m. zaharna clin. chem. 2009 enzymes part 1. m. zaharna clin. chem. 2009 introduction enzymes are...

M. Zaharna Clin. Chem. 2009

EnzymesPart 1


Introduction Enzymes are usually proteins that act as

catalysts, compounds that increase the rate of chemical reactions.

They bind specifically to a substrate, forming a complex.

This complex lowers the activation energy in the reaction: owithout the enzyme becoming consumed o and without changing the equilibrium of the

reaction. A product is produced at the end of the reaction

Introduction The catalyzed reactions are frequently specific

and essential to physiologic functions, such as:o the hydration of carbon dioxide, o nerve conduction, o muscle contraction, o nutrient degradation, o and energy use.

Found in all body tissue, enzymes frequently appear in the serum following cellular injury or, sometimes, in smaller amounts, from degraded cells.



General Properties of Enzymes

Like all proteins 1°, 2°, 3°, and 4° structures

Active site → cavity where substrate interactso Often water-free siteo Reacts with charged amino acid

residues

Allosteric siteo Another site on enzyme where co-

factors or regulatory molecules interact


Isoenzymes

Isoenzymes – are enzymes that differ in amino acid

sequence but catalyze the same chemical reaction.

They have similar catalytic activity, but are different

biochemically or immunologically.

Different forms may be differentiated from each other

based on certain physical properties

o electrophoretic mobility,

o differences in absorption properties

o or by their reaction with a specific antibody


Cofactors

Non-protein molecules required for enzyme activation

• Inorganic Activators– Chloride or magnesium ions, etc.

• Organic Coenzymes– e.g. Nicotinamide adenine dinucleotide (NAD)


Classes of Enzymes

International Union of Biochemistry (IUB)1 = Oxidoreductases (Examples: LDH, G6PD)

• Involved in oxidation - reduction reactions

2 = Transferases (Examples: AST, ALT) • Transfer functional groups

3 = Hydrolases (Examples: acid phosphatase, lipase)• Transfer groups to -OH

4 = Lyases (Examples: aldolase, decarboxylases) • Add across a double bond

5 = Isomerases (Example: glucose phosphate isomerase)• Involved in molecular rearrangements

6 = Ligases Complicated reactions with ATP cleavage• Catalyze the joining of two substrate molecules


Enzyme classification

Plasma vs. non-plasma specific enzymes

a. Plasma specific enzymes have a very definite/specific function in the plasma

1) Plasma is normal site of action

2) Concentration in plasma is greater than in most tissues

3) Often are liver synthesized

4) Examples: cholinesterase, plasmin, thrombin


Enzyme classification

b. Non-plasma specific enzymes have no known physiological function in the plasma

1) Some are secreted into the plasma

2) A number of enzymes associated with cell metabolism normally found in the plasma only in low concentrations.– An increased plasma concentration of these

enzymes is associated with cell disruption or death


Factors Affecting Enzyme Levels in Blood

Entry of enzymes into the bloodo Leakage from cellso Altered production of enzymes

• E.g. increased osteoblastic activity results in increase in enzymes in bone disease

Clearance of enzymesoHalf life vary from few hours to several days

Factors That Influence Enzymatic Reactions

o Substrate Concentration o Enzyme Concentrationo pH o TemperatureoCofactorso Inhibitors



Measuring enzyme activity Enzymes are usually present in very small quantities in

biologic fluids and often difficult to isolate from similar compounds

Therefore, Enzymes are Therefore, Enzymes are notnot directly measured directly measured Enzymes are commonly measured in terms of their

catalytic activity We don’t measure the molecule … But we measure how much “work” it performs (catalytic

activity) That means the rate at which it catalyzes the conversion

of substrate to product The enzymatic activity is a reflection of its concentration Activity is proportional to concentration


Measuring enzyme activity

Enzyme activity can be tested by measuring

o Increase of producto Decrease of substrateo Decrease of co-enzyme

o Increase of altered co-enzyme

If substrate and co-enzyme are in excess concentration, the reaction rate is controlled by the enzyme activity.


NADHNADH ( a common co-enzyme ) ( the reduced form ) absorbs light at 340 NM o NAD NAD does not absorb light at 340 nm

o Increased ( or decreased ) NADH concentration in a solution will cause the Absorbance ( A ) to change.

Measuring enzyme activity


Measurement Conditions

o Excess amounts of substrate and any cofactors or coenzymes

• to handle possible abnormally high patient enzyme levels

o Proper temperature and pHo Inhibitors must be lacking o The temperature should be constant within

±0.1°C throughout the assay at a temperature at which the enzyme is active


Methods for Enzyme measurement

Fixed time methodso the reactants are combined, o the reaction proceeds for a designated time,o the reaction is stopped (usually by inactivating the

enzyme with a weak acid), o a measurement is made of the amount of reaction

that has occurred. o The reaction is assumed to be linear over the reaction

time; the larger the reaction, the more enzyme is present.

o Possible problems with extremely high enzyme levels



Continuous-monitoring methodso multiple measurements, usually of absorbance

change, are made during the reaction, o either at specific time intervals (usually every 30

or 60 seconds) o or continuously by a continuous-recording

spectrophotometer. o These assays are advantageous over fixed-time

methods because the linearity of the reaction may be more adequately verified.


Continuous-monitoring methodso If absorbance is measured at intervals,

several data points are necessary to increase the accuracy of linearity assessment.

oContinuous measurements are preferred because any deviation from linearity is readily observable.



Measurement Units

Reported as “activity” not concentrationo IU = amount of enzyme that will convert 1

μmol of substrate per minute in specified conditions

oUsually reported in IU per liter (IU / L)SI unit = Katal = mol/sec

omoles of substrate converted per secondo enzyme reported as katals per liter (kat / L)o 1 IU = 17nkat


Measurement of Enzyme Mass

Immunoassay methodologies that quantify enzyme concentration by mass are also available and are routinely used for quantification of some enzymes.

Immunoassays may overestimate active enzyme as a result of:

• possible cross-reactivity with inactive enzymes,

• inactive isoenzymes,

• or partially digested enzyme.

Measurement of Enzyme Mass

The relationship between enzyme activity and enzyme quantity is generally linear but should be determined for each enzyme.

Enzymes may also be determined and quantified by electrophoresis techniques which provide resolution of isoenzymes.



Creatine Kinase (CK)

Action – This enzyme is associated with the regeneration and storage of high energy phosphate (ATP).

It catalyzes the following reversible reaction in the body.



High concentrations of CK in: o skeletal muscle, o cardiac muscleo and brain tissue

Increased plasma CK activity is associated with damage to these tissues

CK is especially useful to diagnose:• Acute Myocardial Infarction (AMI)• Skeletal muscle diseases ( Muscular Dystrophy )



CK has 3 isoenzymesEach isoenzyme is composed of two

different polypeptide chains (M & B)– CK - BBCK - BB (CK1) Brain type

– CK - MBCK - MB (CK2) Cardiac type or hybrid type

– CK – MMCK – MM (CK3) Muscle type

o Normal serum consists of approximately 94% to 100% CK-MM

o Cardiac muscle CK is 80% CK-MM and 20% CK-MB


o BB migrates fastest to anode than MB & MMo The major isoenzyme in the sera of healthy people is

the MM form. o Values for the MB isoenzyme range from undetectable

to trace (<6% of total CK). o It also appears that CK-BB is present in small quantities

in the sera of healthy people



The value of CK isoenzyme separation can be found principally in detection of myocardial damage.

Cardiac tissue contains significant quantities of CK-MB, approximately 20% of all CK-MB.

increased CK – MB ( > 6% of the total CK activity ) is a increased CK – MB ( > 6% of the total CK activity ) is a strong indication of AMIstrong indication of AMI

Post AMI CK-MBo CK-MB increases 4 – 8 hours post AMIo Peaks at 12 - 24 hours post AMI o Returns to normal 48 - 72 hours

Diagnostic Significance


CK AssayCK assays are often coupled assays. In the example below, the rate at which

NADPH is produced is a function of CK activity in the first reaction.

Hexokinase and G6PD are auxiliary enzymes

Reverse reaction most commonly performed in clinical laboratory methods

CK Assay

Reference Range for Total CK:oMale, 15-160 U/L (37°C)o Female, 15-130 U/L (37°C)oCK-MB: <6% total CK



CK isoenzymes

For CK isoenzymes, electrophoresis is the reference method.

Other methods include ion-exchange chromatography, and radioimmunoassay.

Rapid assay for CK-MB subforms, uses high voltage electrophoresis on an automated analyzer, the result will be available in 25 min.


Lactate Dehydrogenase (LD) Catalyzes interconversion of lactic and pyruvic acids It is a hydrogen-transfer enzyme NAD is used as coenzyme

High activities in heart, liver, muscle, kidney, and RBC Lesser amounts: Lung, smooth muscle and brain


LDH Isoenzymes

Because increased total LDH is relatively non-specific, LDH isoenzymes can be useful

5 isoenzymes composed of a cardiac (H) and muscle ( M ) component

o LD - 1 ( HHHH ) Cardiac , RBCso LD - 2 ( HHHM ) Cardiac , RBCs o LD - 3 ( HHMM ) Lung, spleen, pancreaso LD - 4 ( HMMM ) Hepatico LD - 5 ( MMMM ) Skeletal muscle

LD-1 is the fastest towards the anode

Diagnostic Significance LDH is elevated in a variety of disorders.

o in cardiac, o hepatic, o skeletal muscle, o and renal diseases, o as well as in several hematologic and neoplastic disorders

The highest levels of LD-1 are seen in pernicious anemia and hemolytic disorders

LD-3 with pulmonary involvement LD-5 predominates with liver & muscle damage



Diagnostic Significance In healthy individuals

o LD-2 is in highest quantity then LD-1, LD-3, LD-4 and LD-5

Heart problems: 2-10 x (Upper Limit of Normal) ULN in acute MI

o If problem is not MI, both LD1 and LD2 rise, with LD2 being greater than LD1

o If problem is MI, LD1 is greater than LD2.

• This is known as a flipped pattern


A sixth LDH isoenzyme has been identifiedLDH-6 has been present in patients with

arteriosclerotic cardiovascular failure Its appearance signifies a grave prognosis

and impending death It is suggested, that LDH-6 may reflect liver

injury secondary to severe circulatory insufficiency



Assay for Enzyme activity

LD

• The reaction can proceed in either a forward or reverse direction

Pyruvate + NAD+ Lactate + NADH + H+

• The optimal pH: – for the forward reaction is 8.3 – 8.9– For the reverse reaction 7.1 – 7.4

• Reference Range : 100-225 U/L (37°C)


Aspartate Aminotransferase (AST, SGOT, GOT)Aspartate Aminotransferase (AST, SGOT, GOT)

Transferase class of enzymes - transaminase Transaminase involved in the transfer of an amino group

between aspartate and -ketoacids.

Pyridoxal phosphate is coenzyme Source is heart, liver, and skeletal muscle

The older terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT)

Aspartate Aminotransferase (AST, SGOT, GOT)Aspartate Aminotransferase (AST, SGOT, GOT)


• The transamination reaction is important in intermediary metabolism because of its function in the synthesis and degradation of amino acids.

• The ketoacids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to provide a source of energy.



The clinical use of AST is limited mainly to the evaluation of hepatocellular disorders and skeletal muscle involvement.

Post AMIo Rises 6 – 8 hourso Peaks at 24 hourso Returns to normal by day 5

AST levels are highest in acute hepatocellular disorders, viral hepatitis, cirrhosis.

o Viral hepatitis may reach 100 x ULN


There are two isoenzyme fractions located in the cell cytoplasm and mitochondria, o the cytoplasmic isoenzyme is predominant in serum o while the mitochondrial one may be increased

following cell necrosis.

Isoenzyme analysis of AST is not routinely performed in the clinical laboratory.



Assay for Enzyme activity

Measurement by Karmen method – use Malate dehydrogenase in second step

Detect change in absorbance at 340 nm

Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD

MD

AST

Reference Range : 5 to 30 U/L (37°C)

m. zaharna clin. chem. 2009 enzymes part 1. m. zaharna clin. chem. 2009 introduction enzymes are...

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