Dietary Antioxidants Against Free Radical Oxidation

A Meta-Analysis
Alexander Han – Korea International School

Background – The drastic difference between antioxidant levels in plasma of AD patients and controls
have been difficult to visualize. I was performed a meta-analysis to assess this association between
antioxidant levels and cognitive impairment.

Methods and Results – A comprehensive search was performed from JSTOR and NCBI databases with
a restriction on the publishing date from 1990 to present. Each study was reviewed independently by 3
investigators. 11 sources with a total of 1,172 subjects (404 AD patients, 768) met the inclusion criteria.
The mean vitamin C level for controls was 46.88μmol/l while the mean for AD patients was drastically
lower at 29.56μmol/l. Similarly, the mean vitamin E level for AD patients (24.93μmol/l) was lower in
comparison to controls (31.44μmol/l).

Conclusions – Vitamin C and E levels are both significantly lower for AD patients in contrast to normal
subjects. The drastic difference of antioxidant level bolsters the notion that Aß peptides induce oxidative
stress, suggesting an intervention backed by dietary antioxidant may be effective for AD.

As the risk for Alzheimer’s disease (AD)
is clearly correlated with advanced age, the
number of those affected by the
neurodegenerative disease is also expected to
increase exponentially [1]. With this imperative
issue in hand, I decided to conduct a report on
dietary antioxidants, a panacea for the hallmark
of AD: amyloid beta (Aß) peptides.
An Aß peptide results from
amyloidogenic processing of amyloid beta
peptides (APP). During amyloidogenic
processing, APP is cleaved sequentially by 𝛽 and
𝛾 secretases, resulting in the abnomal Aß peptide.
Analyzing the prevalence of free radicals in Aß
peptides through electron paramagnetic
resonance (EPR) with the addition of spin traps
indicated that the presence of free radicals was
associated with the primary sequence of Aß
peptides. Addition of varied sequences of Aß
peptides (Aß l-42, Aß l-40, and Aß 25-35) to
metal-chealeated buffer solutions along with spin
trap (N-tert-butyl-a-phenylnitrone), resulted in a
3 and 4 line EPR spectrum, bolstering the
association between free radicals and Aß peptides
[2].
Aß-induced toxicity is especially
observable in the region of the brain as cerebral
tissues are eminently vulnerable to free radical
damage. This susceptibility can be attributed to
the low level of antioxidants, high oxygen
demand, and the neuronal membranes’ large
content of polyunsaturated fatty acids [2]. Aß-
induced oxidative damage largely manifests
through processes of protein oxidation and lipid
peroxidation.
Aß-induced protein oxidation disrupts
the activity of ion-motive ATPases, leading to
disorder in ion homeostasis and rise in
intracellular calcium. These disturbances in
homeostasis culminate in the loss of functionality
in enzymes integral in ATP synthesis, such as
CK, drastically affecting the capacity of cellular
energy [2]. The inability to meet the increasing
energy demands of the neuron to combat
oxidative stress could be detrimental to its
survival. Furthermore, Aß-induced protein
oxidation can also cause loss of activity in
enzyme GS, leading to glutamate excitotoxicity
through over-stimulation of NMDA receptors [3].
In short, Aß-induced protein oxidation can
ultimately result in neuronal death through
cellular energy deficiency and signal pathway
disruption.
 Aß-induced lipid peroxidation increases vitamin E. Through this comprehensive search, a
the prevalence of free fatty acids (FFA) such as list of relevant papers was compiled. These
arachidonic acid. Arachidonic acid is a precursor papers were each independently reviewed with
for cytotoxic species and inflammation-inducing the help of 3 investigators. The reference was
compounds. Cytotoxic species such as 4- utilized to attain additional information missing
hydroxynonenal and malondialdehyde denature from the parent article. Articles from the
the structure and functionality of the cell by reference were also verified by the investigators.
attaching to integral membrane proteins and
reacting with amino acids cysteine, histidine, and Results
lysine [3]. Inflammation-inducing compounds
such as prostaglandins and leukotrienes activate
microglia to produce nitric oxide, which can Plasma Level Comparison
ultimately lead to peroxynitrite damage. The plasma level of vitamins C and E, the two
Non-enzymatic antioxidants which can most accessible dietary antioxidant, were
be obtained through diet is an effective mediator observed. The sources used in the following
for Aß-induced free radical oxidations. Also graphs were identified by their method of AD
known as “free radical scavengers”, these diagnosis. Anomalies such as Bourdel’s paper
antioxidants neutralize free radicals in which justified AD patients as below 14.2 on the
extracellular compartments, hydrophilic cytosols, Mini-Mental State Examination (MMSE) were
and hydrophobic membranes [4]. An example of excluded from the final graphs [6]. Studies
non-enzymatic antioxidant is vitamin E. vitamin included in the graphs not only distinguished
E, a stereoisomeric series of tocopherol, protects controls and AD patients through MMSE but also
polyunsaturated fatty acids located within the through Hachinski's ischaemic score combined
phospholipid bilayer by scavenging pre- with CT brain scanning and NINCDS-ADRDA
oxyradicals such as those from Aß peptides. This (National Institute of Neurological and
allows neurons to maintain their function and Communicative Disorders and Stroke-Alzheimer
structure. Another possible function of vitamin E Disease and Related Disorders Association). The
is that the antioxidant prevents Aß peptide varying units from all sources were uniformed to
formation [2]. The correlation between vitamin E μmol⁄L. Furthermore, the number of subjects
and AD is bolstered by numerous reports. and standard deviations for each study were noted
Plasmas of patients with AD present significantly for standard error.
lower vitamin E levels [5,6,7,8] In addition,
enhanced dietary intake of vitamin E are linked to Vitamin C
reduced AD risk [9,10]. 5 sources were considered to establish a contrast
between vitamin C levels of an AD patient and
control [11-15].
Methods
Study AD n Control n
Study Selection AD Control
As the correlation between free radical oxidation Sinclair 47.1±6.0 25 56.7±4.2 41
and AD has only been extensively studied since Polidori '02 18.1±5.8 35 35.9±6.3 40
the 1990s, I conducted a comprehensive search Polidori '04 25.9±8.9 63 52.4±16.4 55
Schippling 35.0±18.6 29 48.8±18.5 29
from 1990 for JSTOR, and NCBI. For the first Aejmelaeus 21.7±3.5 13 40.6±3.3 14
search, the text key words were “free radical” and
“Alzheimer’s disease.” To narrow into Figure 1. the number that proceeds ± indicates S.D.
antioxidants, the last few searches were focused
on specific non-enzymatic antioxidants such as
Figure 2.

Figure 3.
 Figure 2 indicates that the mean for the 7 sources were considered in identifying the link
vitamin C level in plasma of controls is between vitamin E levels and AD patients [15–
46.88μmol/l. Figure 1 shows that the mean for 21].
the plasma of AD patients was 29.56μmol/l. The
plasma of AD patients has a significantly lower
vitamin C level, evidencing the notion that AD
patients are exposed to a larger amount of free

Control

437
24

37

34

20

37

14
radicals.

n
As all 5 studies indicate to an identical
correlation in which AD patients have a lower
content of the free radical scavenger, it is highly

38.78±10.47

30.03±12.03
unlikely that the data was affected by external

33.10±7.79

31.3±6.3

36.8±2.2

27.9±6.4

22.2±1.8
Control
factors such as nutrition. Additionally, the chi-
values of all of the papers indicate to the rejection
of the null hypothesis, further bolstering the
authenticity of this correlation.
The relation between low vitamin C level
and cognitive impairment is clearly demonstrated

AD
in the study [15]. Their data emphasizes that as

37

44

41

10

42

65

13
n
the magnitude of cognitive impairment of the
patient increases, the vitamin C content in their
plasma decreases.

28.85±11.15

33.91±6.67

18.65±3.62
24.3±8.4

28.7±1.7

25.0±5.6

15.1±1.9
H–C CI–I CI–II CI–III
AD

(n = 14) (n = 9) (n = 13) (n = 14)

Vitamin 40.6±3.3 34.1±4.4 21.7±3.5 17.0±3.3
C

Figure 4. Vitamin C with Varied Cognitive Impairment

Aejmelaeus
Engelhart
Baldeiras
(Aejmelaeus et al Table) unit: μmol/l
Jiménez
Iuliano

Zaman
Study

Mas
H–C: healthy control
CI: cognitive impairment (I is lowest, III is greatest)

Figure 6. the number that proceeds ± indicates S.D.

Figure 5. y axis scaled 16 to 42

Vitamin E
Figure 7.

Figure 8.
 Figure 7 states that the mean for the
vitamin C content in plasma of controls is
31.44μmol/l. Figure 8 shows that the mean for
the plasma of AD patients was 24.93μmol/l.
Analogous to vitamin C, the plasma of AD
patients also has a significantly lower vitamin E
level, further emphasizing the higher prevalence
of free radicals in AD patients. Since the 7 papers
all reject the null hypothesis and collectively
point to the link between AD patients and low
amount of vitamin E, it is extremely improbable
that the data was corrupted by external
influences.
Akin to the previous free radical
scavenger, the correlation between the deficiency
of vitamin E and cognitive impairment is also
clearly displayed in the paper Vitamin E and
Enzymatic/Oxidative Stress Driven Oxysterols in
Amnestic Mild Cognitive Impairment Subtypes
and Alzheimer’s Disease. Their paper indicates
that as cognitive impairment increases, the
vitamin E level decreases.
HC aMCI mdMCI AD
(n = 24) (n = 24) (n = 29) (n = 37)
Vitamin 6.60± 6.16± 5.81± 5.32±
E 1.91 1.49 1.76 1.80
Figure 9. Values of Vitamin E Corrected for Cholesterol
(Iuliano et al Table) unit: μmol/l
HC: healthy control
aMCI: amnestic single-domain mild cognitive impairment
mdMCI: amnestic multi-domain mild cognitive impairment
Figure 5. y axis scaled 5 to 7
Aforementioned, the low level of
antioxidants indicates to the larger amount of free
radicals in the plasma of AD patients. In addition,
the less proportional ratio of free radicals and
antioxidants of AD patients promote free radical
oxidations. This poses the question: could dietary
antioxidants reduce free radical oxidation and
ultimately stall the progress of AD?
Therapeutic Application of Dietary
Antioxidants
As Aß-induced oxidative stress is a major feature
of AD and low antioxidant levels have been
observed in plasma of AD patients,
supplementation of these antioxidants could be
the solution to delay or prevent the advancement
of the neurodegenerative disease. In the last few
years, numerous studies reported positive results
for antioxidant treatments. [22-24]
A randomized clinical trial supplemented
a combination of 400IU vitamin E and 1000
vitamin C or 400 IU vitamin E alone for one
month to 20 AD patients to investigate whether
administration of vitamin E and C could increase
their concentration not only in plasma as well as
the cerebrospinal fluid (CSF) [25].
Administration of vitamin E and C significantly
increased their content in both plasma and CSF.
The abnormally low concentrations of vitamin C
returned to normal after treatment consequently
decreasing the susceptibility of lipoprotein
oxidation. Contrary to the combined supplement,
vitamin E alone had no effect on the vulnerability
of lipoprotein oxidation. This finding highlights
the feasibility of fruits for AD treatments as they
often consist a mixture of different antioxidants.
Fruits have been indicated to be effective
in preventing oxidative damage of nerve growth
factors in a modeled study. In this trial, 46
eighteen months old male F344 rats were fed
either control, blueberry, strawberry, or spinach
to identify the effect of dietary antioxidants on
motor learning [26]. Rats supplemented with
antioxidant sourced diets experienced reversing
effects in age-induced declines in b-adrenergic
receptors of cerebellar Purkinje neurons.
Although this study was based on rats, the
evolutionary similarity between neurons of
humans and rats, allow it to address the notion
that age-related cognitive impairments
(symptoms of early-onset AD) can be inhibited
by nutritional interventions.
 Discussion
Major Finding
The result of my meta-analysis indicates that the
concentration of vitamin C and E for AD patients
is significantly lower in comparison to normal.
Furthermore, the drastically low antioxidant
levels for AD patients validates that Aß-induced
oxidation a major feature of the disease. All these
findings bolster the therapeutic applicability of
dietary antioxidants.
Until now, the substantial difference
between antioxidant levels in plasma of AD
patients and controls have been difficult to
visualize. After compiling multiple reports, the
data not only established a clearer correlation
between antioxidant level and the degree of
cognitive impairment but also substantiated the
efficacy of nutritional interventions.
Study Limitations
My meta-analysis has several limitations. First
and foremost, it only examines the antioxidant
levels for plasma. Without observing the
concentration for CSF, it would be difficult to
conclusively state that the correlation between
antioxidant level and cognitive impairment exist.
Second, the majority of the sources used for
vitamin E and C did not identify prevalence of
APOE4 gene. As the APOE4 gene is known to
have direct correlation to AD, it is plausible that
the distribution of the gene among healthy and
AD patients could have caused disturbances in
plasma levels. Third, in addition to the
inconsideration of the APOE4 gene, the sources
for vitamin E and C did not recognize the
variables of sex, age, and race. As the
distributions of these variables were disclosed, it
is difficult to gauge at its influence on the data as
a whole.
Conclusion
Vitamin C and E levels are both significantly
lower for AD patients in contrast to normal
subjects. The drastic difference of antioxidant
level bolsters the notion that Aß peptides induce
oxidative stress, suggesting an intervention
backed by dietary antioxidant may be effective
for AD.
References
[1] Guerreiro, Rita, and Jose Bras. "The Age Factor In
Alzheimer’S Disease." Genome Medicine, vol 7, no. 1,
2015, Springer Nature.
[2] Butterfield DA, et al. "Vitamin E As An
Antioxidant/Free Radical Scavenger Against Amyloid
Beta-Peptide-Induced Oxidative Stress In Neocortical
Synaptosomal Membranes And ... - Pubmed - NCBI ."
Ncbi.Nlm.Nih.Gov, 2018.
[3] Varadarajan, Sridhar et al. "Review: Alzheimer's
Amyloid Β-Peptide-Associated Free Radical Oxidative
Stress And Neurotoxicity." Journal Of Structural
Biology, vol 130, no. 2-3, 2000, pp. 184-208. Elsevier
BV.
[4] VM, Sardesai. "Role Of Antioxidants In Health
Maintenance. - Pubmed - NCBI ." Ncbi.Nlm.Nih.Gov,
2018.
[5] Rinaldi, P.; Polidori, M.C.; Metastasio, A.; Mariani, E.;
Mattioli, P.; Cherubini, A.; Catani, M.; Cecchetti, R.;
Senin, U.; Mecocci, P. Plasma antioxidants are similarly
depleted in mild cognitive impairment and in
Alzheimer’s disease. Neurobiol. Aging 2003, 24, 915–
919.
[6] Bourdel-Marchasson, I.; Delmas-Beauvieux, M.C.;
Peuchant, E.; Richard-Harston, S.; Decamps, A.;
Reignier, B.; Emeriau, J.P.; Rainfray, M. Antioxidant
defences and oxidative stress markers in erythrocytes
and plasma from normally nourished elderly Alzheimer
patients. Age Ageing 2001, 30, 235–241.
[7] Mangialasche, F.; Xu, W.; Kivipelto, M.; Costanzi, E.;
Ercolani, S.; Pigliautile, M.; Cecchetti, R.; Baglioni, M.;
Simmons, A.; Soininen, H.; et al. Tocopherols and
tocotrienols plasma levels are associated with cognitive
impairment. Neurobiol. Aging 2012, 33, 2282–2290.
[8] Lopes da Silva, S.; Vellas, B.; Elemans, S.; Luchsinger,
J.; Kamphuis, P.; Yaffe, K.; Sijben, J.; Groenendijk, M.;
Stijnen, T. Plasma nutrient status of patients with
Alzheimer’s disease: Systematic review and meta-
analysis. Alzheimer’s Dement. 2014, 10, 485–502.
[9] Mangialasche, F.; Kivipelto, M.; Mecocci, P.; Rizzuto,
D.; Palmer, K.; Winblad, B.; Fratiglioni, L. High plasma
levels of vitamin E forms and reduced Alzheimer’s
disease risk in advanced age. J. Alzheimer’s Dis. 2010,
20, 1029–1037.
[10] Li, F.J.; Shen, L.; Ji, H.F. Dietary intakes of vitamin E,
vitamin C, and β-carotene and risk of Alzheimer’s
disease: A meta-analysis. J. Alzheimer’s Dis. 2012, 31,
253–258
[11] Sinclair, Alan J. et al. "Altered Plasma Antioxidant
Status In Subjects With Alzheimer's Disease And
Vascular Dementia." International Journal Of
Geriatric Psychiatry, vol 13, no. 12, 1998, pp. 840-
845. Wiley, doi:10.1002/(sici)1099-
1166(1998120)13:12<840::aid-gps877>3.0.co;2-r.
[12] Polidori, M. Cristina, and Patrizia Mecocci. "Plasma
Susceptibility To Free Radical-Induced Antioxidant
Consumption And Lipid Peroxidation Is Increased In
Very Old Subjects With Alzheimer Disease." Journal
Of Alzheimer's Disease, vol 4, no. 6, 2002, pp. 517-
522. IOS Press, doi:10.3233/jad-2002-4608.
[13] Polidori, M.Cristina et al. "Plasma Antioxidant Status,
Immunoglobulin G Oxidation And Lipid Peroxidation
In Demented Patients: Relevance To Alzheimer Disease
And Vascular Dementia." Dementia And Geriatric
Cognitive Disorders, vol 18, no. 3-4, 2004, pp. 265-
270. S. Karger AG, doi:10.1159/000080027.
[14] Schippling, Sven et al. "Increased Lipoprotein
Oxidation In Alzheimer’S Disease." Free Radical
 Biology And Medicine, vol 28, no. 3, 2000, pp. 351- [26] Bickford, Paula & Gould, Thomas & Briederick, Lori &
360. Elsevier BV, doi:10.1016/s0891-5849(99)00247- Chadman, Kathryn & Pollock, Amber & Young, David
6. & Shukitt-Hale, Barbara & Joseph, James. (2000).
[15] Aejmelaeus R, et al. "Unidentified Antioxidant Antioxidant-rich diets improve cerebellar physiology
Defences Of Human Plasma In Immobilized Patients: A and motor learning in aged rats. Brain research. 866.
Possible Relation To Basic Metabolic Rate. - Pubmed - 211-7. 10.1016/S0006-8993(00)02280-0.
NCBI .
[16] Iuliano, Luigi & Monticolo, Roberto & Straface,
Giuseppe & Spoletini, Ilaria & Gianni, Walter &
Caltagirone, Carlo & Bossù, Paola & Spalletta,
Gianfranco. (2010). Vitamin E and
Enzymatic/Oxidative Stress-Driven Oxysterols in
Amnestic Mild Cognitive Impairment Subtypes and
Alzheimer's Disease. Journal of Alzheimer's disease :
JAD. 21. 1383-92. 10.3233/JAD-2010-100780.
[17] Jiménez-Jiménez, Félix Javier & Bustos, Fernando &
Molina, Janine & Benito-León, Julián & Tallón-
Barranco, A & Gasalla, Teresa & Ortí-Pareja, M &
Guillamón, F & C Rubio, J & Arenas, Jenifer &
Enríquez de Salamanca, Rafael. (1997). Cerebrospinal
fluid levels of alpha-tocopherol (vitamin E) in
Alzheimer’s disease. Journal of neural transmission
(Vienna, Austria : 1996). 104. 703-10.
10.1007/BF01291887.
[18] Mas, Emilie & Dupuy, Annie & Artero, Sylvaine &
Portet, Florence & Cristol, Jean & Ritchie, Karen &
Touchon, Jacques. (2006). Functional Vitamin E
Deficiency in ApoE4 Patients with Alzheimer’s
Disease. Dementia and geriatric cognitive disorders. 21.
198-204. 10.1159/000090868.
[19] Zaman Z, et al. "Plasma Concentrations Of Vitamins A
And E And Carotenoids In Alzheimer's Disease. -
Pubmed - NCBI ."
[20] Baldeiras, Inês & Santana, Isabel & Teresa Proença,
Maria & Helena Garrucho, Maria & Pascoal, Rui &
Pina Rodrigues, Ana & Duro, Diana & Oliveira,
Catarina. (2008). Peripheral Oxidative Damage in Mild
Cognitive Impairment and Mild Alzheimer's Disease.
Journal of Alzheimer's disease : JAD. 15. 117-28.
10.3233/JAD-2008-15110.
[21] Engelhart MJ, et al. "Plasma Levels Of Antioxidants
Are Not Associated With Alzheimer's Disease Or
Cognitive Decline. - Pubmed - NCBI ."
[22] Kanowski, S & M Herrmann, W & Stephan, K &
Wierich, W & Hoerr, Robert. (1996). Proof of Efficacy
of the Ginkgo Biloba Special Extract EGb 761 in
Outpatients Suffering from Mild to Moderate Primary
Degenerative Dementia of the Alzheimer Type or
Multi-infarct Dementia. Pharmacopsychiatry. 29. 47-
56. 10.1055/s-2007-979544.
[23] Bruce, AJ & Malfroy, B & Baudry, Michel. (1996). β -
Amyloid Toxicity in Organotypic Hippocampal
Cultures: Protection by EUK-8, a Synthetic Catalytic
Free Radical Scavenger. Proceedings of the National
Academy of Sciences of the United States of America.
93. 2312-6. 10.1073/pnas.93.6.2312.
[24] Pappolla, Miguel A. et al. "Melatonin Prevents Death
Of Neuroblastoma Cells Exposed To The Alzheimer
Amyloid Peptide." The Journal Of Neuroscience, vol
17, no. 5, 1997, pp. 1683-1690. Society For
Neuroscience, doi:10.1523/jneurosci.17-05-
01683.1997.
[25] Kontush, Anatol & Mann, Ulrike & Arlt, Sönke &
Ujeyl, Amaar & Lührs, Charlotte & Müller-Thomsen,
Tomas & Beisiegel, Ulrike. (2001). Influence of
vitamin E and C supplementation on lipoprotein
oxidation in patients with Alzheimer's disease. Free
radical biology & medicine. 31. 345-54.
10.1016/S0891-5849(01)00595-0.