© 2016 Wiley Periodicals, Inc.

Received: 3 August 2016 | Accepted: 8 October 2016
DOI: 10.1002/jcla.22090

Background: Alzheimer’s disease (AD) is a neurodegenerative disease, which is associ-
ated with malnutrition and hyperhomocysteine. The current study aimed to analyze
the relationship between malnutrition and hyperhomocysteine in AD patients, and ef-
fects of diet intervention with betaine on the disease.
Methods: The nutritional statuses of the AD patients were assessed by short form
mini nutritional assessment (MNA- SF). The levels of Hcy, tau hyperphosphorylation,
synaptic proteins, blood inflammatory factors were measured by enzymatic cycling
assay, Western blot and ELISA. The cognitive function was measured by AD assess-
ment scale (ADAS- cog).
Results: There was a significant difference in mental status between normal people
and AD patients (P<.05). Overall, malnutrition was reported in a larger proportion of
AD patients and high level of Hcy was closely associated with malnutrition. Betaine
decreased the levels of phosphorylated tau, elevated PP2Ac activity and inhibited Aß
accumulation (P<.05). The levels of IL- lß and TNF- a were significantly higher in the
untreatment group while much lower in the intervention group (P<.05). After interven-
tion of betaine treatment, the expression level of Hcy can be restored and betaine can
effectively suppress inflammation as well as trigger an increase in memory- related
proteins. ADAS- Cog suggested that significant improvement was found after the
intervention of betaine.
Conclusions: AD was associated with both malnutrition and higher levels of Hcy.
Betaine could restore Hcy expression to normal level in AD patient, which might ame-
liorate memory deficits.

K E Y W O R D S

Alzheimer’s disease, betaine, hyperhomocysteine, malnutrition

Department of Neurology, Shandong
Provincial Qianfoshan Hospital, Jinan,
Shandong, China

Correspondence
Jianying Sun, Department of Neurology,
Shandong Provincial Qianfoshan Hospital,
Jinan, Shandong, China.
Email: [email protected]

R E S E A R C H A R T I C L E

Association between malnutrition and hyperhomocysteine in
Alzheimer’s disease patients and diet intervention of betaine

Jianying Sun | Shiling Wen | Jing Zhou | Shuling Ding

1 | I N T R O D U C T I O N

Alzheimer’s disease (AD), a heterogeneous and multifactorial neurode-
generative disease, is characterized by clinical symptoms of irrevers-
ible behavioral alterations as well as learning and memory processes
disorders.1,2 Currently, there are more than 46 million individuals
diagnosed with AD in the world, and the number of AD patients is
estimated to reach 100 million worldwide by 2050. Therefore, AD is
a growing public health concern with great socioeconomic burden.3

Current hypotheses about the pathology of AD include neurodegen-
eration, which is represented in the form of extraneuronal neuritic
plaques, and neuronal deaths due to the excessive production of am-
yloid- ß (Aß) peptide.4 In addition, intraneuronal neurofibrillary tangles
formed by hyperphosphorylated tau proteins are associated with AD.2
As one of the tau protein phosphatases, Protein phosphatase 2AC
(PP2Ac) is able to dephosphorylate tau protein.5 The deactivation of
PP2Ac deprives its function of dephosphorylation and leads to the
formation of neurofibrillary tangle eventually.6

J Clin Lab Anal. 2017;31:e22090. wileyonlinelibrary.com/journal/jcla | 1 of 7
https://doi.org/10.1002/jcla.22090

2 of 7 | SUN et al.

There are multiple risk factors for AD including age, certain genetic
alleles, and some nutritional substances.7 However, pharmacology that
can cure AD is limited, therefore, whether diet could alleviate the risk
and process of AD is of great interest. Although it is challenging to con-
firm whether diet is a contributing factor, many epidemiology studies and
intervention tests have strongly demonstrated that cognitive function
decline is connected to diet.1,7,8 For instance, epidemiological studies
suggest that a low intake of n- 3 fatty acids (n- 3 FA), B- vitamins, and an-
tioxidants are associated with an increased risk of AD. Several nutritional
compounds that can stabilize neurons at normal levels are believed to
play a part in the pathological process of AD.1 For example, the n- 3 long-
chain polyunsaturated fatty acid (n- 3 LC- PUFA) and docosahexaenoic
acid inhibit aberrant Aß and tau- protein progression.9 In addition, anti-
oxidants like vitamin E have the function of protecting neurons against
Aß- induced oxidative stress and stabilizing neuronal membranes.10
Besides lower micronutrient levels, protein/energy malnutrition has been
reported to be associated with disease progression in mild AD patients.11
These evidences indicate that malnutrition is a contributing factor for AD.

A number of researchers have reported that patients with cardiovas-
cular, cerebrovascular diseases and AD usually have an elevated level of
plasma homocysteine (Hcy).12 Hyperhomocysteine (Hhcy) is closely re-
lated to silent brain infarcts, greater cortical atrophy, and longitudinally
more severe cognitive decline.13,14 Hcy is a sulfur- containing amino acid
and a transitional production of the methionine circle, and as a kind of
neurotoxin, normal physiological levels of Hcy are maintained through
its remethylation to methionine, in which common dietary nutrients vi-
tamin B6, folate and B12 are essential.15 Moreover, several studies have
suggested that the uptake of excessive methionine or a deficit of folate
or certain enzymes in the methionine cycle due to genetic alterations
would increase Hcy content in vivo.16 Another potential association
between high Hcy and AD is the alteration of the amyloid precursor
protein metabolic pathway(s). A diet- induced chronic high Hcy results
in a substantial increase in brain Aß levels and deposition in transgenic
mouse models of AD- like amyloidosis.17 Therefore, it is vital to under-
stand the molecular mechanistic relationship between Hcy and AD
pathogenesis as it could offer clues for prevention or treatment of AD.

Betaine, a zwitterionic quaternary ammonium compound, also
called glycine betaine, trimethylglycine, oxyneurine, and lycine, is a
methyl donor that provides the one- carbon units for Hcy remethyla-
tion.18 To date, few studies have investigated the association between
betaine and AD caused by Hhcy. In this study, we aim to study the
possible relationship between mild- to- moderate AD and malnutrition,
and restore cognitive functions through betaine diet.

2 | M AT E R I A L S A N D M E T H O D S

2.1 | Subjects

We recruited 97 AD patients (the mean age was 74.6±9.2 years and
the clinical course was 6- 48 months) who consulted in Shandong
Provincial Qianfoshan Hospital from January 2013 to January 2015.
They were classified as non- malnutrition group (n=36) and malnutri-
tion group (n=61) according to the result of Short form mini nutritional

assessment (MNA- SF). Then, all patients were randomly assigned into
different groups including untreatment group (AD patients without
treatment) and intervention group (AD patients were treated with 50,
100, and 200 µg/kg betaine for 1 month).

Another 65 subjects were enrolled in the control group. They were
healthy volunteers without any mental diseases or neurological dis-
eases consulted in the same hospital. The MNA- SF score of each pa-
tient in the control group was =11.

Inclusion criteria of AD patients were set according to the
Diagnostic and Statistical Manual of Mental Disorders IV (DSM- IV).
Individuals who had acute infection, trauma, and myocardial infarction
were excluded. No treatment was performed in the control or untreat-
ment group. All of the subjects signed the informed consent form. This
study was approved by the ethics committee of Shandong Provincial
Qianfoshan Hospital.

2.2 | Short form mini nutritional assessment (MNA-
SF) Evaluation and Blood measurement

The nutritional status of the patients with senile dementia was as-
sessed by MNA- SF. The total score was 14; a score =11 was consid-
ered normal; malnutrition was defined if the score was <11. All patients
had about 2- 3 mL early morning fasting venous blood taken and blood
samples were placed in anticoagulation tube. The sample was sent
for testing after 30 minutes of mixing. Enzymatic cycling assay was
performed to measure the amount of serum Hcy, hemoglobin, urea,
cholesterol, and serum albumin via an automatic biochemical analyzer
(iChem- 340; icubio Biological Technology Co., Ltd, Shenzhen, China).

2.3 | Western blot

We tested the amount of phosphorylated tau proteins including Thr231
(pT231), Thr205 (pT205) and Ser396 (pS396) in the extracted blood
samples. Equal amounts of proteins were subjected to polyacrylamide
gel electrophoresis. The membrane was incubated in 10 mL of 5% non-
fat milk in m (TBST) solution to block the membrane for 1.5 hours after
transferring the gel to polyvinllidence fluoride membrane. The mem-
brane was incubated with the primary antibodies against Tau1, pT205,
pT231, pS396, Tau5, PP2Ac, p- PP2Ac, NR1, NR2A, NR2B, synap-
totagmin, synaptophysin, synapsin 1, p- synapsin 1 (diluted with 1:800,
1:1000, 1:500, 1:500, 1:800, 1:800, 1:500, 1:800, 1:1000, 1:500,
1:500, 1:800, 1:800, 1:500, respectively; Zhongshan Biology Company,
Beijing, China). After washing the membrane, the secondary antibodies
were used (horseradish peroxidase- conjugated goat anti- goat, 1:2000
dilution; Zhongshan Biology Company) to repeat the procedure again.
Then ECL chemiluminescence reagent kit was added to monitor devel-
opment after washing for three times with TBST.

2.4 | Enzyme linked immunosorbent assay
(ELISA) and ADAS- Cog analysis

ELISA was used to analyze blood inflammatory factors (IL- lß, TNF- a),
Aß40, and Aß42. ELISA was performed with IL- lß, TNF- a, Aß40, and

| 3 of 7SUN et al.

Aß42 ELISAkits from Bnder MedSystems according to the manufac-
turer’s protocol.

The AD assessment scale (ADAS- Cog) includes recall of words, nam-
ing of items and fingers, following instructions, visual- spatial capacity,
intentional- practice skills, directional ability, recognition of dual words,
recall of test instructions, verbal language skills, word finding difficulty,
and attention, and understanding dysfunction. The corresponding
score was divided into five grades. Severity level was evaluated using
selective domains ranging from 0 to 70. A score of 0 means no demen-
tia while a score of 70 points means no cognitive functions.

2.5 | Statistical analysis

Statistical analyses were performed using the SPSS 18.0 statistical
software (SPSS Inc., Chicago, IL, USA). Measurement data were pre-
sented as mean±standard deviation (SD). Enumeration variables were
analyzed by the chi- square (?2) test. Continuous variables were ana-
lyzed by two- tailed t- test or one- way analysis of variance (ANOVA). A
P value <.05 was considered statistically significant.

3 | R E S U LT S

3.1 | Subject characteristics

There was no significant difference between the normal people and
AD patients in terms of age, sex ratio, education degree, tea or alcohol

consumption, smoking, and intake of vitamin B (P>.05). On the con-
trary, significant difference was observed in the results of Mini- Mental
State examination (P<.05, Table 1).

3.2 | Relationship among malnutrition,
Hcy, and betaine

Low cholesterol and albumin levels are signs of malnutrition. As shown
in Table 2, the study of MNA- SF showed that there were 61 malnutri-
tion patients, which accounted for 62.9% of AD patients. Cholesterol
and albumin levels were much lower in the malnutrition patients than
those in the non- malnutrition patients (P<.05). In addition, an Hcy- test
was performed on every subject and the result showed that Hcy level
was significantly elevated in malnutrition patients (P<.05), suggesting
a strong connection between the malnutrition and Hcy.

After betaine diet intervention treatment, betaine regulated Hcy
in a dose- dependent manner, a significant difference in Hcy was iden-
tified between the untreatment group and 200 µg/kg betaine group
(P<.05, Figure 1), indicating that betaine can suppress the level of Hcy.

TABLE 1 Baseline characteristics of two groups

Index AD (n=97) Control (n=65) P

Age (year) 73.3±5.3 74.8±6.3 .104

Sex

Male 52 35 .976

Female 45 30

Education degree (year)

<1 6 4 .992

<6 13 9

<12 52 35

>12 26 17

Alcohol consumption

No 84 60 .257

Yes 13 5

Tea consumption

No 91 62 .669

Yes 6 3

B vitamin supplement

No 86 60 .446

Yes 11 5

MMSE 19.55±2.29 28.21±3.15 <.05

AD, Alzheimer’s disease; MMSE, Mini- Mental State examination.
Data are expressed as n or mean±SD as appropriate. Chi- square test was
used for enumeration variables; the Student t- test was used for continuous
variables.

TABLE 2 Risk factors of mild senile dementia

Factors
Malnutrition
group (n=61)

Non- malnutrition
group (n=36) P

Hemoglobin (g/L) 121.42±9.33 125.15±11.84 .089

Urea nitrogen
(mmol/L)

7.12±2.75 6.84±2.19 .604

Cholesterol
(mmol/L)

4.41±1.36 5.67±1.49 <.05

Plasma albumin
(g/L)

30.38±11.03 42.52±9.15 <.05

Plasma Hcy (g/L) 26.84±9.28 14.72±7.35 <.05

Hcy, homocysteine.
Data are expressed as mean±SD. The Student t- test was used for continu-
ous variables.

FIGURE 1 Effect of betaine on plasma Hcy. Data were expressed
as mean±SD. *P<.05 vs control group; #P<.05 vs untreatment group

P
la
sm
a
H
cy
(µ
m
ol
/L
)

Co
nt
ro
l

Un
tre
at
m
en
t

Be
ta
in
e (
50
µg
/kg
)

Be
ta
in
e (
10
0µ
g/
kg
)

Be
ta
in
e (
20
0µ
g/
kg
)

0

10

20

30

40
*

*
*

#

4 of 7 | SUN et al.

3.3 | Betaine regulates tau phosphorylation and
PP2Ac activity

Hyperphosphorylation of tau protein contributes to neuronal fiber en-
tanglement, which is one of the mechanisms causing neurodegenera-
tion. Western blot was used to determine the levels of phosphorylated
tau proteins. We found that phosphorylated tau proteins including
Thr231 (pT231), Thr205 (pT205), and Ser396 (pS396) all increased
significantly in the untreatment group compared to the control group,
while decreased in intervention group (treated with 200 µg/kg be-
taine) compared with untreatment group (P<.05, Figure 2). The results
indicated that treatment of betaine was able to reduce hyperphos-
phorylation of tau protein.

In addition, we found that intervention of betaine (200 µg/kg) in-
creased protein phosphatase- 2Ac (PP2Ac) expression but decreased
the phosphorylayion of PP2Ac, and thus regulated the activation of
PP2Ac (P<.05, Figure 3).

3.4 | Betaine reverses Aß accumulation

We saw a higher level of Aß40 and Aß42 levels in untreatment
group than control group by ELISA, and a significant decrease
when betaine treatment (200 µg/kg) was given (P<.05, Figure 4).
The result suggests that betaine can reverse Aß aggregation in
AD patients.

3.5 | Analysis of inflammatory factor

The levels of IL- lß and TNF- a in the untreatment group were signifi-
cantly higher than those in the control group, while both reduced sub-
stantially after betaine treatment (200 µg/kg) (P<.05, Figure 5). The
data show that betaine can suppress the inflammatory level and help
relieve the illness.

3.6 | Betaine stimulates memory- related proteins
(NR1, NR2A, and NR2B) levels

The Western blot results of memory- related proteins showed that
NR1 and NR2A levels were lower in untreatment group than those in
the control group, whereas intervention of betaine (200 µg/kg) signifi-
cantly increased the expressions of NR1 and NR2A (P<.05, Figure 6).

FIGURE 2 The phosphorylation levels
of tau proteins. Intervention group was
treated with 200 µg/kg betaine. Data were
expressed as mean±SD. *P<.05 vs control
group; #P<0.05 vs untreatment group

R
el
at
iv
e
In
te
ns
ity

Tau1 pT205 pT231 pS396 Tau5
0

1

2

3
Control
Untreatment

*
#

*

# * #

*

#

Tau1

pT205

pT231

pS396

Tau5

ß-actin

Control Untreatment
A B

Intervention

Intervention

FIGURE 3 The phosphorylation level
of protein phosphatase- 2Ac (PP2Ac).
Intervention group was treated with
200 µg/kg betaine. Data were expressed as
mean±SD. *P<.05 vs control group; #P<.05
vs untreatment group

R
el
at
iv
e
A
ct
iv
ity

PP2Ac p-PP2Ac
0.0

0.5

1.0

1.5

2.0
Control
Untreatment

Control Untreatment

A B

PP2Ac

p-PP2Ac

ß-actin

*
#

*

#
Intervention

Intervention*
*

FIGURE 4 The expression levels of Aß40 and Aß42. Intervention
group was treated with 200 µg/kg betaine. Data were expressed as
mean±SD. *P<.05 vs control group; #P<.05 vs untreatment group

R
el
at
iv
e
Le
ve
l

0

1

2

3
Control
Untreatment

Aß40 Aß42

*

#

*

#

Intervention

*

FIGURE 5 The expression levels of TNF- a and IL- 1ß. Intervention
group was treated with 200 µg/kg betaine. Data were expressed as
mean±SD. *P<.05 vs control group; #P<.05 vs untreatment group

R
el
at
iv
e
Le
ve
l

0

10

20

30

40

50
Control
Untreatment

TNF-a IL-lß

*

#

* #

Intervention

*

| 5 of 7SUN et al.

There were no significant differences between the three groups in
terms of NR2B expression.

3.7 | Betaine regulates synaptic protein expressions

Western blot was used to determine the levels of synaptophysin, syn-
aptotagmin, synapsin I, and phosphorylated synapsin I (p- synapsin I).
As shown in Figure 7, the level of synaptic proteins in the untreat-
ment group was much lower than those in the control group, while the
intervention group (treated with 200 µg/kg betaine) showed an in-
crease in synaptic protein level than untreatment group (P<.05). These
results demonstrated that low level of synaptic proteins, an indicator
of memory deficits in AD, could be restored to normal levels after
betaine intervention.

3.8 | ADAS- Cog analysis

The results of the ADAS- Cog showed no significant difference before
and after intervention (200 µg/kg betaine) in areas such as naming of
items and fingers, instructions, intentional- practice, directional ability,
and verbal language skills and attention (P>.05); while significant im-
provements were identified in recall of words, visual- spatial capacity,
and recognition of dual words (P<.05, Table 3).

4 | D I S C U S S I O N

It has been reported that malnutrition is notably high in patients with
dementia, and malnutrition may be presented even before classi-
cal symptoms of dementia appear.8 Furthermore, it is crucial to ob-
serve malnutrition in AD patients because malnutrition is associated
not only with a faster progression of AD but also with other health
problems.19 In this study, we indicated that the rate of malnutrition

among AD patients was 62.9%, which was consistent with previ-
ously published studies that reported the rate of malnutrition among
community- dwelling AD patients varying between 14% and 80% in
different populations.20 The results of MNA- SF assessment and Hcy
detection showed that malnutrition and Hhcy appeared in patients
with dementia concurrently, which suggests that malnutrition and
Hhcy are closely associated with dementia.

Increasing epidemiology and clinical researches have revealed that
the Hcy level is positively related to the development of AD, and Hhcy
is one of the remarkable risk factors in patients with AD.21 Javier et al.
observed that Aß level can be increased in the brain of an Hhcy mouse
model with amyloidosis.22 Ho et al.23 demonstrated that Hcy impairs
DNA repair in the hippocampus and made hippocampal neurons sen-
sitive to Aß toxity. Moreover, Zhang et al.24 indicated that increase in
Hcy level was highly associated with AD- like Aß accumulation and aug-
mented AD- like tau hyperphosphorylation in brains of rats. Zhang et al.24

FIGURE 6 The expression levels of
NR1, NR2A, and NR2B. Intervention group
was treated with 200 µg/kg betaine. Data
were expressed as mean±SD. *P<.05 vs
control group; #P<.05 vs untreatment group

R
el
at
iv
e
In
te
ns
ity

NR1 NR2A NR2B
0.0

0.5

1.0

1.5 Control
Untreatment

Control Untreatment

A B

NR1

NR2A

NR2B

ß-actin

*

#

*

#

Intervention

Intervention

FIGURE 7 The expression levels of
synaptotagmin, synaptophysin, synapsinI,
and p- synapsin I. Intervention group was
treated with 200 µg/kg betaine. Data were
expressed as mean±SD. * P<.05 vs control
group; #P<.05 vs untreatment group

R
el
at
iv
e
In
te
ns
ity

Sy
na
pt
ot
ag
m
in

Sy
na
pt
op
hy
sin

Sy
na
ps
in
I

p-S
yn
ap
sin
I

0.0

0.5

1.0

1.5 Control
Untreatment

*

#
*

#

*
#

*
#

Control Untreatment

A B

Synaptotagmin

Synaptophysin

Synapsin I

p-Synapsin I

ß-actin

Intervention

Intervention

TABLE 3 The results of ADAS- Cog analysis

Items
Before
treatment

After
treatment P

Recall of words 5.83±1.64 4.27±1.38 <.05

Naming of items and
fingers

0.88±1.13 0.96±0.86 .591

Instructions 1.38±0.82 1.45±0.47 .467

Visual- spatial capacity 1.49±0.92 1.15±0.75 <.05

Intentional- practice 1.31±0.99 1.34±0.96 .831

Directional ability 2.55±1.20 2.41±1.46 .467

Recognition of dual words 5.48±1.18 4.25±1.15 <.05

Verbal language skills and
attention

5.20±1.16 5.32±1.36 .509

ADAS- Cog, Alzheimer’s disease assessment scale.
Data are expressed as mean±SD. The Student t- test was used for continu-
ous variables.

6 of 7 | SUN et al.

have also found that a concurrent supplementation of folate and vitamin
B12 improved the plasma Hcy level moderately, and therefore remark-
ably promotes Aß accumulation, memory damnifications, tau hyperphos-
phorylation, and PP2Ac deactivation. Accordingly, decreasing the plasma
Hcy level could be a novel strategy to inhibit the development of AD.

In this study, we found that intervention of betaine could significantly
reduce the plasma Hcy level and reverse tau protein phosphorylation,
PP2Ac deactivation, as well as Aß accumulation. By down- regulating
serum IL- lß and TNF- a levels, intervention of betaine also attenuated
inflammation and therefore were conducive to the mitigation of demen-
tia. Furthermore, intervention of betaine upregulated expression levels
of memory- related proteins (NR1, NR2A) and synaptic proteins (phos-
phorylated synapsin I, synaptophysin, synaptotagmin), thereby relived
memory ramifications. ADAS- Cog analysis showed that brain cognition
and functionality of AD patients, such as structural links, word memory
and recognition, have been improved after betaine intervention. Our
data were consistent with that of Chai et al.,25 which demonstrated that
betaine could inhibit AD- like pathological changes induced by Hcy.

It is well known that betaine is an endogenous catastate of bursine
containing three effective methyl groups that may act as an active
methyl donor for the remethylation of Hcy, and it is also related to liver
function, cellular reproduction, and carnitine production.26 It has been
reported that betaine contributes to a large number of diseases, such
as heart and liver diseases.27,28 Ganesan et al.29 found that betaine
also plays a protective agonistic role against oxidative injury induced
by stress in Wistar rats. Schiff et al.30 reported that an oral solution of
anhydrous betaine were used to treat homocystinuria, indicating that
betaine could down regulate the levels of Hcy and reverse the effects
of Hcy, including memory impairment.

However, there were some limitations in our study, such as small
sample size and the unclear molecular mechanism. We would pay atten-
tion to the effects of Hcy and malnutrition on AD in further research.

5 | C O N C L U S I O N

As a whole, this study showed that Hcy and malnutrition were closely
related with AD, and betaine intervention can restore the plasma Hcy
level as well as play protective effects in AD patients, with potential
mechanisms that relate to tau hyperphosphorylation, PP2Ac activa-
tion, Aß accumulation, inflammatory reaction, and the stimulations of
memory- related proteins. Our results provided an encouraging pos-
sibility for the treatment of senile dementia.

A C K N O W L E D G M E N T S

We would thank Dr. Tao Zhang (Department of Neurology, Shandong
Provincial Qianfoshan Hospital) for his technical guidance.

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