clinical enzymology.pptx

8/10/2019 Clinical enzymology.pptx

1/84

ClinicalEnzymology


2/84

ENZYMES

Organic catalyst that hasten a chemical reaction without

themselves being consumed or undergoing a chemical change.

Protein in nature with a high degree of specificity for a certain

substrate or class of substrate

SUBSTRATE

Are substances that are acted upon by enzymes


3/84

Enzyme Nomenclature

Enzymes are named acc to the ff:

A. Name of substrate with the addition of the

suffix ase

Ex.

Lipid-lipase

Proteins-protease


4/84

B. Type of reaction they catalyse

Ex.

Transferase

transfer of AMINO GROUP from one substrate to another

Kinase

transfer of PHOSPHATE GROUP from a high energy phosphate

compound to its substrate

Phosphatase effect of hydrolysis on phosphate esters

Dehydrogenase

removal of hydrogen atoms from its substrate


5/84

C. Numerical designation given by the Enzyme

Commission(E.C.)

Ex.

E.C. 1.1.127 Lactate dehydrogenase

E.C. 3.2.1.1 Amylase

1stnumberclass to which the enzyme belongs

2ndand 3rdnumbersub class

4thspecific serial number of the enzyme


6/84

GENERAL CLASSIFICATION OF ENZYMES

Based on the reaction mechanism each enzyme

catalyzes

1. OXIDO-REDUCTASES

removal or addition of electrons

Reduction-oxidation(Redox) reaction

Oxidation-loss of electron

Reduction-gain of electron

Ex.

Removal of hydrogenDehydrogenase

Lactate dehydrogenase

Glucose-6-phosphate dehydrogenase


7/84

Lactate dehydrogenase


8/84

2. TRANSFERASES

Catalyze the transfer of a chemical group from

one substrate to another

Ex.

Aminotransferase -Amino group

Aspartate aminotransferase

Alanine aminotransferase

Kinase/phosphokinase -Phosphate group

Creatine kinase/creatine phosphokinase


9/84

AST-Aspartate Aminotransferase

SGOT-Serum Glutamate Oxaloacetate Transaminase


10/84

ALT-Alanine Aminotransferase

SGPT-Serum Glutamate Pyruvate Transaminase


11/84

3. HYDROLASES

Hydrolyze the splitting of a bond by the addition of

water(hydrolysis reaction)

Ex.: ALP Alkaline phosphatase


12/84

4. Lyases

Removes groups from substrate without hydrolysis

The product contains double bonds

Ex. Aldolase


13/84

5. ISOMERASES

Intramolecular rearrangement of the substrate compound

Same molecular formula but different physical structure

6. LIGASES

Synthethases

Joins two substrate molecules together


14/84

Terms associated with enzymes:

HOLOENZYMES

Active substance formed by the combination of

coenzyme(cofactor) and apoenzyme

HOLOENZYME

APOENZYME COFACTOR

(PROTEIN MOIETY) (NON-PROTEIN MOIETY)

COENZYME

ACTIVATOR

VITAMINS METAL IONS

NAD Zn+2

NADP Ca+2


15/84

APOENZYME

Protein portion of an enzyme

Subject to denaturation, in which enzyme loses its activity

Catalytically inactive protein when cofactor is removed

Heat labile and dialyzable


16/84

COFACTOR

Non-protein subs/compounds needed by an enzyme before

enzymatic activity can be manifested

Thermostable and dialyzable

2 types:

A. Coenzyme

B. Activator

A. Coenzyme

Organic molecule

It hasten enzymatic reaction but undergoes a change or is

consumed to another product

Ex.

NAD-Nicotinamide Adenine Dinucleotide

NADP-Nicotinamide Adenine Dinucleotide Phosphate


17/84

B.Activator

Metal ion

In such, the metal ion may serve as:

A. A bridge to hold the substrate and enzyme together

B. The primary catalytic center

C. As stabilizing agent in the conformation for catalytic activity

Ex.

Amylase Cl-, Br

LDH- Zn+2

Lipase Ca+2

CPK Mg+2


18/84

ISOENZYME

Enzymes present in an individual with similar enzymatic activity

but differ in their physical biochemical and immunological

characteristics

Ex. LDHLactate dehydrogenase

LDH 1

LDH 2

LDH 3

LDH 4

LDH 5

CKCreatine kinase

CK-MB

CK-MM

CK-BB


19/84

METALLOENZYME

Enzyme whose metal ions are intrinsically part of the molecule

such as catalases and cytochrome oxidase

PROENZYME Inactive precursor of enzymes

Also referred to as zymogen

SUBSTRATE Substances acted upon by the enzyme which are specific for ach

of heir particular enzymes


20/84

ENZYME KINETICS

An enzyme(E) catalyses a reaction by combining with its substrate(S)to create an enzyme-substrate complex(ES).

E + S ---------ES

The ES complex according to Michelis and Menten can either:

dissociate back to E + S

breakdown to product(P) and free enzyme(provided that the producthas a low affinity for the enzyme)

K2 E + S ---------ES--------E + S

K3

E + S ---------ES--------P + E


21/84

ENZYME SPECIFICITY

Theories that explain the high degree of specificity of an enzyme for

their particular substrate or class of substrate:

1. Emil Fischers LOCK and KEY THEORY:

Rigid enzyme molecule into which the substrate fits The shape of the key(substrate) must conform into the lock(enzyme)

2. KochlandsINDUCED FIT THEORY

It is based on the attachment of a substrate to the active site of an

enzyme, which then causes conformational changes in the enzyme.

This theory is more acceptable because the protein molecule is

flexible enough to allow conformational changes and also allows

some explanation on the influence of hormones on enzymatic

activity.


22/84


23/84

TYPES OF REACTION ORDER:

1. Zero Order Reaction

The rate of reaction is linear with the time,

independent of concentration of the substrate and

directly proportional to enzyme concentration.

2. First Order Reaction

The rate of reaction is determined by the

concentration of substrate as well as of enzymes(therate of reaction changes continuously with time as the

substrate is consumed.


24/84

FACTORS AFFECTING ENZYME REACTIONS:

1. Enzyme concentration

in enzyme concentration,in rate of reaction

2. Substrate concentration

in substrate concentration, in rate of reaction Upon reaching maximal value of conc. of substrate, it does

not result in increased rate of reaction.

3. Temperature

For every 10 C rise in temp, results to 2-3 times increase inthe rate of reaction

37-40 Coptimum temperature for enzyme activity

>40 C-proteins undergo denaturation and loses its

functional ability


25/84

4. Hydrogen ion concentration or pH

some enzymatic reactions proceed at their fastest rate at an

optimum pH.

Extreme pH causes denaturation of enzymes.

Ex.

Pepsin--- active at pH 2.0

Alkaline phosphataseactive at pH 8.6-10


26/84


27/84

INHIBITORS:

COMPETITIVEINHIBITOR

Substances that compete with the substrate for enzyme binding

because they are chemically analogous to the substrate and bind

to the active sites of the enzyme.

High substrate concentration will overcome the effect of theinhibitor.

NON-COMPETITIVE INHIBITOR

Substances that does not resemble the substrate and bind to the

enzyme in areas other than the active site.

They do not compete with the substrate.

Increasing the substrate concentration will not overcome the

inhibition.


28/84

Units in measuring enzyme

activity International unit(IU)

Amount of enzyme that catalyzes the conversion of

1 micromole of substrate per minute under controlled condition

Katal unit(KU)

Amount of enzyme that catalyzes the conversion of 1 mole of

substrate per second under controlled condition


29/84

Means in measuring enzyme

activity A. change in coenzyme concentration

B. Increase in product concentration

C. Decrease in substrate concentration

Called an inverse technique


30/84

Pitfall in enzyme assays

Hemolysis causes falsely elevated values due to release ofenzymes from RBCs.

Serum rather than plasma is preferred specimen.

Anticoagulants have adverse effect on enzyme activity.

Lactascent or milky serum causes variable absorpton by thespectrophotometer.

Storage:

Most enzymes are stable at 6 for at least 24 hours.

For a longer period of time, temperature of -20 C or

lower are used to ensure preservation of enzyme activity CK must be kept at -70 C to retain activity.

For the LD4 and LD5, room temperature only, because it isinactivated at refrigerator temperature.


31/84

Principles of diagnostic serum

enzymology CAUSES OF INCREASED SERUM ENZYME LEVELS:

Impaired removal of enzymes from plasma.

In pathologic conditions involving tissue necrosis and

degeneration.

Increased permeability of the cell membrane.

Physiological or pathological increase in the number of cells or

increase in the rate of production of cells.


32/84

CAUSES OF DECREASED ENZYME LEVELS:

Increased removal of enzyme from the plasma.

Decreased synthesis due to organ impairment, injury or removal.

Malnutrition leading to decreased enzyme production.


33/84

oxidoreductases

A. LDH

E.C.: 1.1.1.27

Recommended Name: Lactate dehydrogenase

Systematic name: L-Lactate: NAD+ oxidoreductase

Action:

Conversion of lactic and pyruvic acid

Hydrogen-transfer enzyme

Coenzyme: NAD


34/84

EQUATION:


35/84

Tissue source:

Heart, liver, skeletal muscle, kidney and RBCs

Lungs, smooth muscles, brain

Diagnostic Significance: Cardiac, hepatic, skeletal muscle, renal and hematologic dse.

Highest level is seen in pernicious anemia and Hematologic

disorders

In AMI, LDH rise within 12-24 hrs

peak within 48-72 hrs

remains elevated for 10 days


36/84

Ldh isoenzymes

ISOENZYME TISSUE DISORDERS:

LDH-1

(heat

stable)

HHHH 14-26% Heart

RBC

MI

Hemolytic anemia

LDH-2

(heatstable)

HHHM 29-39% Heart

RBCRenal cortex

Megaloblastic anemia

Acute renal infarctionhemolysis

LDH-3 HHM

M

20-26% Lung

Lymphocytes

SpleenPancreas

Brain

Embolism

Pneumonia

LymphocytosisPancreatitis

CA

LDH-4

(heat labile)

HMM

M

8-16% Liver Hepatic injury

LDH-5

(heat labile)

MMM

M

6-16% Skeletal muscle Skeletal muscle injury


37/84

LDH-6 Alcohol dehydrogenase

Increased in Arterosclerotic cardiovascular failure

Elevated level signifies grave prognosis and impeding death

Techniques in measuring LD isoenzymes: A. Physical

Electrophoresis

Selective absorption on Diethylaminoethyl cellulose(DEAE)Solvent precipitation technique

Heat denaturation at 65 C for 30 mins

B. Chemical Substrate-product relationship

Coenzyme affinty

Differential inhibition of LD activity C. Immunological test


38/84

Sources of error:

Hemolysis

Unstable serum

Spx should be stored at 25 C and analyzed within 48 hrs

LDH-5most labile

Clinical Significance:

LD levels are markedly increased in the ff:

Megaoblastic anemia Pulmunary infarctionGranulocytic leukemia Hodgkins Dse

Hemolytic anemia Infectious mnonucleosis

Progressive Muscular Dystrophy(PMD)

l h h


39/84

Glucose-6-phosphate

dehydrogenase E.C.: 1.1.1.49

Recommended name: Glucose-6-Phosphate Dehydrogenase

Systematic name: D-glucose-6-phosphate: NADP+ 1-

oxidoreductase

Action: oxidation of glucose-6-phosphate to 6-

phosphogluconate


40/84

Tissue Source:

Adrenal cortex, spleen, thymus, lymph nodes, lactating

mammary glands and RBCs

Diagnostic Significance:

RBC

NADPH is converted into glutathione

Glutathione

Anti-oxidant

protects hgb from oxidation

Dec. G6PD=dec. NADPH=dec. Gluthathione

Dec. Gluthathionemay cause hemolysis/damage to cell membrane


41/84

Decreased G6PD

G6PD deficiency

Inherited sex-linked trait

May cause drug-induced hemolytic anemia/exposure to oxidizing

substances

Increased G6PD

Myocardial Infarction and Megaloblastic anemias


42/84

Assays:

Specimen used:

Deficiency of enzymered cell hemolysate

Elevation of enzyme levelserum

Reference Range:

10-15 u/g hgb


43/84

TRANSFERASE

AST

E.C.: 2.6.1.1

Recommended name: Aspartate aminotransferase

Systematic name: L-aspartate:2-oxaloglutarate

aminotransferase

Action:

Transaminase-synthesis and degradation of amino acid

Transfer of amino group between aspartate and a-ketoglutarate


44/84


45/84

Tissue source:

Cardiac muscle, liver, skeletal muscle

Kidney, pancreas, RBC


Hepatocellular and Skeletal Dse.

AMI

AST rise at 6-8 hrs

Peak at 24 hrs

Return to normal within 5 days

Decreased in

pregnant women


46/84

Assays: Rietmann-Frankel Method

Substrate: aspartic alpha ketoglurate

Color developer: 2,4 dinitrophenyl hydrazine

End Product: glutamic acid and oxaloacetic acid

End color: brown

Karmen Method

Indicator: Malate dehydrogenase

Absorbance: 340 nm

pH: 7.3-7.8

Babson and Read Method

Color developer: diazonium salt

End color: violet

Source of error:

Hemolysis Falsely increased results

Alcohol lowers AST values

Muscle trauma like intramuscular injectiions, exercise, or surgical operation can significantly increase ATS levels

Reference Range: 5-30 u/L


47/84

ALT

E.C.: 2.6.1.2

Recommended name: Alanine aminotransferase

Systematic name: L-alanine: pyruvate aminotransferase


48/84

Tissue source:

Liver

Liver-specific enzyme

Diagnostic significxanc:

Hepatic Disorders


49/84

Assay:

Reitman and Frankel Method

Substrate: alanine alpha ketoglutarate

End Product: glutamate and pyruvic acid

Walker method


50/84

Assay:

Indicator: LDH

Absorbance:340 nm

pH: 7.3-7.8

Source of error:

Stable for 3-4 days at 4 C

Reference

range: 6-37 u/L


51/84

CK

E.C.: 2.7.3.2

Recommended name: Creatine kinase, creatinephosphokinase

Systematic name: ATP: Creatine N-phosphotransferase

Action:

Associated with ATP regeneration in contractile and transportsystems

Physiologic Function:

Muscle cells, creatine phosphate

Production of ATP


52/84


53/84

Ck isoenzymes

ISOENZYME LOCATION

CK-1 CK-BB(BRAIN) BRAIN, BLADDER, LUNG,

PROSTATE, UTERUS,

COLON, STOMACH,

THYROID

CK-2 CK-MB(HYBRID) CARDIAC, SKELETALMUSCLE

CK-3 CK-MM(MUSCLE) CARDIAC, SKELETAL

MUSCLE


54/84

CK-BB

Highest concentrations in CNS, GI tract and uterus

Elevated in px with CA

Useful tumor-associated marker

Increased in CNS damage, tumors, child-birth

CK-MB

Major isoenzyme found in striated muscle and normal serum

Elevated in cardiac disorders

In AMI, CK rise at 4-8 hours

Peak in 12-24 hrs

Return to normal within 48-72 hours


55/84

Macro-CK

Midway between CK-MM and CK-MB

CK-Mi(mitochondrial CK)

muscle, brain, liver

Assay:

Electrophoresis

Reference method used for measurement of isoenzymes

Ion-exchange Chromatography

Radio Immunoassay(RIA)

Immuno-inhibition


56/84


57/84

GGT

E.C.: 2.3.2.2

Recommended name: Gamma-glutamyl transferase

Systematic name: (5-glutamyl) peptide: amno acid-5-glutamyl

transferase

Action: transfer of gamma-glutamyl residue from gamma-glutamyl peptides to amino acid, H2O and other smallpeptides

Involved in peptide and protein synthesis, regulation of tissuegluthathione levels and transport of amino acid across cellmembrane


58/84

Tissue source:

Kidney, brain, prostate, pancreas and liver

Diagnostic significance

Liver

Biliary ductules

Hepatobiliary disorders

Increased in patients taking drugs such as warfarin, phenobarbitaland phenytoin

Alcoholism

GGT assays:

Useful in monitoring the effects of abstention from alcohol and areused by alcohol treatment centers


59/84

Assay:

Substrate: gamma glutamyl-p-nitroanilide

Absorbance: 405 to 420 nm

Source of error:

Stable for 1 week at 4 C

Reference Range:

Male: 6-45 u/L

Female: 5-30 u/L


60/84


61/84

Optimum pH: 9.0-10.0 Activator: Mg 2+

Tissue source:

Intestines, liver, bone, spleen, placenta, kidney, RBC


Increased in:

Hepatobiliary and Bone disorders

Obstructive conditions

Pagets dse, Osteomalacia, Ricketts

Pregnancy

Decreased in:

Hypophosphatasia

Malnutrition


62/84

Considerations in ALP assays:

Physiologically elevated in growing children and in pregnant

women in the third trimester

EDTA inactivates ALP owing to chelation of magnesium ions


63/84

Alp isoenzymes

ISOENZYME

LIVER FASTEST

BONE HEAT LABILE

PLACENTA HEAT STABLE

INHIBITED BY PHENYLALANINE

INTESTINES SLOWEST


64/84

Regan-Nagao Isoenzyme

Carcino-placental ALP

Detected in various CA(lung, breast, ovarian and colon)

Assay:


65/84

Assays:

A. Substrate: beta-glycerophosphate

End Product: inorganic phosphate+glycerol

1. Bodansky

2. Shinowara3. Jones

4. Reinhart

B. Substrate: phenylphosphae

End product: phenol

1. King and Armstrong


66/84

C. Substrate: p-nitrophenyl phosphate

Product: p- nitrphenol or yellow phenoxide ion

1. Bessey, Lowry and Brock

2. Bowers and Mc comb

D. Substrate: phenolphthalein diphosphateProduct: phenolphthalein red

1. Huggins and Talalay

E. Substrate: alpha naphthol phosphate

Product: alpha naphtol

F. Substrate: Buffered phenolphthalein phosphate

End product: free phenolphthalein

1. Klein, Babson and Read


67/84

Klein, Babson and Read

Substrate: Buffered phenolphthalein phosphate

End product: free phenolphthalein

Color developer: NaOH

End color: pink

Bessey, Lowry and Brock

Substrate: p-nitrophenyl phosphate

Product: p- nitrphenol or yellow phenoxide ionColor developer: 0.02 N NaOH

End color: yellow


68/84

ACP

E.C. : 3.1.3.2

Recommended name: Acid phosphatase

Systematic name:


69/84

Tissue source:

Prostategland

RBC, Platelet, liver, spleen, milk, bone marrow

To differentiate Prostatic ACP and RBC ACP inhibitors areadded:

L-tartrate ions; Prostatic ACP

Formaldehyde and Cupric ions: RBC and Platelet ACP

Total ACP-ACP after the addition of L-tartrate= Prostatic ACP

Optimum pH: 4.9-5.0


70/84

Assays:

Methods: Substrate End product

1. Bodansky Beta-glycerophosphate Inorganic phosphate

2. Gutman and Gutman Phenyl phosphate phenol

3. Fishman and Lerner Phenyl phosphate phenol

4. King Armstrong PNPP P-nitrophenol

5. Shinowara PNPP

6. Bessy, Lowry annd

Brock

PNPP

7. Babson, Read and

Phillips

Alpha naphthyl

phosphate

Alpha-napthol

8. Roy and Hillman Thymolphthalein

monophosphate

Free thymolphthalein


71/84

Roy and Hillman

Thymolphthalein monohosphate

Most specific substrate for ACP

Color developer: Na OH- Na Carbonate solution

End color: blue

Babson, Read and Phillips

Color developer: tetra azotized orthodianisidine

End color: brown


72/84

Source of error:

Serum ACP is higher than plasma ACP

Hemolysis

Diagnostic significance:

Increased in:

Metastatic prostate carcinoma

A. moderate elevation of TOTAL ACP

Female breast CA

Pagets Dse

Hyper parathyroidism

B. Non-Prostatic ACP elevation

Niemann-Picks dse

Gauchersdse

Myelocytic leukemia

Also present in seminal fluid Used in forensic cases such as in rape


73/84

Reference range:

Prostatic ACP

0-35 IU/L

Total ACP Male: 2.5-117 IU/L

Female: 0.2-3.5 IU/L


74/84

AMS

E.C. : 3.2.1.1

Recommended name: a-amylase

Systematic name: 1,4-D-glucan glcanohydrolase

Action:

Breakdown of starch and glycogen


75/84

2 isoenzymes:

1. Salivary

Ptyalin(fast)

2. Pancreatic

Amylopsin(slow)

Starch/glycogen glucose, maltose, dextrins

Activators:

Ca2+

Cl-


76/84

Tissue source:

Pancreas, salivary glands

Skeletal muscles, small intestine, fallopian tube


Acute pancreatitis

Levels reaches 4-6 times the normal value and normalizes

within 3-4 days

Mumps, perforated petic ulcers, appendicitis, ruptured ectopicpregnancy, dissecting aortic aneurysm, biliary tract disease


77/84

Assay for enzyme activity: Amyloclastic

Decrease in substrate concentration/disappearance of starch

Saccharogenic

Amount of reducing sugars produced

Chromogenic

Measures increasing color from production of product coupled withchromogenic dye

Continous monitoring

Coupling of several enzyme system to monitor amylase activity


78/84

Reference Range:

Serum:

25-130IU/L

Urine

1-15 IU/L

Considerations in AMS assays:

Mouth pipetting is not done

Lipemic specimens cause a reduction in amylase activity


79/84

LPS

E.C. : 3.1.1.3

Recommended name: Lipase

Systematic name: Triacyl glycerol acylhydrolase

Action:

Hydrolisis of esters in the alpha-position to yield beta-

monoglycerides and fatty acids


80/84

Tissue Source: Pancreas, stomach and small intestine


Acute pancreatitis

Assay for enzyme Activity:

Cherry-Crandal

Substrate: olive oil

Indicator: phenolphthalein

End color: pink

Incubation period: 24 hrs


81/84

Tietz and Templaton Indicator: Thymolphthalein

End coor: blue

Incubation period: 4-6 hrs

Source of error:

Hemolysis

Falsely decreased result

Hgbacts as inhibitor for enzymatic activity

Reference Range:

0-1.0 IU/mL

1.0-1.5 cherry-crandal unit

l


82/84

lyases

ALDOLASE

E.C.: 4.1.2.13

Recommended name: Fructose diphophate

Systematic name: D-D-fructose-1, 6-bidiphosphate, D-

glyceraldehyde phosphate lyase Action:

Spitting of D-fructose di phosphate to D-glyceraldehyde

phosphate and dihydroxy acetone phosphate


83/84

3 isoenzymes:

A. Aldolase A- skeletal muscle

B. Aldolase B- liver, kidney and WBC

C. Aldolase C- brain

Assay:

Pinto, Kaplan and Van Dreal

Sibley and Lehninger


84/84


Severe:

Muscle degradation

Viral hepatitis

Moderate:

Gangrene

Megaloblastic anemia

Granulocytic leukemia

Metastatic liver CA

Psychosis

Trichinosis

clinical enzymology.pptx

Documents