Development and Validation of a Two-Site Immunoradiometric assay for Glypican...
Dual-Enzyme CatalyCEST
1. Simultaneous Detection of
Two Enzyme Activities with
catalyCEST MRI
GARY TYREE, GABRIELA FERNANDEZ-CUERVO, DR.
MARK PAGEL
MINORITY HEALTH DISPARITIES 2014
2. 1,600,000 new instances of cancer each year
600,000 deaths caused by cancer each year
The estimated financial costs of cancer on the US
economy was $216.6 billion in 2009
Our best tool to fighting cancer is early and
precise diagnostic techniques
Cancer
3. Chemical Exchange Saturation
Transfer (CEST) MRI scanning
Hydrogen atoms in substances are constantly
swapping between different molecules back and
forth, unless tightly bonded to the molecule in
question.
CEST sends pulses of radiation at various frequencies
measuring the amount of Hydrogen atom
exchanges in a sample. Different substances have
different frequencies at which they respond to the
CEST radiation.
For CEST scans, there needs to be a contrast agent,
a molecule that has one or more Hydrogens that
are able to transfer, so that it can show a CEST
signal.
N
H H H
O
H
H H
O
H H
O
H H
O
H H
O
H H
O
4. CEST MRI Scanning Continued
The data from a CEST scan looks like this:
The scale is inverted so that the peaks are at
the lowest points and the Saturation
Frequency gets more negative the farther
right you travel.
The large peak is water, and it is typically
shifted from its starting position at 4 ppm to 0
ppm. It’s size means that the concentration
of water molecules is high.
The other peak is the contrast agent we’re
looking for.
CEST Spectra
5. CatalyCEST MRI Scanning
In our project, we used a specific form of CEST that measures
enzyme activity, or CatalyCEST. Enzyme activity is a distinct
biomarker of cancerous cells, and if it can be detected, then it can
be utilized by finding micro-tumors indistinguishable by the human
eye and more deeply understanding the causes of the tumors
present, allowing for an improved treatment plan.
By creating a sample with an enzyme and a substance that will turn
into our contrast agent with the help of the enzyme, the strength of
the CEST signal will tell us how much contrast agent there is in the
sample, and as a result, how much enzyme must have been present
to create the contrast agent.
6. Dual-Enzyme CatalyCEST
In our project, we performed CatalyCEST, but instead
of only using one enzyme reaction to create the
contrast agent, we used two.
Our experiment was designed to test the feasibility of
a new diagnostic technique that could potentially be
more specific to identifying tumor traits than the
original CatalyCEST technique by testing for the
presence of two enzymes as opposed to one.
7. Methods
Our contrast agent was a commonly used contrast agent for
CatalyCEST, Salicylic Acid.
The enzymes were Pig-Liver Esterase (PLE) and Amino Peptidase (APEP),
which should have created Salicylic Acid from our synthesized
compound. The compound is unnamed, so we simply called it GFC-
ENZ-005.
Our reactions were always within the physiological conditions of the
enzymes (pH 7 and 37 degrees Celcius) to ensure their proper function.
We experimented with two buffer solutions, TRIS-HCL and PBS, and
determined that samples in TRIS-HCL were more soluble and continued
using TRIS-HCL for the remainder of the experiment.
There were also numerous control reactions to see where error would
arise in any step of the process
Additionally, we ran several CEST scans under different conditions for
each sample and control to optimize the data collected
9. Results
After we finished the experiment, we performed a Mass Spectrometry
(to confirm the presence of Salicylic Acid) and CEST Scan on the result.
11. Conclusions
The Mass Spec of our results shows that Salicylic Acid is present
However, the CEST scans do not detect the Salicylic Acid, and so
there is only one peak (the water)
This could have been for several reasons, but the most likely was
that the Salicylic Acid was in too low of a concentration to be
detected by CEST but high enough to be detected by mass spec.
12. Second Methods
Under our previous logic, we repeated the experiments two more
times. Once with the optimal concentration of enzyme, to allow for
the enzymes to produce the most amount of contrast agent as
possible per unit enzyme. The other time with a higher concentration
of GFC-ENZ-005, to allow for more maximal production of contrast
agent.
Everything else was the same, including control reactions, pH,
temperature, and types of CEST scans on samples.
14. Conclusions Continued
Even after optimizing the reaction, the Salicylic Acid still did not
show on the CEST scan.
If our hypothesis were true, the results should have looked like this
CEST scan of pure Salicylic Acid:
15. In the Future
For an unknown reason, the CEST scan will not show the Salicylic
Acid. We will be looking for a way to fix this error before moving on
to optimizing the reactions to produce the strongest CEST scan
possible.
If Dual-Enzyme CatlyCEST MRI is deemed plausible, then possible
follow-up research can be done for the several uses of this
technique hypothesized by the CAMEL team.
16. Special Thanks
Dr. Mark Pagel
Gabriela Fernandez-Cuervo
Stephanie Adamson
Cindy Neal
17. Works Cited
American Cancer Society. Cancer Facts & Figures 2014. Atlanta: American Cancer
Society; 2014.
Pagel, Marty. Picturing the Future of Health Care with CEST MRI. Tucson: The University of
Arizona, n.d. PPT.
Photo of cancerous tumor. Digital image. John Hopkins Department of Pathology, n.d.
Web. 1 Aug. 2014. <http://pathology.jhu.edu/pc/images/adenoca.gif>.
Hi, I’m Gary Tyree. Thanks to the Minority Health Disparities Program, I’ve been working in the Contrast Agent Molecular Engineering Laboratory, or CAMEL, under Dr. Mark Pagel and alongside Gabriela Fernandez-Cuervo. We’ve focused our efforts on determining the effectiveness of a novel cancer detection technique.
Cancer is currently one of the greatest medical challenges to overcome. Even when diagnosed, it is never a simple matter to treat a patient with cancer, as there is no cure. Without a cure, the most effective tool we have to improve the survivability of cancer is early and precise diagnostic techniques. With this in mind, our project hopes to improve current diagnostic techniques of cancer.
Our lab is working with a particular diagnostic technique called CEST MRI. This technique measures a phenomena called chemical exchange, or the transfer of Hydrogen atoms in molecules. Using the contrast agent, which can transfer its Hydrogen(s) to the water molecules near it, a CEST signal can be found at the agent’s specific energy frequency.
