This presentation is on the topic of "Developing Diagnostic Tools for Alzheimer’s Disease Using Iron Biomarkers"

1. Developing Diagnostic Tools for
Alzheimer’s Disease Using Iron
Biomarkers
By Shayan Waseh
Masters in Public Health
Thomas Jefferson University
Dr. Michael Garrick, Lin Zhao, Dr. Zohi Sternberg, Daniel Sternberg

2. Background Information

3. Alzheimer’s Disease: A Big Problem1
• More than five million elderly Americans have Alzheimer’s disease
• The population of America with Alzheimer’s disease is larger than the
entire population of Norway
• The estimated economic cost of Alzheimer’s disease for the United
States is over 200 billion dollars a year
• The estimated cost of Alzheimer’s disease in the United States is more
than the gross domestic product of Ukraine and Cuba combined

4. How Do We Cure Alzheimer’s Disease?
• First we need to be able to diagnose it:
• Currently, physicians cannot definitively diagnose
Alzheimer’s disease until a patient dies and they look at his
or her brain
• This is too late to be helpful for the patient. Even if we had a
cure, we need to know who actually has Alzheimer’s disease
to use it effectively
• Once Alzheimer’s disease damages the brain, nothing will
restore function. Even a cure would only prevent more
damage, not repair it
• It is clear that we need a way to diagnose Alzheimer’s
disease early onHealthy | Alzheimer’s

5. We may be able to use the
body’s iron homeostasis to
help diagnose Alzheimer’s
disease early on

6. 1. Long before Alzheimer’s disease
damages the brain, there is
inflammation
2. Inflammation is often caused by
infection, so the body reacts as if it is
being invaded by microbes
The body responds by
hiding much of its iron
inside body cells, where
microbes cannot easily
reach the iron and use it
to grow
3. Even though the
inflammation in early
Alzheimer’s disease is not
caused by microbes, the body
responds as if it is. Therefore,
we can test blood proteins
and hormones to see if this
response is occurring.
It may mean that the patient
has early Alzheimer’s disease
Introduction to
the Body’s Iron
Homeostasis

7. Introduction to
the Body’s Iron
Homeostasis #2
Here is a brief and simplified overview of exactly how the body
responds to inflammation by storing iron inside body cells2
Fe
1. Iron in cells is stored by binding with a protein
called ferritin while iron in the circulation is
bound with a protein called transferrin.
2. Iron enters the cell through transferrin receptors
and leaves the cell through ferroportin
3. When there is inflammation, the body
uses a hormone called hepcidin to stop
iron from leaving the cell through
ferroportin
4. This means that iron is entering cells but not
leaving them – resulting in storage inside cells

8. So What Does This Have To Do With
Alzheimer’s Disease?
• There is neuroinflammation in the brain, even decades before
Alzheimer’s disease begins to impair daily function
• Since there is neuroinflammation, we should be able to look at the
patient’s blood to see if the iron-storing reaction is occurring
• If a patient has their blood tested over several years and this process
is chronically occurring with no acute cause, then it may point
towards early Alzheimer’s disease as the cause

9. Research Methods

10. Origination of Samples and Patient Data
• Blood samples and longitudinal clinical data were collected from a
Layton Aging & Alzheimer’s Research Center patient cohort
• 57 samples from 44 Alzheimer’s patients
• 31 samples from 21 control patients
• Clinical data included socioeconomic status, age of death, diagnosis,
and mini-mental state exam (MMSE) score
• The MMSE tracks cognitive decline. It is scored out of thirty points, and asks
thirty questions and actions such as “what is the year?” or name three objects.
As mental ability deteriorates, MMSE score also declines.

11. Our Experimental Data from Patient Samples
• Blood samples were thawed at room temperature and then
centrifuged to separate the plasma which was pipetted into 96-well
plates. The plasma is where the iron-related biomarkers reside.

12. • We used quantitative colorimetric determination to measure iron
levels and unbound iron-binding capacity
• Ferrozine Stanbio kit with neocuproine to prevent copper interference,
hydroxylamine to reduce iron to its ferrous form, and hydrochloric acid to
release iron so that it can be measured.
• BioTek EL808 absorbance reader
The clear plasma sample
changes colors depending on
how much iron is in the sample –
more iron, more color
The absorbance reader
measures color intensity, so we
can calculate biomarker levels

13. • We were able to experimentally measure iron levels and unbound
iron-binding capacity.
• Samples with sufficient volume were tested twice to ensure reliability of
measurements
• Those measurements allowed us to calculate total iron-binding
capacity (TIBC), which is an indirect measure of transferrin.
• Total iron-binding capacity = serum iron + unbound iron-binding capacity
• We combined these biomarker measurements and calculations with
serum hepcidin and ferritin values from another lab and with the
longitudinal patient data we gathered.

14. • Once all of this data was gathered together, we performed exploratory
statistics to better understand how Alzheimer’s disease impacts iron
homeostasis and the body’s iron-storing response to inflammation

15. Data and Results

16. Result #1 – Serum Iron Levels Drops
As MMSE scores drop
(Alzheimer’s disease and
cognition worsen), serum
iron levels drop
This makes sense since
inflammation is
worsening, causing more
iron storage in cells

17. Result #2 – More Transferrin in Alzheimer’s
260
270
280
290
300
310
320
330
340
350
TransferrinConcentration(ug/mL)
Alzheimer's
Control
Patients with Alzheimer’s
disease had higher levels of
transferrin compared to controls
(344 ug/mL vs. 288 mg/uL)
This makes sense since iron
binds transferrin in the blood
stream, allowing it to enter cells
through transferrin receptors
Ferritin (TIBC)

18. Result #3 – More Ferritin in Alzheimer’s
Patients with Alzheimer’s
disease had more ferritin
compared to controls.
Additionally, the ratio of
serum iron to ferritin was
lower (3.17 vs 8.03)
This makes sense since iron in
the cells is stored with ferritin,
so more ferritin means more
storage is occurring

19. Result #4 – Hepcidin Is Higher in Alzheimer’s
To tie it all together, we
found that patients with
Alzheimer’s disease have
higher hepcidin levels.
These hepcidin levels
increase as MMSE score
decreases.
This is likely why we see
all the iron storing effects
in Alzheimer’s disease

20. Discussion and Conclusion

21. Model for Our Results
1
Patients with Alzheimer’s
disease have mild inflammation,
which gets worse as there is
more and more damage and
loss of brain function
2
More and more hepcidin is
released as brain damage
continues
3
This increases transferrin in the
blood and ferritin in the cells
The transferrin allows iron to enter
the cells, while the ferritin keeps
the iron in cellular storage
4
Overall cellular storage of iron

22. What Does This Mean For Alzheimer’s Patients?
• While differences in hepcidin, ferritin, and transferrin between
healthy people and people that will develop Alzheimer’s disease
are small in the early stages, they slowly become more different.
• This may allow us to use these differences to predict if someone
will develop Alzheimer’s disease or not
• This research may serve as a foundation for developing future
diagnostic tools

23. Acknowledgements and
Citations

24. Acknowledgements
• I would like to thank Dr. Michael Garrick and Lin Zhao for their
support and guidance throughout my research project
• This research was made possible through the Center for
Undergraduate Research and Creative Activities (CURCA)
Undergraduate Research Grant from the State University of New York
at Buffalo
• I would also like to thank my family and loved ones for their support,
particularly Nooshin Asadpour, as well as my parents Mandana and
Shahdad Waseh

25. Citations
1. "Latest Alzheimer's Facts and Figures." Latest Facts & Figures
Report. Alzheimer's Association, 29 Mar. 2016. Web. 22 Feb.
2017.
2. Garrick, Michael D., and Laura M. Garrick. "Cellular iron
transport." Biochimica et Biophysica Acta (BBA)-General
Subjects 1790.5 (2009): 309-325.

26. Thank You!