The study of disease
The use of laboratory tests for the diagnosis of disease
The study of biochemical reactions of body fluids for the diagnosis of disease
What are the two types of tests that usually require serum or plasma samples?
1. Tests which detect and quantify the concentration of a certain substance (i.e. measure amount of glucose in plasma or serum) 2. Tests which detect and quantify the activity of enzymes in a sample
What are the tests which detect and quantify the activity of enzymes in a sample, known as collectively
Clinical chemistry tests are an important part of the ______ process so they must provide accurate and reliable results. In order to ensure this list the three things tthat must be correct:
-diagnostic 1. The correct sample 2. Handle the sample correctly 3. Correct testing (quality assurance)
What is enzymology?
Branch of chemistry concerned with the properties and actions of enzymes.
It has been demonstrated that changes in the activity level of specific enzymes are associated with specific ______ and as a result play a major role in ______
A protein which increases the rate of a particular reaction withour itselfbeing consumed or altered
What is the structure of an enzyme?
Consists of a chain of amino acids whose order, number and type determine the enzyme structure.
What is denaturation?
Any alteration in structure results in a loss of activity
What is the function of enzymes?
To catalyze metabolic reactions in the body
To be of clinical value enzyme testing have what three characteristics?
1. Be readily available 2. Be economically feasible 3. Reflect pathological changes in a specific organ or group of organs
It is difficult to measure enzyme levels in the ____ of interest, so we measure the enzymes that have entered the ______
What causes enzymes to enter the plasma? How are they removed?
-The breakdown of tissue cells -Removed by macrophage catabolism
Why do plasma levels of enzymes become elevated?
When a tissue is damaged or diseased, more cells die and break open, releasing more enzymes into the plasma, elevating their plasma levels
Enzymes found in the plasma can be categorized into what two groups?
1. Plasma Specific Enzymes 2. Non- Plasma Specific Enzymes
Plasma specific enzymes have high levels in the plasma and low levels in the ______. These are aka ____ ______. They are synthesized by the ____ which maintains their constant level. What might decreased levels of these enzymes in the plasma indicate (3)?
-Tissues -coagulation factors 1. Impaired liver function 2. Genetic defect 3. Toxins such as vitamin K antagonists
Non-Plasma Specific Enzymes have no known function in the ______. They are present in zero to low levels in the plasma and high levels in the _____. They are produced with the cells of specific ____ or _____ and are released into the plasma as a result of _____ _________
-Plasma -Tissues -Organs or tissues -Cell breakdown
Some enzymes may be produced by very ____ ______ or _____. Others may be produced by a ____ of _____ or _____
-specific tissues or organs -variety of tissues or organs
T or F Enzymes can have a number of different names
T (most have at least two)
Enzyme names usually end in -____ and are based on the _____ they break they work on. (There are exceptions)
Enzymes might have a _________ name which describes their reaction and include the _____ and type of reaction. Enzyme names might also be ________ to ____ letters
-systematic -substrate -abbreviated -capital
Enzymes have been classified into classes based on the types of ____ they _______. Name the three of the six that are relevant to this course.
-reactions -catalyze 1. Oxidoreductases 2. Transferases 3. Hydrolases
What reactions do the class oxidoreductases catalyse? Give an example of an enzyme.
-They catalzye oxidation reactions -Oxidases
What reactions do the class transferases catalyse? Give an example of an enzyme.
-They catalyse the transfer of a functional group between two substrates -Transaminases
What reactions do the class hydrolases catalyse? Give an example of an enzyme.
-They catalyze the hydrolysis of carbohydrates, esters and proteins -Amylase
Which class of enzymes is often found in mitochondria?
Which class of enzyme is often found in lysosomes?
When we measure enzymes we report their quantity in _____. Because enzymes come in very ____ quantities these are based on enzyme _____. They are a measure of a ____ at which the reaction proceeds.
-units -small -activity -rate
Enzyme activity is usually measured by determining a decrease in the amount of _______ or an increase in the amount of ________
For each enzyme there are often many different units why? Does this cause an issue? How was it resolved?
-This is due to different methods used to measure a specific enzymes activity level. -Causes difficulty in correlating values from one method to another. - A common unit called the International Unit is now used
Define the International Unit used to measure enzymes. How is it expressed?
-The quantity of enzyme that will catalyze the reaction of one micromole of substrate per minute at specified conditions. -Expressed as IU/L or U/L or mmol/L
Enzymes are highly specific what two respects?
1. The type of reaction they catalyze 2. The type of substrate involved
The substrate is bound to a specific region of the enzyme called the ____ site to form an ____-____ _______ (__)
-active -enzyme-substrate complex (ES)
How do enzymes actually serve as catalysts?
By reducing the activation energy of chemical reactions
Enzymes can be inhibited by specific small _____ or ____ which link to the enzyme and block the ______ site. these are known as ______ _______
-molecules or ions -active site -Enzyme Inhibitors
Some enzymes are synthesized in an inactive precursor form called a _______ and are activated at a _________ approriate time and place
What are the five factors that affect enzyme activity?
1. pH (Hydrogen Ion Concentration) 2. Temperature 3. Substrate concentration 4. Cofactors 5. Inhibitors
All enzymes are active between certain specific pH values. Maximum activity is obtained at a certain pH value and this is called the _______ __. At extremes of pH the enzyme may be _______. The test may have a _____ whose function is to keep the solution at the ______ ___
-Optimum pH -Denatured -Buffer -Optimum pH
Each enzyme has a unique optimum temperature at which it is maximally active under a given set of conditions. For most enzymes an increase in the temperature up to ___ degrees will result in an ______ rate of reaction. If the temperature is too cool then the reaction would be ____ than normal.
-45 degrees -increased -slower
When collecting and handling samples what is important to remember?
That enzymes are unstable and samples must be handled with care, if not denaturation will occur leading to inaccurate test results
At what temperature should an enzyme assay sample be stored and for how long? What if you have to store it longer?
-Refrigerate serum at 4 degrees C for up to 24 hours -If you need to store it longer freeze it at -20 degrees C to -70 degrees C
With a low substrate concentration is the reaction rate higher or lower
Lower (The reaction rate is proportional to substrate concentration until a maximum rate is reached)
As the substrate concentration _______ , more enzyme sites are _____ and the reaction rate stays at a _______ constant, in some cases if you allow the assay to run for double the time it will ______ the amount of product
-increases -filled -maximum constant -double
The reaction rate is proportional to ______ _______ until a ______ ______ is reached
-substrate concentration -maximum rate
What are cofactors? What are the two types?
-They are substances that when present with the enzyme further increase the rate of reaction 1. Coenzymes 2. Enzyme Activators
What are coenzymes? Are they essential? Give examples.
-One of the two types of cofactors. They are organic compounds required by some enzymes to catalyze reactions. -Essential to those certain enzymes which require them -Many vitamins function as coenzymes
What are Enzyme Activators? Are they essential? Give examples.
-Inorganic substances which spped up the reaction, usually through binding weakly to the enzyme. -They can be essential or nonessential -Metal ions
What is the difference between essential enzyme activators and non essential ones?
-Essential: The enzyme has no activity without the activator - Nonessential: The enzyme is active without the activator but the enzyme's activity is increased if the activator is present
What are Enzyme Inhibitors? What are the two types?
-Substances which bind to the active site of the enzyme and reduce the rate of reaction 1. Reversible 2. Irreversible
What is the difference between Reversible and Irreversible Inhibitors?
Reversible Inhibitors bind weakly tot he active site, and can be unbound, which then restores the enzyme's activity. Irreversible Inhibitors destroy the active site and permanently end the enzyme's activity.
If plasma or serum is frozen what must we be sure to do before testing?
-Mix well after thawing to redistribute constituents
What are some tips for proper sample collection (4)?
1. Careful collection of blood sample into appropriate tubes 2. Spin off plasma or serum immediately 3. Perform chemical tests within one hour of collection 4. Use sterile tubes and equipment to avoid contamination
How do you prepare a plasma sample (5)?
1. Collect in container with appropriate anti-coagulant and mix with a gentle rocking motion 2. Make sure container is covered to prevent evaporation during centrifugation then centrifuge within an hour of collection at 2000 to 3000 rpm for 10 mins 3. Carefully remove the fluid plasma layer from bottom layer of cells with a capillary pipette and transfer to a properly labelled container 5. Process immediately or refrigerate or freeze as appropriate
How long and at what rpm do we centrifuge plasma and serum samples for?
At 2000 to 3000 rpm for 10 mins
How is a sample container properly labelled(4)?
1. Date 2. Time of collection 3. Patient's name 4. Case or clinic number
How do you prepare a serum sample (7)?
1. Collect a whole blood sample in a container that contains no anticoagulant 2. Allow the blood to clot in it's container at RT for 20 -30 mins 3. Gently seperate the clot from the container by running a wooden applicator stick around the wall of the container between the clot and the wall 4. Cover sample and centrifuge 5. With a capillary pipette remove the serum from the clot 6. Transfer the serum to a properly labelled container 7. Run, refrigerate or freeze the sample as appropriate
When preparing a serum sample how long do we wait for the clot to form?
20 - 30 mins
Which sample always collected in a container containing no anticoagulant, plasma or serum? Why?
-Serum -Because we want the whole blood collected to clot so we can obtain the serum which is essentially plasma without the clotting factors.
A serum sample is being evaluated for a specific enzyme. Explain how the
enzyme level would be affected if the sample sat at room temperature for 8 hours
before being assayed. How should this sample be stored?
-Level decreased since enzymes unstable at room temperature -It should be refrigerated (4ºC).
Explain how each of these factors will affect enzyme activity a) Increasing substrate concentration b) pH not at optimum c) Temperature at 25ºC d) Temperature at 50ºC e) Running test for 10 minutes instead of 15 minutes f) Enzyme activator absent:
a) Increased with substrate concentration up to a maximum. b) Decreased activity as move away from optimum pH. c) Decreased activity, maximum activity at 37-42ºC with decreased
activity as move away from this. d) Decreased activity due to enzyme denaturation. e) No change in activity, however final amount of product formed will
be decreased due to decreased incubation time. f) Decreased activity, some enzymes require activators to speed up the
Explain why the timing of tests is critical when dealing with enzymes.
The longer the time reaction occurs, the more product formed and
the higher the results, but over time the rate of reaction will decrease.
Describe the levels of enzymes in blood and tissue when discussing plasma specific
and non-plasma specific enzymes.
Plasma specific: blood high, tissue low.
Nonplasma specific: blood low, tissue high.
State the effect of increased tissue damage on the blood level of nonplasma specific
Nonplasma specific enzymes contained within tissues; upon destruction
of tissue cells these enzymes are released into blood stream and level in blood
Using the enzyme summary chart in your module, state the significance of an increase
in the blood concentration of these nonplasma specific enzymes. a) Alkaline Phosphatase (ALP) and Aspartate Aminotransferase (AST) b) Aspartate Aminotransferase (AST) and Creatine Phosphokinase (CPK) c) Amylase and Lipase
a) Liver damage. b) Striated muscle damage. c) Pancreatitis.