enzymes ppt

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


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


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


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


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


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


Enzymes

II. General Properties and DefinitionsB. Terms associated with enzymes

i. Substratesii. Cofactorsiii. Isoenzymeiv. Apoenzymev. Holoenzymesvi. Proenzyme or Zymogensvii. Allosteric enzymesviii. Inhibitors

Enzymes


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

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


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

http://upload.wikimedia.org/wikipedia/commons/e/e9/Enzymes.JPG

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

http://upload.wikimedia.org/wikipedia/commons/e/e9/Enzymes.JPG

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


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 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


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.

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CHARACTERISTICS AND PROPERTIES OF ENZYMES

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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


i. Relationship between Enzyme, Substrate and Product

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


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


1. Substrate Concentration First order kinetics (Michaelis- Menten hypothesis)

Reaction rate is proportional to the substrate concentration.

http://upload.wikimedia.org/wikipedia/commons/0/09/KinEnzymo(en).svg

Chapter Outline


2. Enzyme Concentration Zero-order kinetics

Only a fixed number of substrate (in excess) is converted to product per second


Chapter Outline


Substrate and Enzyme Concentration First order and Zero Order kinetics


Chapter Outline


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


5. Cofactors Activators: Metalic (Ca2+) and Non Metallic (Cl- ) Coenzymes (prosthetic groups): 2nd substrates (NAD)

Chapter Outline


6. Inhibitors

Chapter Outline


1. Substrate Concentration2. Enzyme Concentration3. pH4. Temperature5. Cofactors6. Inhibitors

Chapter Outline


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


Measurement of catalytic activity1. ↑ in product concentration2. ↓ in substrate concentration3. ↓ or ↑ in coenzyme concentration (NADH)4. ↑ in altered enzyme concentration

Chapter Outline


Measurement of catalytic activitya. Dependent on enzyme concentrationb. Performed in zero-order kinetics (linear phase)

Chapter Outline


General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay2. Continuous-monitoring or kinetic assays

Chapter Outline


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


General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay

Reagents are combined and the amount of reaction is measured.


Chapter Outline


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


General methods of measuring enzymatic reaction1. Fixed time (Two point) Assay2. Continuous-monitoring or kinetic assays

Chapter Outline


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


Chapter Outline

IV. Enzyme Kineticsv. Measurement of Enzyme Mass

Immunoassays Electrophoresis

Chapter Outline


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


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


Enzymes


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


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


Enzymes


1. Creatinine Kinase (CK)b. Methods of Determination

i. Forward Reaction (Tanzer-Givarg)ii. Reverse Reaction (Oliver-Rosalki)

Enzymes



i. Forward Reaction (Tanzer-Givarg)

Auxiliary enzyme

Indicator enzyme

Coupled-enzyme assay

Enzymes



i. Forward Reaction (Tanzer-Givarg)• Measure ↓ in absorbance at 340 nm• Optimum pH is 9.0

Enzymes



ii. Reverse Reaction (Oliver-Rosalki)

Enzymes



ii. Reverse Reaction (Oliver-Rosalki)• ↑ in absorbance at 340 nm• 6x faster than forward reaction• Optimum pH: 6.8

Enzymes



i. Forward Reaction (Tanzer-Givarg)ii. Reverse Reaction (Oliver-Rosalki)

Enzymes


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




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


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




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


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



Chapter Outline


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


2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total AST

Enzymes


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


2. Aspartate Aminotransferase (AST)

Enzymes


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


2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AST

Enzymes


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


2. Aspartate Aminotransferase (AST)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AST

Chapter Outline




Enzymes


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


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



Enzymes


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



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



b. Wrobleuski - La Due Reverse Reaction (Pyruvate Lactate) ↓ in absorbance is monitored at 340 nm Optimal pH is 7.1 to 7.4

Enzymes



c. α-hydroxybutyrate dehydrogenase (α-HBD) Has greater affinity of H subunits Represent LDH-1

Enzymes



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



Enzymes


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


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


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


3. Lactate Dehydrogenase (LDH)d. Methods of Determination of LDH Isoenzymes

Correspondence Between CPK and LDH Isoenzyme Patterns

Enzymes


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


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

MI Profile

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Chapter Outline




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


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


1. Alanine Aminotransferase (ALT)

Enzymes


1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total ALT

Enzymes


1. Alanine Aminotransferase (ALT)b. Methods of Determination of Total ALT

i. Walker Methodii. Reitmann-Frankel

Enzymes



i. Walker Method Uses LD and monitors ↓ in absorbance (340 nm) Reference Range: 6-37 U/L

Enzymes



ii. Reitmann-Frankel Reagent: 2,4 dinitrophenyl hydrazine (2,4-DNPH) End Color: Brown

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


AST/SGOT ALT/SGPTAST/SGOT ALT/SGPTOrgan Affected Heart Liver

AST/SGOT ALT/SGPTOrgan Affected Heart Liver

Substrate Aspartateα-ketoglutarate

Alanineα-ketoglutarate




End Products Glutamic acidOxaloacetic acid

Glutamic acidPyruvic acid




End Products Glutamic acidOxaloacetic acid

Glutamic acidPyruvic acid

Test KarmenReitman-Frankel

WalkerReitman-Frankel

Enzymes


1. Alanine Aminotransferase (ALT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of Total AST

Chapter Outline




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


2. Alkaline Phosphatasea. Function, Tissue Source and Clinical Significance

Catalyze the hydrolysis of phosphomonoesters Requires Mg2+ activator Evaluation of hepatobiliary and bone disorders.

Enzymes


2. Alkaline Phosphatase

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


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


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


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


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


2. Alkaline Phosphatase (ALP)c. Diagnostic Significance of ALP Isoenzyme

1. Liver ALP2. Bone ALP3. Placental ALP4. Intestinal ALP

Enzymes



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



3. Placental ALP ↑ in pregnancy

4. Intestinal ALP Blood groups B or O, ↑ in fatty meal ↑ GIT disorders

Enzymes



1. Liver ALP2. Bone ALP3. Placental ALP4. Intestinal ALP

Enzymes



Enzymes


2. Alkaline Phosphatase (ALP)d. Methods of Determination for ALP Isoenzymes

i. Difference by Heat Stabilityii. Chemical Inhibitioniii. Electrophoresis

Enzymes



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



i. Difference by Heat Stabilityii. Selective Chemical Inhibitioniii. Electrophoresis

Enzymes



ii. Selective Chemical Inhibition Placental and Intestinal ALP are inhibited by

phenylalanine (chemical inhibition)

Enzymes




Enzymes


2. Alkaline Phosphatased. Methods of Determination for ALP Isoenzymes

iii. Electrophoresis

Enzymes



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




Chapter Outline




Enzymes


3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination for GGT

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


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

Enzymes


3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination for GGT

Enzymes


3. Gamma-Glutamyltransferase (GGT)b. Methods of Determination for GGT

Szaz Assay Absorbance of p-Nitroaniline is measured at

405-420 nm

Enzymes


3. Gamma-Glutamyltransferase (GGT)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of GGT

Chapter Outline



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


1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AMS

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


1. Amylase (AMS)a. Function, Tissue Source and Clinical Significance

Enzymes


1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determination of AMS

Enzymes


1. Amylase (AMS)b. Methods of Determination of AMS

Amylase Methodologies1. Amyloclastic2. Chromogenic3. Saccharogenic4. Continuous monitoring

Enzymes



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



Amylase Methodologies

3. ChromogenicMeasures the ↑ in colorstarch - dye starch-dye fragments

4. Continuous Monitoring

Coupling of several enzyme systems to monitor amylase activity

Enzymes


1. Amylase (AMS)b. Methods of Determination

Amylase Methodologies4. Continuous Monitoring

Coupling of several enzyme systems to monitor amylase activity

Enzymes



Amylase Methodologies1. Amyloclastic2. Chromogenic3. Saccharogenic4. Continuous monitoring

Enzymes



Amylase Isoenzymesi. Salivary Amylase • ptyalin • fast moving

ii. Pancreatic Amylase • amylopsin • slow moving

Enzymes



Amylase Isoenzymes

http://www.clinchem.org/cgi/reprint/30/3/387.pdf

Enzymes


1. Amylase (AMS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations

Chapter Outline



D. Other Enzymes1. Acid Phosphatase (ACP)2. Glucose-6-Phosphate Dehydrogenase (G-6-PD)

Enzymes


2. Lipase (LPS)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations

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


2. Lipase (LPS)

Enzymes



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



Chapter Outline




Enzymes


1. Acid Phosphatase (ACP)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations

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


1. Acid Phosphatase (ACP)

Enzymes


1. Acid Phosphatase (ACP)a. Function, Tissue Source and Clinical Significanceb. Methods of Determinations

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


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




Chapter Outline


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!

enzymes ppt

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