1. Alzheimer remedy on amyloid plaque formation
Oscar Heller (9th grade)
Pittsburgh Central Catholic High School

2. Existing Problem Alzheimer’s disease affects:
44 million people around the world
5.5 million Americans
200,000 Americans with early on-set Alzheimer’s
10% of all Americans aged 65 and older.
Research indicates that there is a high correlation between Alzheimer’s disease and metals such as aluminum, calcium, etc.

3. Background: Amyloid plaques
Beta amyloid = protein fragment snipped from an amyloid precursor protein (APP.)
In a healthy brain, these protein fragments are broken down and eliminated.
In Alzheimer's disease, the fragments accumulate to form plaques.
Why this occurs is unknown

4. Background: past studies
Metal-induced neurotoxicity has been associated with multiple neurological diseases in humans, including Alzheimer’s disease
Chinese and American researchers conducted research to determine if there is any valid relationship between higher blood levels of various heavy metals and the risk for developing Alzheimer’s.
How does one detect the concentration of metals in human brain tissue?

5. Background: Using 8-hydroxiquinolline /KBr (potassium bromide)
-8-hydroxiquinolline = complexing agent used to detect metals by molecular structure
-Contains a nitrogen functional & a carboxyl group
-Attracts metals through electromagnetic forces
-KBr was used to buffer the 8- hydroxiquinolline to avoid mismeasurement.

6. Background: Using an ft-iR
A Fourier Transform Infrared Spectrophotometer (FT-IR) was used.
An unknown liquid/solid sample is placed in path of infrared (IR) light.
Produces spectrum of light absorbed & frequency of IR light
Can give hints about the chemical structure of the unknown substance.

7. Background: using an ft-ir (cont.)
Frequencies emitted from a sample (brain tissue) can identify substances.
Done by analyzing the frequencies, and comparing those frequencies
Offers a practical way to evaluate if common metals are present in the brain tissue of Alzheimer’s patients

8. purpose
To determine if there is a statistically significant correlation between metals (such as aluminum) deposited in brain tissue and Alzheimer’s disease.
Question: Is there a link between the presence of Alzheimer’s disease and metals?

9. Null: There is no statistically significant difference in metal concentrations in brain tissue between Alzheimer’s patients and non-Alzheimer’s patients.
Alternative: There is a substantial statistical difference in metal concentrations in brain tissue between Alzheimer’s patients and non- Alzheimer’s patients.
Hypothesis

10. Materials Example of 3 Different FT-IR machines Materials:
Latex gloves
Dry ice (storage)
Sterile test tubes (storage)
Brain tissue samples from 6 patients with Alzheimer’s disease
Brain tissue samples from 2 patients without Alzheimer’s disease
An FT-IR
Windows laptop (to display data)
Wrenches
Bolt and nuts
Vice
8-hydroxiquinolline
KBr
A bowl and mortar
Example of 3 Different FT-IR machines

11. procedure
While wearing latex gloves, extracted mg of brain tissue from a deceased human patient. (for 2 trials)
Mixed brain tissue sample with 2 mg of 8-hydroxiquinolline and 25 mg of KBr.
Used a bowl and mortar to mix the three items into a fine substance.
Inserted a screw into a bolt and pour the powder into it.
Inserted another screw on the opposite side and exerted extreme amounts of pressure on the powder substance until the powder became a glassy substance.
Placed the bolt (with the glassy substance in the middle) into the FT-IR and ran analysis to obtain a graph that displayed peaks of the frequencies found in the sample.
Analyze the peaks by comparing the observed spectra to a spectrum generated from a sample of pure aluminum
Repeat steps 1 through 7 on samples from 3 patients with pathologically proven Alzheimer’s disease and 1 patient without Alzheimer’s disease (control group) for Trial 1.
Repeat steps 1 through 7 on samples from 3 additional patients with pathologically proven Alzheimer’s disease and 1 additional patient without Alzheimer’s disease (control group) for Trial 2.

12. Trial one (superimposed graphs)
Concentration of Metals in an Alzheimer Patient’s Brain Tissue vs Concentration of Metals in a non-Alzheimer’s Patient Brain Tissue (Control Group)
P-value: E-05, indicates significant variation
Key:
--: non-Alzheimer patient (control)
--: Alzheimer patient 1
--: Alzheimer patient 2
--: Alzheimer patient 3

13. Trial two (superimposed graphs)
Concentration of Metal in an Patient with Alzheimer’s Amyloid Plaque vs. Concentration of Metal in the Brain Tissue of a non-Alzheimer’s Patient (Control Group)
P-value: E-06, indicates significant variation
Key:
--: non-Alzheimer patient (control)
--: Alzheimer patient 1
--: Alzheimer patient 2
--: Alzheimer patient 3

14. Analysis of graphs
How does one know that the peaks indicate metals? Solution: Aluminum exhibits a specific peak. Peaks can be seen in Alzheimer’s amyloid plaque

15. Analysis of graphs (cont.)
Spectrum of Pure Aluminum

16. Analysis of graphs (cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 1, Alzheimer patient 1)
Aluminum sample
Alzheimer Patient 1

17. Analysis of graphs(cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 1, Alzheimer patient 2)
Aluminum sample
Alzheimer Patient 2

18. Analysis of graphs (cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 1, Alzheimer patient 3)
Aluminum sample
Alzheimer patient 3

19. Analysis of graphs (cont.)
Comparison between spectrum from pure Aluminum sample vs. Control Group (non-Alzheimer Brain Tissue)
No distinct similarity in peaks
Aluminum sample
Non-Alzheimer Patient 1

20. Analysis of graphs (cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 2, Alzheimer patient 4)
Aluminum sample
Alzheimer patient 4

21. Analysis of graphs (cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 2, Alzheimer patient 5)
Aluminum sample
Alzheimer patient 5

22. Analysis of graphs (cont.)
Similarities between spectrum from pure aluminum sample and spectrum observed from Alzheimer Brain Tissue (Trial 2, Alzheimer patient 6)
Aluminum sample
Alzheimer patient 6

23. Analysis of graphs (cont.)
Comparison between spectrum from pure Aluminum sample vs. Control Group (non-Alzheimer Brain Tissue)
No distinct similarity in peaks
Aluminum sample
Non-Alzheimer Patient 2

24. Know this because of similar peaks
Aluminum is observable in brain tissue of Alzheimer patients (reject null)
Know this because of similar peaks
Aluminum is not detected in brain tissue of non-Alzheimer patients (control group)
ANOVA and Dunnett’s test show statistically significant variation in peaks between control group and Alzheimer patients
Variation in peaks shows that there is a link between aluminum and Alzheimer’s disease
Conclusion

25. Inconsistencies in measurement due to human error
Through FT-IR testing, aluminum was the only metal that was clearly present in the brain tissue of Alzheimer’s patients
Finding other metals within the large number of peaks would require technology beyond this experiment
Inconsistencies in measurement due to human error
Limitations

26. Further experimentation
More experimentation is needed in order to determine if metals help cause Alzheimer’s disease or if the presence of metals is a side effect of Alzheimer’s disease.
To determine if other metals are present with more precise and advanced technology
Further experimentation

27. Works cited
Brkovich , Zachary. “Alternative Treatments.” Alzheimer's Association, Alzheimer's Association, 2016,
Kepp, Kasper. “Alzheimer's Disease: How Metal Ions Define β-Amyloid Function.” NeuroImage, Academic Press, 20 May 2017,
Miller, Lindsey. “New Study Shows Potential Link between Brain Metals and Alzheimer's Disease.” Alzheimer's Society, 2018, between-brain-metals-and-alzheimers-disease.
Ramsey, Lydia. “The 10 Most Popular Prescription Drugs in the US.” Business Insider, Business Insider, 28 Dec. 2017,
Shantz, David. “Treatments.” Alzheimer's Association, Alzheimer's Association, Mar. 2016,

28. Anova test for trial one
P-value: E-05, indicates significant variation
Trial One Raw Data (T%)
Control Group
Amyloid Plaque 1
Amyloid Plaque 2
Amyloid Plaque 3 30 15 16 1 25 19 20
1.5 44
15.3 23 17 12
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1 4
3.5
0.875
Column 2
122
30.5
Column 3
66.3
16.575
Column 4 63
15.75
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
3.2703E-05
Within Groups 12
Total 15

29. Anova test for trial two
P-value: E-06, indicates significant variation
Trial two raw data (T%)
Control Group
Amyloid Plaque 1
Amyloid Plaque 2
Amyloid Plaque 3
0.1
0.7 1
0.5
0.9
0.4
0.6
0.8
0.2
0.82
0.95
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1 4
0.3
0.075
Column 2
3.02
0.755
Column 3
3.65
0.9125
Column 4
2.2
0.55
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
1.2404E-06
Within Groups 12
Total 15

30. If t > t crit (3.618): reject null (significant variation)
Dunnett’s tests
If t > t crit (3.618): reject null (significant variation)
If t < t crit (3.618): fail to reject null (insignificant variation)
Trial one:
Amyloid Plaque 1: – 0.875/sqrt 2 x 26.09/4 = 8.202, significant
Amyloid Plaque 2: /sqrt 2 x 26.09/4= 4.435, significant
Amyloid Plaque 3: – 0.875/sqrt 2 x 26.09/4 = 4.118, significant
Trial two:
Amyloid Plaque 1: /sqrt 2 x 0.013/4 = 8.434, significant
Amyloid Plaque 2: – 0.075/sqrt 2 x 0.013/4 = , significant
Amyloid Plaque 3: – 0.075/sqrt 2 x 0.013/4 = 5.892, significant