymes
III.

2.

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.

2.

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.

3.

Hydrolases
Catalyze hydrolysis of various bonds AB + H2O AOH + BH E.g: Amylase (AMY), Lipase (LPS), Phosphatase (ALP, ACP)
Enzymes
III.

4.

Lyases
Catalyze the removal of groups from substrates without hydrolysis; the product remain double bonds ATP cAMP + PPi Fructose biphosphate aldolase (ALS)
Enzymes
III.

5.

Isomerases
Catalyze the interconversion of geometric, optical or positional isomers AB E.g. Triphosphate isomerase (TPI)
6.
Ligases
Catalyze the joining of two substrate molecules, coupled with breaking of pyrophosphate bond in ATP Ab + C AC + b Glutathione Synthetase (GSH-S)
Chapter Outline
III.

1.
2. 3.
4.
5. 6.
Oxidoreductases Transferase Hydrolases Lyases Isomerases Ligases
Chapter Outline
I.
II.
III. IV. V.
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 substrateby 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 Enzymesubstrate (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 (H) 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
MODELS OF ENZYME ACTION
1.Lock and key hypothesis
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 Kinetics
i.
ii. iii.
iv.
v.
Catalytic Mechanism of Enzymes Factors that Influence Enzymatic Reactions Measurement of Enzyme Activity Calculation of Enzyme Activity Measurement of Enzyme Mass
Enzymes
IV.
Enzyme Kinetics
i.
Catalytic Mechanism of Enzymes

Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach
Enzymes
IV.
Enzyme Kinetics
i.
i.

Relationship between Enzyme, Substrate and Product
Enzymes
IV.
Enzyme Kinetics
i.
i.

Relationship between Enzyme, Substrate and Product
Enzymes
IV.
Enzyme Kinetics
i.
i.

Absolute specificity Combines with only one substrate and catalyzes only one reaction (E.g. CK, LD) Group specificity Combine with all substrates containing a particular chemical group (E.g. ACP, ALP) Bond specificity Specific to chemical bonds (E.g. AMY, LPS) Sterioisometric specificity Combine with one optical isomer (E.g. LDH, G6PD)
ii.
iii.
iv.
Chapter Outline
IV.
Enzyme Kinetics
i.
ii. iii.
iv.
v.
Chapter Outline
IV.
Enzyme Kinetics
ii.
1. 2.
3.
4. 5. 6.
Factors that Influence Enzymatic Reactions Substrate Concentration Enzyme Concentration pH Temperature Cofactors 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 Kinetics
ii.
1.
Factors that Influence Enzymatic Reactions Substrate Concentration
First order kinetics (Michaelis- Menten hypothesis) Reaction rate is proportional to the substrate concentration.
Chapter Outline
IV.
Enzyme Kinetics
ii.
2.
Factors that Influence Enzymatic Reactions

Enzyme Concentration Zero-order kinetics Only a fixed number of substrate (in excess) is converted to product per second
Chapter Outline
IV.
Enzyme Kinetics
ii.
Factors that Influence Enzymatic Reactions Substrate and Enzyme Concentration
First order and Zero Order kinetics
Chapter Outline
IV.
Enzyme Kinetics
ii.
3.
Factors that Influence Enzymatic Reactions pH

Common range 7.0-8.0 Controlled by buffers Temperature Within 0.1C Enzyme is active at 25C, 30C, 37C.
4.
Optimum pH of the Different Enzymes

Enzymes Sucrase Ribonuclease - Glucosidase Sources Intestine Pancreas Yeast Substrates Sucrose 3-50-Cytidylyl adenine Methyl--Dglucoside Acetylcholine Optimum pH 6.2 7.0 5.4 7.5
Acetylcholinester Erythrocytes ase Enolase Rabbit Muscle Arginase Pepsin Beef Liver Gastric mucosa
2-Phospho-DGlycerate L-Arginine Acetyl LPhenylalanine , L-phenylalanine
6.8
8.4-9.7 1.5-2.5
Chapter Outline
IV.
Enzyme Kinetics
ii.
5.
Factors that Influence Enzymatic Reactions Cofactors
Activators: Metalic (Ca2+) and Non Metallic (Cl- ) Coenzymes (prosthetic groups): 2nd substrates (NAD)
Chapter Outline
IV.
Enzyme Kinetics
ii.
6.
Factors that Influence Enzymatic Reactions

Inhibitors
Chapter Outline
IV.
Enzyme Kinetics
ii.
1. 2.
3.
4. 5. 6.
Factors that Influence Enzymatic Reactions Substrate Concentration Enzyme Concentration pH Temperature Cofactors Inhibitors
Chapter Outline
IV.
Enzyme Kinetics
i.
ii. iii.
iv.
v.
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity Measurement of catalytic activity 1. in product concentration 2. in substrate concentration 3. or in coenzyme concentration (NADH) 4. in altered enzyme concentration
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity Measurement of catalytic activity 1. in product concentration 2. in substrate concentration 3. or in coenzyme concentration (NADH) 4. in altered enzyme concentration
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity Measurement of catalytic activity a. Dependent on enzyme concentration b. Performed in zero-order kinetics (linear phase)
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity General methods of measuring enzymatic reaction 1. Fixed time (Two point) Assay 2. Continuous-monitoring or kinetic assays
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity General methods of measuring enzymatic reaction 1. Fixed time (Two point) Assay Reagents are combined and the amount of reaction is measured (AMS, LPS, ACP, ALP)
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity General methods of measuring enzymatic reaction 1. Fixed time (Two point) Assay Reagents are combined and the amount of reaction is measured.
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity General methods of measuring enzymatic reaction 2. Continuous-monitoring or kinetic assays Measurements at specific time intervals Rate of change in substrate, cofactor, product.
Chapter Outline
IV.
Enzyme Kinetics
iii.
Measurement of Enzyme Activity General methods of measuring enzymatic reaction 1. Fixed time (Two point) Assay 2. Continuous-monitoring or kinetic assays
Chapter Outline
IV.
Enzyme Kinetics
i.
ii. iii.
iv.
v.
Chapter Outline
IV.
Enzyme Kinetics
iv.
Calculation of Enzyme Activity 1. IU (EC)
Amount of enzyme that will catalyze the reaction of 1 mol of substrate per minute (mol /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 Kinetics
i.
ii. iii.
iv.
v.
Chapter Outline
IV.
Enzyme Kinetics
v.
Measurement of Enzyme Mass Immunoassays Electrophoresis
Chapter Outline
IV.
Enzyme Kinetics
i.
ii. iii.
iv.
v.
Chapter Outline
I.
II.
III. IV. V.
Chapter Outline
V.
Enzymes of Clinical Significance

A.
1. 2.
3.
B.
1. 2. 3. 4.
MI Profile CK AST LDH Liver Enzymes ALT ALP LDH GGT
C.
1.
2.
Pancreatic Enzymes AMS LPS Other Enzymes ACP G-6-PDH
D.
1.
2.
Chapter Outline
V.

A.
1. 2.
3.
B.
1. 2. 3.
MI Profile Creatinine Kinase (CK) Aspartate Aminotransferase (AST) Lactate Dehydrogenase (LDH) Liver Enzymes Alanine Aminotransferase (ALT) Alkaline Phosphatase (ALP) Gamma glutamyl transferase (GGT)
Chapter Outline
V.

A.
MI Profile
Enzymes
V.

A.
1.
MI Profile Creatinine Kinase (CK)

a.
b.
c. d.
Function, Tissue Source and Clinical Significance Methods of Determination of Total CK Diagnostic Significance of CK Isoenzyme Methods of Determination of CK Isoenzymes
Enzymes
V.

A.
1.
MI Profile 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.

A.
MI Profile
Enzymes
V.

A.
1.

a.
b.
c. d.
Enzymes
V.

A.
1.
MI Profile Creatinine Kinase (CK) b. Methods of Determination i. Forward Reaction (Tanzer-Givarg) ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V.

A.
1.
MI Profile Creatinine Kinase (CK) b. Methods of Determination i. Forward Reaction (Tanzer-Givarg)

Coupled-enzyme assay
Auxiliary enzyme
Indicator enzyme
Enzymes
V.

A.
1.
MI Profile 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.

A.
1.
MI Profile Creatinine Kinase (CK) b. Methods of Determination ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V.

A.
1.
MI Profile 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.

A.
1.
MI Profile Creatinine Kinase (CK) b. Methods of Determination i. Forward Reaction (Tanzer-Givarg) ii. Reverse Reaction (Oliver-Rosalki)
Enzymes
V.

A.
1.
MI Profile 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.

A.
1.

a.
b.
c. d.
Enzymes
V.

A.
1.
MI Profile
Creatinine Kinase (CK) c. Diagnostic Significance of CK Isoenzymes CK- 2 / CK-MB /
Hybrid Type
CK-3 / CK-MM /
Muscle type
CK-1 / CK-BB /
Brain Type
Slowest mobility
2nd fastest
Migrate fastest
Major isoenzyme in striated muscle and normal serum
Significant quantities in heart tissues
Highest concentration in CNS, GI tract and uterus (pregnancy)
Enzymes
V.

A.
1.
MI Profile
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.

A.
1.

a.
b.
c. d.
V.

A.
1.
MI Profile
Creatinine Kinase (CK) d. Methods of Determination of CK Isoenzymes
Reference Values: 94-100% CK-MM 0-6% CK-MB
Normal Control
Myocardial infarction
Separation of CK isoenzymes by electrophoresis
Enzymes
V.

A.
1.
MI Profile
Creatinine Kinase (CK) Other CK Isoenzymes a. Macro-CK Migrate midway CK-MM and CK-MB CK-BB complexed with IgG/IgA
b.
CK-MM with LPP Mitocondrial CK (CK-Mi) Migrates cathodal to CK-MM Bound to mitochondrial membranes
Enzymes
V.

A.
1.

a.
b.
c. d.
Chapter Outline
V.

A.
1. 2.
MI Profile
Creatinine Kinase (CK) Aspartate Aminotransferase (AST) Lactate Dehydrogenase (LD)
3.
B.
1. 2. 3. 4.
Liver Enzymes
Alanine Aminotransferase (ALT) Alkaline Phosphatase (ALP) Lactate Dehydrogenase (LDH) Gamma glutamyl transferase (GGT)
Enzymes
V.

A.
2.
MI Profile Aspartate Aminotransferase (AST)

a.
b.
Function, Tissue Source and Clinical Significance Methods of Determination of Total AST
Enzymes
V.

A.
2.

a.
Function, Tissue Source and Clinical Significance Serum glutamic-oxaloacetic transaminase (SGOT) Transfer of amino group in aspartate to -keto. Involved in the synthesis and degradation of AA. Highest activities in cardiac, liver and skeletal muscle.
Enzymes
V.

A.
2.
Enzymes
V.

A.
2.

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.

A.
2.

a.
b.
Function, Tissue Source and Clinical Significance Methods of Determination of AST
Enzymes
V.

A.
2.

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.

A.
2.

a.
b.
Function, Tissue Source and Clinical Significance Methods of Determination of AST
Chapter Outline
V.

A.
1. 2.
MI Profile
3.
B.
1. 2. 3.
Liver Enzymes
Alanine Aminotransferase (ALT) Alkaline Phosphatase (ALP) Gamma glutamyl transferase (GGT)
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total LDH c. Diagnostic Significance of LDH Isoenzyme d. Methods of Determination of LDH Isoenzymes
Enzymes
V.

A.
3.
MI Profile 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.

A.
3.
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) b. Methods of Determination of Total LDH a. Wrobleuski Cabaud or Wacker method
b.
Forward Reaction (Lactate Pyruvate) Wrobleuski La Due Reverse Reaction (Pyruvate Lactate)
Enzymes
V.

A.
3.
MI Profile 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.

A.
3.
MI Profile 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.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) b. Methods of Determination of Total LDH c. -hydroxybutyrate dehydrogenase (-HBD)
Has greater affinity of H subunits Represent LDH-1
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) b. Methods of Determination of Total LDH LD begin to rise within 10-24 hrs, peak at 4872 hrs, and remains elevated for 10 days. Reference Range: 100-225 U/L
Enzymes
V.

A.
3.
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase Isoenzymes (LDH Isoenzymes) c. Diagnostic Significance of LDH Isoenzyme Tetramer containing two active sub-units
Disorder () MI, Hemolytic anemia RI, Megaloblastic anemia Pulmonary embolism Hepatic injury Skeletal muscle injury
Isoenzyme Tissue LDH-1 (HHHH) Heart, RBC LDH-2 (HHHM) LDH-3 (HHMM) Lung, Spleen, Pancreas LDH-4 (HMMM) Liver, Skeletal Muscle LDH-5 (MMMM)
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total LDH c. Diagnostic Significance of LDH Isoenzyme d. Methods of Determination for LDH Isoenzymes
Enzymes
V.

A.
3.
MI Profile 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.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) d. Methods of Determination of LDH Isoenzymes
Correspondence Between CPK and LDH Isoenzyme Patterns
Enzymes
V.

A.
3.
MI Profile Lactate Dehydrogenase (LDH) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total LDH c. Diagnostic Significance of LDH Isoenzyme d. Methods of Determination for LDH Isoenzymes
Enzymes
V.

A.
1. 2.
3.
MI Profile CK AST LD
CK-MB AST 6-8 hrs 24 hrs 5 days LDH 10-24 hrs 48-72 hrs 10 days
Appearance Peak Stay Elevated
4-8 hrs 12-24 hrs 3 days
MI Profile
Appearance Myoglobin
Troponin (cTN) Troponin I (TnI) CK-MB AST LDH
Peak 6-9 hrs

12-48 hrs 12-18 hrs 12-24 hrs 24 hrs 48-72 hrs
Stay Elevated 18-24 days

4-10 days 6 days 3 days 5 days 10 days
1-4 hrs
4-10 hrs 4-6 hrs 4-8 hrs 6-8 hrs 10-24 hrs
Troponin T (TnT) 4-10 hrs
2 & 4 days 7-10 days
MI Profile
Chapter Outline
V.

A.
1. 2.
MI Profile
3.
B.
1. 2. 3.
Liver Enzymes
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total AST
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT) a. Function, Tissue Source and Clinical Significance Serum glutamic-pyruvic transaminase (SGPT) Transfer of an amino group between alanine and -ketoglutarate in hepatocellular disorders
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT)
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total ALT
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT) b. Methods of Determination of Total ALT i. Walker Method ii. Reitmann-Frankel
Enzymes
V.

B.
1.
Liver Enzymes 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.

B.
1.
Liver Enzymes 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.

B.
1.
Liver Enzymes 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.

B.
Liver Enzymes
AST/SGOT Heart Aspartate -ketoglutarate Glutamic acid Oxaloacetic acid Karmen Reitman-Frankel ALT/SGPT Liver Alanine -ketoglutarate Glutamic acid Pyruvic acid Walker Reitman-Frankel
Organ Affected Substrate End Products Test
Enzymes
V.

B.
1.
Liver Enzymes Alanine Aminotransferase (ALT) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of Total AST
Chapter Outline
V.

A.
1. 2.
MI Profile
3.
B.
1. 2. 3.
Liver Enzymes
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase a. Function, Tissue Source and Clinical Significance b. Methods of Determination for ALP c. Diagnostic Significance of ALP Isoenzyme d. Methods of Determination for ALP Isoenzymes
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase a. Function, Tissue Source and Clinical Significance Catalyze the hydrolysis of phosphomonoesters Requires Mg2+ activator Evaluation of hepatobiliary and bone disorders.
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase a. Function, Tissue Source and Clinical Significance b. Methods of Determination of ALP c. Diagnostic Significance of ALP Isoenzyme d. Methods of Determination for ALP Isoenzymes
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase b. Methods of Determination of ALP Bowers and McComb
Based on molar absorptivity of p-Nitrophenol Absorbance is measured at 405 nm
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) b. Methods of Determination of ALP

End Product Inorganic PO4 + Glycerol
p-nitrophenol
phenol phenol
Methods Substrate 1-4. Bodansky, Shinowara, -glycero-phosphate Jones, Reinhart 5. Bessy, Lowry & Brock p-nitrophenyl phosphate 6. Bowers & McComb 7. King and Armstrong Phenyl phosphate Phenolpthalein diphosphate 8. Huggins & Talalay 9. Moss -napthol phosphate
-napthol
Enzymes
V.

B.
2.
Liver Enzymes 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.

B.
2.
Liver Enzymes Alkaline Phosphatase a. Function, Tissue Source and Clinical Significance b. Methods of Determination for ALP c. Diagnostic Significance of ALP Isoenzyme d. Methods of Determination for ALP Isoenzymes
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) c. Diagnostic Significance of ALP Isoenzyme 1. Liver ALP 2. Bone ALP 3. Placental ALP 4. Intestinal ALP
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) c. Diagnostic Significance of ALP Isoenzyme 1. Liver ALP

2.
in liver diseases Fractions: Major liver and fast liver (1) band Bone ALP in bone disease, healing of bone fractures and physiologic bone growth
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) c. Diagnostic Significance of ALP Isoenzyme 3. Placental ALP
4.
in pregnancy Intestinal ALP Blood groups B or O, in fatty meal GIT disorders
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) c. Diagnostic Significance of ALP Isoenzyme 1. Liver ALP 2. Bone ALP 3. Placental ALP 4. Intestinal ALP
Enzymes
V.

B.
2.
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) d. Methods of Determination for ALP Isoenzymes i. Difference by Heat Stability ii. Chemical Inhibition iii. Electrophoresis
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) d. Methods of Determination for ALP Isoenzymes i. Difference by Heat Stability Serum is heated at 56C 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.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) d. Methods of Determination for ALP Isoenzymes i. Difference by Heat Stability ii. Selective Chemical Inhibition iii. Electrophoresis
Enzymes
V.

B.
2.
Liver Enzymes 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.

B.
2.
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase d. Methods of Determination for ALP Isoenzymes iii. Electrophoresis
Enzymes
V.

B.
2.
Liver Enzymes Alkaline Phosphatase (ALP) d. Methods of Determination for ALP Isoenzymes
Heat (56C for 15 Order of Migration Heat : 15m) mins or Urea (3M) (Anodal) Urea (3M)
Source of Enzyme L-Phenylalanine Liver Bone Intestine Intestine Placenta Placenta Regan (Carcinoma) Regan (Carcinoma) 10 10 10 75 75 80 80 80 80
60 90 90 60 60 0 0 0 0
1 2 4 3 3
Enzymes
V.

B.
2.
Enzymes
V.

B.
2.
Chapter Outline
V.

A.
1. 2.
MI Profile
3.
B.
1. 2. 3.
Liver Enzymes
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT) a. Function, Tissue Source and Clinical Significance b. Methods of Determination for GGT
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT) a. Function, Tissue Source and Clinical Significance Catalyze the transfer of the -glutamyl residue from -glutamyl peptides to amino acids Diagnosis hepatobiliary disorders (obstructive liver disease) and chronic alcoholism
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT) a. Function, Tissue Source and Clinical Significance
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT)

a.
b.
Function, Tissue Source and Clinical Significance Methods of Determination for GGT
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT) b. Methods of Determination for GGT Szaz Assay
Absorbance of p-Nitroaniline is measured at 405-420 nm
Enzymes
V.

B.
3.
Liver Enzymes Gamma-Glutamyltransferase (GGT)

a.
b.
Function, Tissue Source and Clinical Significance Methods of Determination of GGT
Chapter Outline
V.

A.
1. 2.
MI Profile
3.
B.
1. 2. 3.
Liver Enzymes
Alanine Aminotransferase (ALT) Alkaline Phosphatase (ALP) Gamma glutamyl transferase (GGTP)
Chapter Outline
V.

C.
1. 2.
Pancreatic Enzymes
Amylase (AMS) Lipase (LPS)
D.
1. 2.
Prostate Enzymes
Acid Phosphatase (ACP) Glucose-6-phosphate dehydrogenase (G-6-PDH)
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of AMS
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) a. Function, Tissue Source and Clinical Significance Breakdown of starch via , 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.

C.
1.
Pancreatic Enzymes Amylase (AMS) a. Function, Tissue Source and Clinical Significance
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) a. Function, Tissue Source and Clinical Significance b. Methods of Determination of AMS
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) b. Methods of Determination of AMS
Amylase Methodologies
1. 2. 3. 4.
Amyloclastic Chromogenic Saccharogenic Continuous monitoring
Enzymes
V.

C.
1.
Amylase Methodologies Measures the disappearance of starch substrate 1. Amyloclastic Starch-iodine comp (dark-blue) color intensity
2. Saccharogenic
Measures the appearance of the product Starch reducing sugars
Enzymes
V.

C.
1.
Amylase Methodologies Measures the in color 3. Chromogenic starch - dye starch-dye fragments 4. Continuous Coupling of several enzyme systems to monitor Monitoring amylase activity
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) b. Methods of Determination
Amylase Methodologies 4. Continuous Coupling of several enzyme systems to monitor Monitoring amylase activity
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) b. Methods of Determination
Amylase Methodologies
1. 2. 3. 4.
Amyloclastic Chromogenic Saccharogenic Continuous monitoring
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) b. Methods of Determination Amylase Isoenzymes i. Salivary Amylase
ptyalin fast moving ii. Pancreatic Amylase amylopsin slow moving
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) b. Methods of Determination Amylase Isoenzymes
http://www.clinchem.org/cgi/reprint/30/3/387.pdf
Enzymes
V.

C.
1.
Pancreatic Enzymes Amylase (AMS) a. Function, Tissue Source and Clinical Significance b. Methods of Determinations
Chapter Outline
V.

C.
1. 2.
Pancreatic Enzymes
D.
1. 2.
Other Enzymes
Acid Phosphatase (ACP) Glucose-6-Phosphate Dehydrogenase (G-6-PD)
Enzymes
V.

C.
2.
Pancreatic Enzymes Lipase (LPS) a. Function, Tissue Source and Clinical Significance b. Methods of Determinations
Enzymes
V.

C.
2.
Pancreatic Enzymes 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.

C.
2.
Pancreatic Enzymes Lipase (LPS)
Enzymes
V.

C.
2.
Enzymes
V.

C.
2.
Pancreatic Enzymes Lipase (LPS) b. Methods of Determinations

Cherry Crandall Tietz
Substrate Titrating agent Indicator Endpoint End Color
50% olive oil 0.4N NaOH Phenolpthalein FA (Oleic Acid) Pink
50% olive oil (triolein) 0.4N NaOH

Thymolpthalein + Veronal
FA (Oleic Acid) Blue
Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.


Enzymes
V.

C.
2.
Chapter Outline
V.

C.
1. 2.
Pancreatic Enzymes
D.
1. 2.
Other Enzymes
Enzymes
V.

B.
1.
Liver Enzymes Acid Phosphatase (ACP) a. Function, Tissue Source and Clinical Significance b. Methods of Determinations
Enzymes
V.

B.
1.
Liver Enzymes 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.

B.
1.
Liver Enzymes Acid Phosphatase (ACP)
Enzymes
V.

B.
1.
Liver Enzymes Acid Phosphatase (ACP) a. Function, Tissue Source and Clinical Significance b. Methods of Determinations
Enzymes
V.

B.
1.
Liver Enzymes Acid Phosphatase (ACP) b. Methods of Determinations Phosphatase inhibitors i. 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.

B.
1.
Liver Enzymes Acid Phosphatase (ACP) b. Methods of Determinations Assay for Enzyme Activity Reference Range: Prostatic ACP: 0 -3.5 ng/ml
Methods 1. Quantitative end point 2. Continuous monitoring
Substrate Thymolpthalein monophosphate -napthyl phosphate
Chapter Outline
V.

C.
1. 2.
Pancreatic Enzymes
D.
1. 2.
Other Enzymes
Chapter Outline
V.

A.
1. 2.
MI Profile
CK AST LDH
C.
1.
Pancreatic Enymes
2.
AMS LPS
3.
B.
1. 2. 3. 4.
Liver Enzymes
ALT ALP LDH GGT
D.
1.
2.
Other Enzymes
ACP G-6-PDH

Enzymes

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Enzymes

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INTRODUCTION TO CLINICAL CHEMISTRY

Measure the concentration of a particular constituent (the analyte) in body fluids

Role of the Medical Technologist

Role of the Medical Technologist

Must understand the tools:

General Properties and Definitions A. Components of an Enzyme

General Properties and Definitions A. Components of an Enzyme

General Properties and Definitions B. Terms associated with enzymes

General Properties and Definitions B. Terms associated with enzymes

General Properties and Definitions B. Terms associated with enzymes

General Properties and Definitions B. Terms associated with enzymes

General Properties and Definitions B. Terms associated with enzymes

Two kinds of allosteric enzymes:

Two kinds of allosteric enzymes:

Examples of competitive inhibitors

Enzyme Classification and Nomenclature

Oxidoreductases Transferase Hydrolases Lyases Isomerases Ligases

Enzyme Classification and Nomenclature

1. Oxido reductase 2. Transferase

-Amylase 4. Lyase Aldolase

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Enzyme Classification and Nomenclature

Oxidoreductases Transferase Hydrolases Lyases Isomerases Ligases

MODELS OF ENZYME ACTION

1.Lock and key hypothesis

2. Induced Fit Hypothesis

CHARACTERISTICS AND PROPERTIES OF ENZYMES

Catalytic Mechanism of Enzymes

Catalytic Mechanism of Enzymes

Catalytic Mechanism of Enzymes

Catalytic Mechanism of Enzymes

LOWERING ACTIVATION ENERGY

Factors that Influence Enzymatic Reactions Substrate Concentration

Factors that Influence Enzymatic Reactions

Factors that Influence Enzymatic Reactions Substrate and Enzyme Concentration

First order and Zero Order kinetics

Factors that Influence Enzymatic Reactions pH

Optimum pH of the Different Enzymes

2-Phospho-DGlycerate L-Arginine Acetyl LPhenylalanine , L-phenylalanine

Factors that Influence Enzymatic Reactions Cofactors

Factors that Influence Enzymatic Reactions

Calculation of Enzyme Activity 1. IU (EC)

Measurement of Enzyme Mass Immunoassays Electrophoresis

Enzymes of Clinical Significance

MI Profile CK AST LDH Liver Enzymes ALT ALP LDH GGT

Pancreatic Enzymes AMS LPS Other Enzymes ACP G-6-PDH

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

MI Profile Creatinine Kinase (CK)

Enzymes of Clinical Significance

MI Profile Creatine Kinase (CK)

Enzymes of Clinical Significance

Enzymes of Clinical Significance

MI Profile Creatinine Kinase (CK)

Enzymes of Clinical Significance

Enzymes of Clinical Significance

MI Profile Creatinine Kinase (CK) b. Methods of Determination i. Forward Reaction (Tanzer-Givarg)