GenChem
Week 10
Patient Project: Kinetic tests
Today’s Agenda:
 Review patient project.
 Review kinetic clinical tests.
 Develop the day’s problem.
 Perform the challenge: determine CK
and LD activity in sample.
The Patient Project
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 Patient case studies
 Patient interview data
 Urine analysis (test strips)
 CK and LD tests
Glucose tests
Ion-selective electrodes
 Diagnosis and presentation as a poster
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Testing for lactate
dehydrogenase and
creatine kinase
The question posed to you is
“how much of each enzyme is in
your samples?” You will use two
different tests to get your answer.
What do you know about these tests:
 How do they work?
 What information do they provide?
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Kinetic
Enzyme
Pre-lab
Let′s think!
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Representing
enzyme-mediated
reactions
Kinetic testing
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What determines the rate of this reaction?
With lots of substrate, the
rate is only dependent on
the amount of enzyme
Kinetic testing
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What will the data look like?
With lots of substrate the rate
is proportional to the [LDH]
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Lactate
Dehydrogenase
What do you know about this enzyme?
How much do you expect to find in plasma and/or
urine?
What factors might cause this value to vary?
Measured activity/
Normal
activity
Figure 19-1
Serum Values following MI for CK and LD
7
6
5
4
3
2
1
0
CK
LD
0
1
2
3
4
5
6
Days following a MI
7
8
9
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Creatine Kinase
What do you know about this
enzyme?
How much do you expect to find in
plasma and/or urine?
What factors might cause this value
to vary?
Measured activity/
Normal
activity
Figure 19-1
Serum Values following MI for CK and LD
7
6
5
4
3
2
1
0
CK
LD
0
1
2
3
4
5
6
Days following a MI
7
8
9
Let′s think!
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Measuring Reaction Rates
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Let′s think!
Using Spectroscopy to measure concentrations
In your groups:
A   bC
 Discuss how you can use Beer’s Law to
determine the rate of a reaction.
Given values for  and b, A can be
measured and C calculated.
Let′s think!
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Using Spectroscopy to measure reaction rates
One U/L activity is defined as the amount of
enzyme that will catalyze the formation of one
micromole (10–6 mol) of product in one minute.
Activity:
U/L = (6590) X Rate (absorbance/min)
A proportionality constant
that takes into account the
molar absorptivity and rate
constant of the reaction.
Let′s think!
Measuring
Absorbance
In your groups review your
responses to the following
pre-lab question and come up
with a consensus answer.
Describe how you expect to go
about setting up Logger Pro to make
absorption measurements and how
to zero the spectrometer against a
standard solution.
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Let′s think!
Testing a Sample
In your groups review your
responses to the following
pre-lab question and come up
with a consensus answer.
Using the Summary of Steps given
in chapter 11 of the lab book as a
guide prepare a set of directions for
performing the reaction.
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Divvying up tasks
Your group will need data
from both procedures on
your patient samples. Each
procedure is best
performed by a pair of
students. Decide in your
group who is going to do
what.
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Performing the reactions safely.
CK & LDH test reagents - The reagents used in
these reactions are not dangerous, (unless you
drink them).
Patient Samples - These are actual human
samples. To protect yourself from inadvertent
contact you MUST wear gloves while performing
these reactions.
Liquid Waste disposal – All reagents (both used and unused) and all
patient samples must be disposed of in the “Biohazard Waste” bottle in
the waste hood.
Solid Waste disposal – All solids that have come in contact with the
samples must be disposed of in the blue bucket labeled “Biohazard
Waste” found in the waste hood. This includes all pipet tips and cuvets.
Using the Pipets
For this experiment you will be using a
Wheaton Socorex 100 uL fixed volume
pipette. Your instructor will now
demonstrate its use. Pay close attention.
Remember that the used tips are biohazard
waste.
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Let′s explore!
Your Challenge
Perform the reactions
Measure their absorbances.
Available resources:
 CK test reagent, obtained from
instructor.
 LD test reagent, you prepare.
Design and
implement an
experimental
procedure!
 automatic pipet.
 various glassware.
 Spectrometer.
You have
90 minutes
Wrap-up
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Determine the CK and LD activities in your patient
samples
Activity:
U/L = (6590) X Rate
Remember: the rate is
absorbance/minute
Progress Report
You will now complete
and submit the
progress report
covering this week and
last.
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These are to be individual efforts although
consultation is highly encouraged.
Identification: name, TA name and group
members.
Urine Test Strip Results: values and what they
mean
Precision of pH measurement: cite data,
especially measured pH values versus the
estimated values from the color chart
Precision of glucose measurement. Same as pH
Effect of fructose & galactose. What was
learned?
Effect of Bleach. The questions are in the
worksheet.
CK and LD activity: calculated answer and what
can be concluded about patient condition.