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2.2 Clinical Chemistry > Enzymes > Flashcards

Enzymes Flashcards

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1
Q

How can the activity of an enzyme be monitored?
(2)

A

By following the concentration of substrate or product change with time

Following the progress of the reaction the substrate will decrease and the product will build up

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2
Q

What do we often use to measure enzymes?

A

Artificial substrates

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3
Q

Why do we use artificial substrates
(2)

A

As enzymes work so fast we use artificial substrates

This slows the reaction down so we can easily measure the rate

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4
Q

Why do we use artificial substrates
(2)

A

As enzymes work so fast we use artificial substrates

This slows the reaction down so we can easily measure the rate

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5
Q

Comment on the initiation of an enzyme reaction
(3)

A

The reaction may not start immediately when the reagents are mixed

There may be a lag time required to permit formation of a measurable product

Typically, the most accurate value is obtained when the reaction just gets going i.e. initial velocity

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6
Q

What are we measuring in a kinetic enzyme assay?

A

We are measuring the change of absorbance over time or the rate at which the absorbance changes

It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present (first order of kinetics)

This is the linear phase of the progress curve

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7
Q

What is the first order kinetics

A

It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present

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8
Q

Define velocity

A

Reaction progress per unit time

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9
Q

What are the three different types of progress curves?

A

A = rate is constant
B = rapidly becomes non-linear
C = there is a bigger lag phase

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10
Q

What are coupled enzyme assays?
(2)

A

Reactions linked together such that a second reaction generates a compound which can be measured spectrophotometrically

The indicator reaction needs to be directly proportional to the rate of reaction (product formation) in the primary reaction

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11
Q

Give an example of a coupled enzyme assay

A

Measurement of AST

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12
Q

What are the two reactions in a coupled enzyme assay

A

Primary assay reaction
Indicator reaction

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13
Q

Give some examples of enzymes used

A

Urease
Uricase
Glucose Oxidase
Perioxidase
Hexokinase
Cholesterol Oxidase
Lipase
Alkaline phosphatase
Reverse transcriptase

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14
Q

Write about measuring enzyme substrates
(3)

A

Can also exploit the specificity of an enzyme to measure the concentration of a substance

Typically the enzymes substrate

e.g. a common method to measure glucose based on glucose oxidase - peroxidase (GOD-PAP)

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15
Q

Write about the GOD-PAP method
(6)

A

An end point method/equilibrium method

Using a coupled system to produce a colour

The glucose oxidase gives the specificity

The peroxidase generates the colour

A trapping reaction is necessary to ensure that the enzyme will quantitatively convert substrate to product

This traps the product and makes the reaction go to completion

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16
Q

Give three examples of indicator reactions in coupled assays

A

The production of H2O2 by an oxidase is followed by production of the red compound quinoneimine (cholesterol, TAGs, glucose)

The consumption of NADH is followed by a decrease in absorbanace at 340nm (ALT, AST)

Release of the yellow coloured nitroanilide (GGT, ALP)

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17
Q

What are the five advantages of enzyme assays?

A

Specificity -> contrast with chemical methods

Sensitivity -> nmol and pmol amounts easy to do

Versatility -> using coupled systems we can assay virtually anything

Reproductibility -> as long as the same amount of enzyme is added to the tube the reaction rate will be the same

Ease of use -> enzymes like mild conditions: most chemical methods need harsh ones

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18
Q

Write about immobilised enzymes

A

Enzymes can also be immobilised onto surfaces

Allow for the measurement of the enzymes substrate (specific analytes)

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19
Q

What are some clinical applications of immobilised enzymes?

A

Immobilised enzyme: used for the detection of abnormal substances in urine

e.g. Paper coated with GOD-PAP for glucose in urine

20
Q

What 7 factors affect enzyme reactions?

A

Enzyme concentration

Substrate concentration

pH

Temperature

Inhibitors and activators

Coenzymes and prosthetic groups

Timing

21
Q

Write about enzyme assay standardisation
(3)

A

One analyte may have a large number of different methods or different assay conditions used to measure it

This makes it difficult to compare results from different labs

This also makes it more difficult for clinicians to interpret results

22
Q

Why is standardisation of assay conditions critical?
(4)

A

Comparable results

Common units

Common reference ranges

Indication of trueness (accuracy)

23
Q

How is enzyme assay standardisation done?
(4)

A

Enzyme preparation is generated with known catalytic activity

Used as a standard (reduces variability)

Leaves a path of ‘traceability’ patient results can be traced back to the original standard

Requires co-operation between international bodies, manufacturers, labs

24
Q

How do manufacturers standardise assay conditions?

A

By optimising:
-pH
- Temperature
- Substrate conditions
- Method parameters (kinetic versus endpoint)
- Units

25
Q

How do we standardise assays in the lab?

A

We ensure that:
- assay protocols are followed
- control material is assayed
- instrumentation is maintained and in proper working order

26
Q

Why do we need enzyme units?
(3)

A

Enzymes are not always available in pure form: they are often purified from tissues

e.g. trypsin from the intestine; the greater the activity per unit of protein the more pure the enzyme preparation

As the preparation may not be 100% pure you need to know what the activity of your preparation is i.e. how active the enzyme is

27
Q

What is the enzyme unit of measurement?

A

The katal

28
Q

What is the katal?
(3)

A

The SI unit for catalytic activity

Based on two other SI units: the mole (amount) and the second (unit of time)

The katal is the amount of enzyme which will catalyse the transformation of one mole of substrate to product per second or moles/sec

29
Q

What do we usually use instead of the katal and why?

A

The katal is usually very small numbers e.g. nanokatals or microkatals

Instead we use international units which are more practical

30
Q

What is the international unit

A

The amount of enzyme which will catalyse the transformations of 1 micromole of the substrate to product per minute per litre, under standard conditions: umol/L/min

31
Q

What is functional serum enzyme?

A

Responsible for reaction taking place in blood e.g. clotting of blood

32
Q

What are non functional serum enzymes?

A

Do not have their function in blood but they are found in low concentrations in serum due to wear and tear of tissues and normal cell turnover

33
Q

What are the three types of enzymes in serum?

A

Functional serum enzyme
Non functional serum enzymes
Obstruction to secretory pathway

34
Q

What causes enzymes in serum due to obstruction to secretory pathway?

A

Reflux back into blood because of blockage

35
Q

Comment on the clinical significance of enzymes
(2)

A

Enzymes are normally intracellular and injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream

Serum enzyme level may be increased by disease that cause increase in its rate of release or decrease in their rate of excretion

36
Q

Comment on the clinical significance of enzymes
(2)

A

Enzymes are normally intracellular and injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream

Serum enzyme level may be increased by disease that cause increase in its rate of release or decrease in their rate of excretion

37
Q

What are 9 commonly assayed enzymes

A

Transaminases - ALT and AST
Alkaline phosphate
Acid phosphatase
Gamma-glutamyl transferase
Amylase
Lipase
Creatinine kinase
Lactic dehydrogenase
Prostate specific antigen

38
Q

What are isoenzymes?
(5)

A

Enzymes that occur in different molecular forms which differ in their physiochemical properties but catalyse the same chemical reaction

They vary with respect to their kinetic parameters, electrophoretic mobility and localisation

They all have independent action

They can be used to identify the specific affected tissues

They can be differentiated from each other and can be clinically quantified in the lab

39
Q

What are the three main problems with measuring enzyme activity

A

Sometimes can be common to more than one tissue

Timing of sample collection is critical

Macroenzymes -> rarely pathological but can cause confusion

40
Q

How can enzymes being more common to more than one tissue cause problems?

A

An increase in the activity of the enzyme could reflect damage to one of several different tissues

Two ways of overcoming this problem
- measuring test profiles (more than one analyte associated with the disease)
- measuring specific isoforms or isoenzymes

41
Q

Why might timing of sample collection cause problems?

A

Enzymes will rise and then fall as they are cleared

42
Q

Why might macroenzymes cause problems

A

Enzymes can form complexes causing reduced clearance

43
Q

How is greater specificity is achieved in what three ways?
(3)

A

Interpreting investigations in the light of clinical features e.g. jaundice patient with high ALT in hepatitis and therefore not muscle injury

Combining measurement with other analytes
- ALP up in cholestasis and bone diseases
- confirmed by increased GGT in cholestasis

Isoenzyme determination
- CK up in muscle injury
- CK-MB up in cardiac muscle injury

44
Q

What enzyme is defective in albinism

A

Tyrokinase

45
Q

What enzyme is defective in Glucose 6 phosphate dehydrogenase

A

G6PD deficiency

46
Q

What three areas can enzymes be used in therapy?

A

Substitution of missing production of digestive enzymes (e.g. digestive enzymes) e.g. pepsin, trypsin

Removal of deposits of death tissue or fibrin (e.g. in lungs or eyes) treatment of skin defects -> proteinases, nucleases, collagenase

Clot lysis in myocardial infarction
- streptokinase, urokinase

2.2 Clinical Chemistry (28 decks)

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