Developing Diagnostic Tools for Alzheimer’s Disease Using Iron Biomarkers

By Shayan WasehMasters in Public Health

Thomas Jefferson University

Dr. Michael Garrick, Lin Zhao, Dr. Zohi Sternberg, Daniel Sternberg

Background Information

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

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 on

Healthy | Alzheimer’s

We may be able to use the body’s iron homeostasis to help diagnose Alzheimer’s

disease early on

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

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

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

Research Methods

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.

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.

• 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

• 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.

• 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

Data and Results

Result #1 – Serum Iron Levels DropsAs 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

Result #2 – More Transferrin in Alzheimer’s

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Alzheimer'sControl

Tran

sfer

rin C

once

ntra

tion

(ug/

mL)

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)

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

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

Discussion and Conclusion

Model for Our Results1

Patients with Alzheimer’s disease have mild inflammation,

which gets worse as there is more and more damage and

loss of brain function

2More and more hepcidin is released as brain damage

continues

3This 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

4Overall cellular storage of iron

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

Acknowledgements and Citations

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

Citations1. "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.

Thank You!