Available via license: CC BY 4.0
Content may be subject to copyright.
The Journal of Nutrition
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Citicoline and Memory Function in Healthy
Older Adults: A Randomized, Double-Blind,
Placebo-Controlled Clinical Trial
Eri Nakazaki,1Eunice Mah,2Kristen Sanoshy,2Danielle Citrolo,3and Fumiko Watanabe1
1Research & Innovation Center, Kyowa Hakko Bio Co., Ltd, Tsukuba, Ibaraki, 305–0841, Japan; 2Biofortis Innovation Services, Addison,
IL 60101, USA; and 3Scientic and Regulatory Affairs, Kyowa Hakko USA Inc., New York, NY 10016, USA
ABSTRACT
Background: Supplementation of citicoline (CDP-choline), a naturally occurring mononucleotide, has shown benecial
effects on memory function and behavior in populations with a wide range of impairments. However, few studies have
investigated its effect in healthy older populations.
Objective: The objective of this study was to investigate the effects of citicoline (Cognizin®), on memory in healthy
elderly populations with age-associated memory impairment (AAMI).
Methods: A total of 100 healthy men and women aged between 50 and 85 y with AAMI participated in this randomized,
double-blind, placebo-controlled trial. Participants were randomized to receive placebo (n=51) or citicoline (n=49;
500 mg/d) for 12 wk. Memory function was assessed at baseline and end of the intervention (12 wk) using computerized
tests (Cambridge Brain Sciences, Ontario, Canada). Safety measurements included adverse events query, body weight,
blood pressure, and hematology and metabolic panel. Intent-to-treat analysis was conducted using ANCOVA for the
primary and secondary outcome variables with Bonferroni correction for multiple comparisons.
Results: A total of 99 out of 100 participants completed the study in its entirety. After the 12-wk intervention, participants
supplemented with citicoline showed signicantly greater improvements in secondary outcomes of episodic memory
(assessed by the Paired Associate test), compared with those on placebo (mean: 0.15 vs. 0.06, respectively, P=0.0025).
Composite memory (secondary outcome), calculated using the scores of 4 memor y tests, also signicantly improved
to a greater extent following citicoline supplementation (mean: 3.78) compared with placebo (mean: 0.72, P=0.0052).
Conclusions: Dietary supplementation of citicoline for 12 wk improved overall memory performance, especially
episodic memory, in healthy older males and females with AAMI. The ndings suggest that regular consumption
of citicoline may be safe and potentially benecial against memory loss due to aging. This trial was registered at
clinicaltrials.gov as NCT03369925. J Nutr 2021;151:2153–2160.
Keywords: 5-cytidine diphosphate choline, citicoline, aging, brain, memory loss
Introduction
The world’s population is aging with almost every country
experiencing a growth in the number and proportion of older
persons. Projections of the World Population Prospects by
the United Nations suggest that 1 in 6 people (16%) in the
world will be aged over 65 y by 2050 (1). An expected
challenge of an aging population is the increased rates of
health issues associated with aging, such as age-related cognitive
decline. Although age is the most important predictor of
cognitive decline, this process is a complex interplay of many
contributing factors including demographic, genetic, socioe-
conomic, environmental, and nutritional parameters (2). No
effective pharmaceutical treatments for age-related cognitive
decline are available, emphasizing the importance of prevention
strategies against cognitive decline. There is extensive evidence
on how nutrients and bioactive compounds may impact on
cognitive decline due to aging (3). Some evidence points to
specic dietary patterns (e.g., ketogenic or Mediterranean diets)
as having strong potential to combat age-related cognitive
decline (4). Additionally, individual nutrients such as B vitamins,
antioxidant polyphenols, selenium, vitamin D, medium-chain
triglycerides, and long-chain omega-3 fatty acids have been
investigated for their potential cognitive-enhancing properties
(5).
An important nutrient that is known to affect brain
development and aging is choline. Choline is essential for
several biological functions of cells. For example, choline
metabolites acetylcholine and phosphatidylcholine, respectively,
contribute to proper signaling functions for normal cholin-
ergic neurotransmission and the structural integrity of cell
membranes (6). Brain choline uptake is decreased in older
C
The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the
terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction
in any medium, provided the original work is properly cited.
Manuscript received November 30, 2020. Initial review completed January 8, 2021. Revision accepted April 5, 2021.
First published online May 12, 2021; doi: https://doi.org/10.1093/jn/nxab119. 2153
adults (7) and the lower supply of extracellular choline may
contribute to aging-related cognitive decline. Not surprisingly,
higher dietary choline is associated with better cognitive
performance in elderly individuals (8,9). One method to
increase dietary choline is through supplementation with
citicoline (CDP-choline or cytidine-5-diphosphate choline),
choline salts, such as choline chloride and choline bitartrate,
glycerophosphocholine, as well as phosphatidylcholine. Com-
pared with choline moiety obtained through other dietary
sources such as phosphatidylcholine, choline in citicoline has
been suggested to be less prone to conversion to trimethylamine
(TMA) and trimethylamine N-oxide (TMAO), which have been
implicated in the pathogenesis of cardiovascular disease (10).
Citicoline is a naturally occurring mononucleotide comprised
of cytosine, ribose, pyrophosphate, and choline. It is produced
by the body as an intermediate product of phosphatidylcholine
and sphingomyelin biosynthesis (11,12). The neuroprotective
actions of citicoline include activating the biosynthesis of
structural phospholipids in the neuronal membranes, increasing
cerebral metabolism, noradrenaline, and dopamine levels in
the central nervous system, preventing the loss of cardiolipin
(an exclusive inner mitochondrial phospholipid enriched with
unsaturated fatty acids), and protecting cell membranes by
accelerating re-synthesis of phospholipids (13). Unsurprisingly,
citicoline supplementation has shown benecial effects on
memory function and behavior in populations with a wide range
of impairments such as those with mild to moderate vascular
cognitive impairment, vascular dementia, or senile dementia
(14).
In healthy populations, oral intake of a citicoline supplement
(Cognizin), improved attention compared with placebo in
middle-aged women [250 mg/d for 1 mo, age 40–60 y
(15)] and in adolescent males [250 and 500 mg/d for 28 d,
n=24/group (16)]. Additionally, oral citicoline supplementa-
tion (1 g/d for 3 mo) improved logical memory score compared
with placebo in men and women (n=49/group, age 50–
85 y) with relatively inefcient memory [i.e., scored below
average of all recruited participants (17)]. Another open-
label clinical trial demonstrated a signicant improvement
in word and object recalls after citicoline supplementation
(1 g/d) for 28 d (18). To date, no studies have investigated
the effects of citicoline supplementation at 500 mg/d on
memory in healthy elderly adults with age-associated memory
impairment (AAMI). Thus, the objective of this study was
to investigate the effects of citicoline supplementation at
500 mg/d on memory in healthy elderly populations with
AAMI.
This research was sponsored by Kyowa Hakko Bio Co., Ltd. This research did
not receive any specic grant from funding agencies in the public, commercial,
or not-for-prot sectors. Kyowa Hakko Bio Co., Ltd funded the study and
participated in study design, but was not involved in the intervention, data
collection, and statistical analysis. The views expressed in this manuscript are
those of the authors and do not necessarily reect the position or policy of
Kyowa Hakko Bio Co., Ltd.
Author disclosures: EN and FW are current employees of Kyowa Hakko Bio Co.,
Ltd. EM and KS are current employees of Biofortis Innovation Ser vices. DC is a
current employee of Kyowa Hakko USA, Inc.
Supplemental Tables 1 and 2 are available from the “Supplementar y data” link
in the online posting of the article and from the same link in the online table of
contents at https://academic.oup.com/jn.
Address correspondence to EN (e-mail: eri.nakazaki@kyowa-kirin.co.jp).
Abbreviations used: AAMI, age-associated memory impairment; AE, adverse
event; CFR, Code of Federal Regulations; GCP, Good Clinical Practices; ITT,
intent-to-treat; TMA, trimethylamine; TMAO, trimethylamine N-oxide.
Methods
Study design
This randomized, double-blind, placebo-controlled trial of citicoline
was conducted at Biofortis Inc. (Addison, IL) between January 2018
and December 2018. This study was carried out in compliance with
the protocol and in accordance with Good Clinical Practices (GCP), the
applicable US Code of Federal Regulations (CFR), and the Declaration
of Helsinki (2013 Version). The study protocol was approved by an
Institutional Review Board (IntegReview, Austin, TX). Signed written
informed consent for participation in the study was obtained from all
participants before protocol-specic procedures were carried out. This
trial was registered at ClinicalTrials.gov with identier NCT03369925.
Study participants
Healthy men and women (aged 50 to 85 y) with AAMI were recruited
by the Biofortis Clinical Research team by using an established database
of volunteers and local advertisements. While there is no agreed-upon
denition for AAMI, we based our recruitment on the criteria for
AAMI suggested by the US National Institutes of Health (19). Thus, the
inclusion criteria for this study included: age 50–85 y, scored ≥24 on the
Mini-Mental State Examination, ≥85 on the Kaufman Brief Intelligence
Test - Second Edition, ≤5 on the Geriatric Depression Scale, and 4, 3, or
2 on the Spatial Span test, and no health conditions that would prevent
him or her from fullling the study requirements on the basis of medical
history and routine laboratory test results. Exclusion criteria included
color blindness, abnormal laboratory test results that fell outside of
the normal range as dened by the analytical laboratory (Elmhurst
Memorial Reference Laboratory, Elmhurst, IL), major medical or
neurological illness including, but not limited to, hyperparathyroidism,
type 1 or 2 diabetes mellitus, hypoglycemia, myocardial infarction,
peripheral arterial disease, uncontrolled asthma, Alzheimer’s disease,
Parkinson’s disease, stroke, intracranial hemorrhage, and local brain
lesions, females who were pregnant or planning to be pregnant during
the study period, and medications that may have interfered with the
interpretation of the study results (e.g., medications known to affect
cognition).
Study product and treatment
Eligible participants were randomly assigned 1:1 to oral citicoline
(500 mg/d) or placebo in a double-blind design using a randomization
sequence prepared by the lead study statistician. The randomization
sequence was designed such that it allowed approximately equal
distribution of baseline spatial span test score and sex among the 2 test
groups. A randomization number was assigned to eligible participants
using an electronic randomization module (Medrio, Inc., San Francisco,
CA). Participants, research staff, and outcome assessors were blinded to
group allocations until data analyses had been completed.
Study supplements consisted of encapsulated microcrystalline cellu-
lose (placebo), or 250 mg/capsule of citicoline (Cognizin; Kyowa Hakko
Bio Co., Ltd). Placebo and citicoline capsules were identical in color and
size. Participants were instructed to consume 2 capsules with breakfast
for 12 wk; thus participants in the citicoline group consumed a total of
500 mg/d of citicoline. Compliance was documented as a percentage of
study product consumed calculated based on scheduled product intakes
and number of returned study product. Non-compliance was dened as
consumption of <80% or >120% of the scheduled intake upon study
completion. Overall compliance was determined at Week 12.
Study procedures
This study consisted of 1 screening visit, 2 test visits (Week 0 and
Week 12), and 1 compliance visit (Week 6). To minimize the impact of
lifestyle changes on cognition, participants were instructed to maintain
their habitual diet, exercise routines, and sleep duration throughout
the study and any major change/life stress event that could impact
cognition was inquired and documented. At screening, participants were
screened for inclusion and exclusion criteria. Additionally, evaluations
of medical history, prior/current medication/supplement use, height,
body weight measured using a digital scale (Health-O-Meter 349KLX;
Sunbeam Products, Inc., Boca Raton, FL), vital signs including systolic
2154 Nakazaki et al.
and diastolic BP measured using an automated blood pressure monitor
(Welch Allyn 53000; Hill-Rom Holdings, Inc., Chicago, IL), Mini-
Mental State Examination, Kaufman Brief Intelligence Test - Second
Edition, Geriatric Depression Scale, and sleep and smoking habits were
assessed. The 24-h diet record was collected and reviewed to compare
food and beverage consumption up to the day before test visits (Weeks 0
and 12) for consistency.BMI was calculated as kg/m2. At baseline (Week
0) and end of study (Week 12), participants arrived at the clinic fasted
(10–14 h), consumed a standard breakfast, and completed the cognitive
assessments test battery. Fasting (10–14 h) blood samples were collected
at the screening and end of study visits for hematology and metabolic
panel assessments.
Cognitive assessments
Cognitive performance was assessed using Cambridge Brain Sciences
(Toronto, Ontario, Canada) computerized tests. This computerized
testing battery was publicly available and was validated by the Medical
Research Council and Brain Sciences Unit [Cambridge, UK (20)]. The
testing battery was used to assess working memory (Monkey Ladder
task), short-term spatial memory (Spatial Span), short-term verbal
memory (Digit Span task), episodic memory (Paired Associate task),
selective attention (Feature Match and Interlocking Polygons tasks), and
sustained attention (Sustained Attention to Response Task). Normative
data from >74,000 participants aged between 11 and 100 y provided
by Cambridge Brain Sciences showed a decrease in Spatial Span score
with age. Thus, it was used at screening to identify participants with
compromised memory function dened as scoring at least 1 standard
deviation below the mean established for young adults (based on
normative data provided by Cambridge Brain Sciences). Additionally,
screening Spatial Span scores were also used during randomization
whereby participants were stratied into 3 Spatial Span test score strata
(score of 2, 3, or 4). All 7 tasks were administered at Week 0 and
Week 12, but not administered at Week 6. Participants were given the
opportunity to practice all cognitive tasks during the screening visit.
Safety assessments
Safety was assessed by adverse events (AEs) reported by participants,
as well as assessment of vital signs, body weight, and hematology and
metabolic panels. Inquiry of AEs was conducted using an open-ended
question at Weeks 0, 6, and 12, and during phone calls between each
visit. The Clinical Investigator evaluated all AEs with respect to their
severity,according to the World Health Organization Adverse Reaction
Terminology (WHO-ART) dictionary (21). The Clinical Investigator
also judged the likelihood that the AE was related to the study product
in accordance with Reviewer Guidance on Conducting a Clinical
Safety Review of a New Product Application and Preparing a Report
on the Review (22). Finally, metabolic and hematology panels were
evaluated by Elmhurst Memorial Reference Laboratory (Elmhurst, IL)
from heparin plasma samples using the Dimension Vista 500 System
(Siemens Medical Solutions USA, Inc., Malvern, PA) and from whole
blood samples using the Sysmex XN-3100 Automated Hematology
System (Sysmex America, Inc., Lincolnshire, IL), respectively.
Statistical analysis
Sample size calculations were performed using G∗Power (Version
3.1.9.2), for ANCOVA (xed effects, main effects, and interactions)
with the following parameters: αerr prob =0.05, power (1-βerr
prob) =0.80, numerator df =1, number of groups =2, number of
covariates =2. Sample size calculations also used proprietary normative
data (obtained from >74,000 adults) for Spatial Span provided by
Cambridge Brain Sciences and estimated improvement difference of
9.9% and distribution in scores following citicoline supplementation
between groups based on using data from a previous study on citicoline
and memory (17), while taking into account 2 covariates (age and
baseline scores). An evaluable sample size of 82 was needed to detect
a signicant difference between groups and a total of 100 participants
were randomized to account for possible attrition.
The primary outcome variable was the raw change in Spatial
Span scores, calculated as the difference in baseline score and end
of the test period (Week 12) score for each participant. Secondary
outcome variables included the raw change in test scores for the
remaining 6 cognitive tasks. Additionally, the composite memory score
was calculated for overall memory function based on the Spatial Span
(SSscore), Monkey Ladder (MLscore), Paired Associates (PAscore), and
Digit Span (DSscore) raw scores using the following formula provided
by Cambridge Brain Sciences:
Com pos it eM emor yS core =˜
Q·15+100
where
˜
Q= SSscore−6.11
1.07 ·0.69+ MLscore−7.8
1.16 ·0.69+ PAscore−5.24
1.11 ·0.58+ DSscore−7.15
1.48 ·0.26
(4·0.39)
All statistical analyses were conducted using SAS for Windows
(version 9.4, Cary, NC) on the intent-to-treat (ITT) population,
which included all participants who were randomized into the study.
All tests of signicance, unless otherwise stated, were performed at
alpha =0.00625 (2-sided), which accounted for multiple comparisons
with Bonferroni correction. ANCOVA was used to assess differences
between test groups for the primary and secondary outcome variables.
Initial ANCOVA models contained terms for test group, Spatial Span
screening score (i.e., 4, 3, 2), sex, BMI, 2-way interaction terms “test
group by sex,” “test group by second Spatial Span screening score,”
“test group by BMI,”and “test group by age,”with age and baseline test
scores as covariates. Models were reduced using a backward selection
method until only terms for test group, Spatial Span screening score (i.e.,
4, 3, 2), sex, age, baseline test scores, and signicant 2-way interaction
terms (if there are any) remained in the model. Data of unadjusted
means ±SEM were presented for each test group. Assumption of
normality of residuals was investigated for each outcome variable at
the 5% level of signicance with the Shapiro-Wilk test (23).
For hematology and metabolic panels, continuous measures were
compared between groups with the Wilcoxon rank sum test. At each
time point, the Pvalues were adjusted for multiple comparisons with
the false discovery rate. Data of unadjusted means ±SD are presented
for each test group.
Finally, differences between groups for baseline characteristics
(Tab l e 1), were analyzed using independent t-test for continuous
variables and Fisher’s exact for categorical variables.
Results
Participant characteristics
A CONSORT diagram illustrating participant recruitment and
attrition during the trial is presented in Figure 1. A total
of 426 participants were screened for participation and 100
participants were randomized. A total of 99 participants
completed the study in its entirety. One participant in the
citicoline group withdrew from the study due to a headache,
which was judged as possibly related to study product. As
dened in the study protocol, the ITT sample population
consisted of all randomized participants. In the ITT sample
population, a total of n=100 participants contributed to data
at Week 0 and n=99 participants contributed to data at Week
12. No premature unblinding occurred during the study. There
were no differences in selected demographics, and baseline
characteristics for sample populations are listed in Tabl e 1 .
Compliance over the 12-wk supplementation period was 99.2 ±
0.5% for the 99 participants who completed the study.
Cognitive performance
The results on cognitive function tests at Weeks 0 (baseline),
and 12 (end of study) are shown in Tabl e 2 . Within-
group analysis indicated that the citicoline group, but not
Citicoline on memory in healthy older adults 2155
TABLE 1 Characteristics of the 100 study participants enrolled in this randomized controlled trial1
Placebo Citicoline
(n=51) (n=49) P
Age,2y 65.5 ±1.13 63.2 ±1.12 0.53
Gender,3n(%)
Male 16 (31.4) 19 (38.8)
Female 35 (68.6) 30 (61.2) 0.16
Race,3n(%) 0.17
White 47 (92.2) 41 (83.7)
Black/African American 3 (5.88) 7 (14.3)
American Indian or Alaskan Native 0 (0.00) 1 (2.04)
Asian 0 (0.00) 0 (0.00)
Native Hawaiian or other Pacic Islander 1 (1.92) 0 (0.00)
Ethnicity,3n(%) 1.00
Not Hispanic/Latino 49 (96.1) 48 (98.0)
Hispanic/Latino 2 (3.92) 1 (2.04)
BMI,2kg/m229.6 ±0.96 30.7 ±0.91 0.35
Systolic BP,2mm Hg 127 ±2.06 130 ±1.83 0.25
Diastolic BP,2mm Hg 74.4 ±1.64 76.1 ±1.38 0.44
MMSE227.6 ±0.22 27.8 ±0.21 0.50
KBIT-22101 ±1.45 98.9 ±1.26 0.30
Geriatric Depression Scale20.784 ±0.144 0.796 ±0.124 0.95
Spatial Span score (at screening)23.71 ±0.08 3.80 ±0.06 0.35
Hours of sleep (at Week 0),2h7.32±0.12 7.20 ±0.11 0.47
1Values are means ±SEMs or frequency (%). Differences between placebo and citicoline groups for all characteristics were analyzed using independent t-test for continuous
variables and Fisher’s exact test for categorical variables. BP, blood pressure; kg, kilogram; KBIT-2, Kaufman Brief Intelligence Test, Second Edition; m, meter; mm Hg,
millimeter mercury; MMSE, Mini-Mental State Examination.
2Independent t-test.
3Fisher’s exact test.
the placebo group, had a statistically signicant improvement
in Spatial Span, Feature Match, and Composite Memory
scores from baseline. Between-group analysis indicated that
the changes from baseline scores were statistically signif-
icantly different (P<0.00625) between test groups for
Paired Associates and Composite Memory whereby the citi-
coline group demonstrated greater improvements in these
tests compared with the placebo group (Figures 2 and
3). No additional statistically signicant cognitive effects
were detected in Monkey Ladder, Digit Span, Interlocking
Polygon Task, and Sustained Attention to Response Task
according to multiple comparisons with Bonferroni correc-
tion.
Safety assessments
No AEs were judged to be serious. Eight mild AEs were
judged to be “possibly” related to 1 of the study products,
with 2 occurring in the placebo group and 6 occurring
in the citicoline group. All 6 of the AEs in the citicoline
group [increased appetite, weight gain, increased atulence
(2 instances), headache, and increased burping] were mild and
transient, and were not unexpected based on previous studies
with the product (15,16). No AEs were deemed “denitely”
or “probably” related to the ingestion of study product.
Data from the hematology and metabolic panels obtained
at baseline and end of study are presented in Supplemental
Tabl e 1 and Supplemental Table 2, respectively. There were
no statistically signicant differences in mean values between
groups at baseline or at end of study. None of the values fell
outside the normal range.
Discussion
To our knowledge, this is the rst randomized, double-blind,
placebo-controlled parallel study to evaluate the effects of
chronic (12 wk) supplementation of 500 mg/d of citicoline
in healthy adults with AAMI on memory performance.
Compared with those taking placebo, participants taking
citicoline demonstrated a signicant improvement in episodic
memory assessed using the Paired Associate task and overall
memory assessed by the composite memory score. Although
each cognitive test assessed distinct components of memory and
tapped into different processes, composite score provides for
a single outcome variable combining each cognitive test. Our
observations are consistent with previous studies demonstrating
benecial effects of citicoline on memory (17,18).
Episodic memory describes the ability to remember and
recall specic events, paired with the content in which they
occurred, such as identifying when and where an object was
encountered (24). A decline in episodic memory often manifests
as the inability to recall past events or retrieve lessons from past
experiences, which can lead to repeat error. Episodic memory
is more vulnerable than other memory systems to decreases
due to aging (25). Kinugawa et al. (26) reported that middle-
aged (48–62 y) and aged (71–83 y) participants showed lower
episodic memory score as compared with the young (21–45 y)
participants with longitudinal studies demonstrating a decline
after age 60 y (27,28). To the best of our knowledge, our
study is the rst to demonstrate a benecial effect of citicoline
supplementation in maintaining and/or improving episodic
memory that may decline with age.
In addition to a statistically signicant improvement in
Paired Associates, we also observed a tendency toward
improvement in the citicoline group for the Spatial Span task.
2156 Nakazaki et al.
FIGURE 1 CONSORT participant ow diagram.
Owen (20) reported activation of the mid-ventrolateral frontal
cortex during the Spatial Span and Paired Associates tasks.
Citicoline has been shown to improve frontal lobe bioenergetics
and alter phospholipid membrane turnover in humans (29).
Age-related declines in cognitive abilities, particularly related
to function in frontal lobe has been demonstrated in humans
(30,31). Taken together, the ndings suggest that activation of
the mid-ventrolateral frontal cortex is a possible mechanism of
action by which citicoline improved cognition. Future studies
may consider assessing regionally specic neuronal activation
following citicoline supplementation to better understand its
mechanism of action and effect on brain function.
We also observed an improvement in selective attention
(assessed using Feature Match task) in the citicoline group
compared with baseline; however, there was no signicant
difference between groups. The primary objective of this
study was to assess the effect of citicoline on short-term
spatial memory, and thus, is likely underpowered to assess
difference in attention between groups. However, the within-
group improvement that we observed along with the positive
effect reported by a previous study (16), suggests a promising
benecial effect of citicoline on attention that warrants further
investigation.
Citicoline is well known to increase the synthesis of
phosphatidylcholine, which is the primary phospholipid of
neuronal membrane. Studies in rodents have demonstrated
increased phosphatidylcholine levels in the brain following
repeated citicoline supplementation (32,33). A clinical study
in healthy participants consuming 500 mg/d of citicoline for
6 wk demonstrated increased levels of phosphodiesters, a
noninvasive biomarker of phospholipid synthesis in the brain,
thus supporting the ability of citicoline to increase brain phos-
phatidylcholine synthesis in humans (34). Phosphatidylcholine
is essential for cell membrane integrity and repair (35,36,37),
and is normally reduced in brain as a result of aging (38).
Plasma phosphatidylcholine levels are positively associated with
cognitive exibility in healthy older adults, and the inferior
prefrontal cortex mediates the relationship between plasma
phosphatidylcholine and cognitive exibility (39). Increase in
phosphodiesters correlated with improvement on the California
Verbal Learning Test, an assessment of verbal learning and
memory decits, in healthy older adults (34). Taken together,
these ndings suggest that citicoline may slow or prevent AAMI
by inuencing specic structures within the brain.
Citicoline is naturally present in humans (40), and is a
nontoxic material determined by animal toxicology studies (41,
42,43). In our study, we did not observe any serious adverse
events following daily consumption of citicoline for 12 wk.
Consistent with our ndings, previous clinical trials involving
oral citicoline supplementation of Alzheimer’s disease patients
for 6 wk at a dose of 500 mg/d or for 12 wk at a dose of
1 g/d reported no serious adverse drug reactions (44,45). Thus,
based on our ndings and that of others, oral intake of citicoline
at amounts up to 1 g/d is safe and well tolerated. Future
investigations are required to determine the acute and longer-
term effects of citicoline, in addition to the extent to which
the benecial effects of citicoline on memory last following
cessation of citicoline supplementation.
Citicoline on memory in healthy older adults 2157
TABLE 2 Results for cognitive performance of the citicoline supplementation group and the placebo group in the ITT population at
Weeks 0 and 121
Tes t Baseline (Week 0) End of study (Week 12)
Spatial Span
Placebo 4.75 ±0.11 4.67 ±0.13
Citicoline 4.65 ±0.09 4.90 ±0.12∗
Monkey Ladder
Placebo 6.43 ±0.12 6.51 ±0.13
Citicoline 6.65 ±0.14 6.88 ±0.17
Digit Span
Placebo 5.94 ±0.18 6.22 ±0.18
Citicoline 5.88 ±0.16 5.94 ±0.17
Paired Associate
Placebo 3.86 ±0.13 3.92 ±0.14
Citicoline 3.92 ±0.11 4.04 ±0.11†
Composite Memory
Placebo 74.8 ±1.5 75.5 ±1.8
Citicoline 75.6 ±1.4 79.1 ±1.7∗†
Feature Match
Placebo 83.8 ±3.0 88.6 ±3.3
Citicoline 86.7 ±2.8 96.8 ±3.5∗
Interlocking Polygon Task
Placebo 26.2 ±2.3 28.0 ±2.3
Citicoline 22.8 ±2.4 25.3 ±2.8
Sustained Attention to Response Task
Placebo 174 ±7 185 ±2
Citicoline 181 ±4 177 ±6
1Values are means ±SEMs of test score. n=51 for placebo and n=49–48 for citicoline. An increase composite memory score and all of each individual cognitive tests score
indicates an improvement. ∗P<0.05 within-group difference (baseline vs. end of study) using paired t-test. †P<0.05 between-group difference for the raw change score
analyzed using ANCOVA with Bonferroni correction for multiple comparisons. The raw change was calculated as the difference in scores at baseline to the end of the test
period for each participant.
The study has some limitations worth noting. Participants
were specically screened for AAMI, and thus, the effects
observed may not be generalizable to young adults, and those
with cognitive diseases such as dementia and Alzheimer’s
disease. Disparities in cognitive functioning by race/ethnicity
have been suggested by several studies (46,47). Thus, future
FIGURE 2 Composite memory scores after 12 wk of citicoline
supplementation of the citicoline supplementation group and the
placebo group in the ITT population. Participants were healthy older-
aged males and females with AAMI. Data shown are unadjusted
means ±SEMs for each test group. n=51 for placebo and n=49–
48 for citicoline. An increased score indicates improvement. The
Pvalue shows between-group difference (vs. placebo) for the raw
change score using ANCOVA with Bonferroni correction for multiple
comparisons. The raw change was calculated as the difference in
scores at baseline to the end of the test period for each participant.
studies with a wider range of ethnicity are warranted to
understand if the benecial effects of citicoline on memory
observed in our mostly white Caucasian population may be
extended to other ethnic and racial groups. In total, there were
fewer males than females in this study (35:65 males:females)
although effort was made to balance the number of males
FIGURE 3 Paired Associates scores after 12 wk of citicoline
supplementation of the citicoline supplementation group and the
placebo group in the ITT population. Participants were healthy older-
aged males and females with AAMI. Data shown are unadjusted
means ±SEMs for each test group. n=51 for placebo and n=49–
48 for citicoline. An increased score indicates improvement. The
Pvalue shows between-group difference (vs. placebo) for the raw
change score using ANCOVA with Bonferroni correction for multiple
comparisons. The raw change was calculated as the difference in
scores at baseline to the end of the test period for each participant.
2158 Nakazaki et al.
and females by intervention group to limit any confounding
factors due to sex. Additionally, participants were instructed
to maintain their habitual diet and lifestyle in an effort to
minimize other confounding factors. The day before test visits
(Weeks 0 and 12),information on diet intake and sleeping hours
was collected to conrm whether participants maintained their
habits. Care was taken to ensure that participants completed
all cognitive assessments in a supervised environment where
lighting and noise were controlled.
Taken together, dietary supplement of citicoline improves
overall memory performance, especially episodic memory in
healthy males and females with AAMI. The ndings suggest that
regular consumption of citicoline (Cognizin) may be safe and
potentially benecial against memory loss due to aging.
Acknowledgments
The authors’ contributions were as follows—EN, EM, DC, and
FW: designed research; KS: conducted research; EM: analyzed
data; EN: wrote the rst draft of the manuscript; EN: had
primary responsibility for nal content; and all authors: read
and approved the nal manuscript. The authors would also
like to thank Dr Libertie Mantilla for statistical analysis and
consultation.
References
1. United Nations, Department of Economic and Social Affairs, Population
Division. World Population Prospects 2019: highlights. New York:
United Nations; 2019.
2. Dominguez LJ, Barbagallo M. Nutritional prevention of cognitive
decline and dementia. Acta Biomed 2018;89(2):276–90.
3. Prince M, Albanese E, Guerchet M, Prina M. World Alzheimer Report
2014: dementia and risk reduction. London: Alzheimer’s Disease
International (ADI); 2014.
4. Jennings A, Cunnane SC, Minihane AM. Can nutrition support healthy
cognitive ageing and reduce dementia risk? BMJ 2020;369:m2269.
5. Vauzour D, Camprubi-Robles M, Miquel-Kergoat S, Andres-Lacueva
C, Bánáti D, Barberger-Gateau P, Bowman GL, Caberlotto L, Clarke R,
Hogervorst E, et al. Nutrition for the ageing brain: towards evidence for
an optimal diet. Ageing Res Rev 2017;35:222–40.
6. Zeisel SH. Choline: critical role during fetal development and dietary
requirements in adults. Annu Rev Nutr 2006;26:229–50.
7. Cohen BM, Renshaw PF, Stoll AL, Wurtman RJ, Yurgelun-Todd
D, Babb SM. Decreased brain choline uptake in older adults.
An in vivo proton magnetic resonance spectroscopy study. JAMA
1995;274(11):902–7.
8. Poly C, Massaro JM, Seshadri S, Wolf PA, Cho EY, Krall E, Jacques
PF, Au R. The relation of dietary choline to cognitive performance and
white-matter hyperintensity in the Framingham Offspring Cohort. Am
J Clin Nutr 2011;94(6):1584–91.
9. Nurk E, Refsum H, Bjelland I, Drevon CA, Tell GS, Ueland PM, Vollset
SE, Engedal K, Nygaard HA, Smith DA. Plasma free choline, betaine
and cognitive performance: the Hordaland Health Study. Br J Nutr
2013;109(3):511–9.
10. Christopher P, Margret M, Aouatef B. Trimethylamine N-oxide
in relation to cardiometabolic health—cause or effect? Nutrients
2020;12(5):1330.
11. D’Orlando KJ, Sandage Jr BW. Citicoline (CDP-choline): mechanisms
of action and effects in ischemic brain injury. Neurol Res 1995;17: 281–
4.
12. Michel V, Yuan Z, Ramsubir S, Bakovic M. Choline transport for
phospholipid synthesis. Exp Biol Med 2006;231:490–504.
13. Gareri P, Castagna A, Cotroneo AM, Putignano S, De Sarro G, Bruni
AC. The role of citicoline in cognitive impairment: pharmacological
characteristics, possible advantages, and doubts for an old drug with
new perspectives. Clin Interv Aging 2015;10:1421–29.
14. Fioravanti M, Yanagi M. Cytidinediphosphocholine (CDP choline)
for cognitive and behavioural disturbances associated with chronic
cerebral disorders in the elderly. Cochrane Database Syst Rev 2005,
18;(2):CD000269, Available from: https://pubmed.ncbi.nlm.nih.gov/15
846601/.
15. McGlade E, Locatelli A, Hardy J, Kamiya T, Morita M, Morishita
K, Sugimura Y, Yurgelun-Todd D. Improved attentional performance
following citicoline administration in healthy adult women. Food Nutr
Sci 2012;3:769–73.
16. McGlade E, Agoston AM, DiMuzio J, Kizaki M, Nakazaki E, Kamiya
T, Yurgelun-Todd D. The effect of citicoline supplementation on
motor speed and attention in adolescent males. J Atten Disord
2019;23(2):121–34.
17. Spiers PA, Myers D, Hochanadel GS, Lieberman HR, Wurtman
RJ. Citicoline improves verbal memory in aging. Arch Neurol
1996;53(5):441–8.
18. Alvarez XA, Laredo M, Corzo D, Fernández-Novoa L, Mouzo R, Perea
JE, Daniele D, Cacabelos R. Citicoline improves memory performance
in elderly subjects. Methods Find Exp Clin Pharmacol 1997;19(3):201–
10.
19. Crook T, Bartus RT, Ferris SH, Whitehouse P, Cohen GD, Gershon
S. Age-associated memory impairment: proposed diagnostic criteria
and measures of clinical change – report of a National Institute
of Mental Health work group. Dev Neuropsychol 1986;2(4):
261–76.
20. Owen AM. The functional organization of working memory processes
within human lateral frontal cortex: the contribution of functional
neuroimaging. Eur J Neurosci 1997;9(7):1329–39.
21. The Uppsala Monitoring Centre (UMC). The WHO Adverse Reaction
Terminology (WHO-ART). 2016.
22. Center for Drug Evaluation and Research (CDER). Reviewer Guidance:
Conducting a Clinical Safety Review of a New Product Application and
Preparing a Report on the Review. U.S. Dept. of Health and Human
Services, Food and Drug Administration. Rockville (MD): Center for
Drug Evaluation and Research; 2005.
23. Shapiro SS, Wilk MB. An analysis of variance test for normality
(complete samples). Biometrika 1965;52:591–611.
24. Tulving E. Episodic memory: from mind to brain. Annu Rev Psychol
2002;53:1–25.
25. Nyberg L. Functional brain imaging of episodic memory decline in
ageing. J Intern Med 2017;281(1):65–74.
26. Kinugawa K, Schumm S, Pollina M, Depre M, Jungbluth C, Doulazmi
M, Sebban C, Zlomuzica A, Pietrowsky R, Pause B, et al. Aging-related
episodic memory decline: are emotions the key? Front Behav Neurosci
2013;7:2.
27. Rönnlund M, Nyberg L, Bäckman L, Nilsson LG. Stability, growth, and
decline in adult life span development of declarative memory: cross-
sectional and longitudinal data from a population-based study. Psychol
Aging 2005;20(1):3–18.
28. Josefsson M, de Luna X, Pudas S, Nilsson LG, Nyberg L. Genetic and
lifestyle predictors of 15-year longitudinal change in episodic memory.
J Am Geriatr Soc 2012;60(12):2308–12.
29. Silveri MM, Dikan J, Ross AJ, Jensen JE, Kamiya T, Kawada
Y, Renshaw PF, Yurgelun-Todd DA. Citicoline enhances frontal
lobe bioenergetics as measured by phosphorus magnetic resonance
spectroscopy. NMR Biomed 2008;21:1066–75.
30. Grieve SM, Williams LM, Paul RH, Clark CR, Gordon E. Cognitive
aging, executive function, and fractional anisotropy: a diffusion
tensor MR imaging study. AJNR Am J Neuroradiol 2007;28:
226–35.
31. Hazlett EA, Buchsbaum MS, Mohs RC, Spiegel-Cohen J, Wei TC,
Azueta R, Haznedar MM, Singer MB, Shihabuddin L, Luu-Hsia C,
et al. Age-related shift in brain region activity during successful memory
performance. Neurobiol Aging 1998;19:437–45.
32. Agut J, Lopez GCI, Ortiz JA, Wurtman RJ. Oral cytidine 50-diphosphate
choline administration to rats increases brain phospholipid levels. Ann
N Y Acad Sci 1993;695:318–20.
33. Lopez-Coviella I, Agut J, Savci V, Ortiz JA, Wurtman RJ. Evidence that
5-cytidinediphosphocholine can affect brain phospholipid composition
by increasing choline and cytidine plasma levels. J Neurochem 1995;
65:889–94.
34. Babb SM, Wald LL, Cohen BM, Villafuerte RA, Gruber SA, Yurgelun-
Todd DA, Renshaw PF. Chronic citicoline increases phosphodiesters
in the brains of healthy older subjects: an in vivo phosphorus
magnetic resonance spectroscopy study. Psychopharmacology (Berl)
2002;161:248–54.
Citicoline on memory in healthy older adults 2159
35. Marcucci H, Paoletti L, Jackowski S, Banchio C. Phosphatidylcholine
biosynthesis during neuronal differentiation and its role in cell fate
determination. J Biol Chem 2010;285(33):25382–93.
36. Paoletti L, Elena C, Domizi P, Banchio C. Role of phosphatidylcholine
during neuronal differentiation. IUBMB Life 2011;63(9):714–20.
37. Skripuletz T, Manzel A, Gropengießer K, Schäfer N, Gudi V, Singh V,
Salinas Tejedor L, Jörg S, Hammer A, Voss E, et al. Pivotal role of choline
metabolites in remyelination. Brain 2015;138(2):398–413.
38. Babb SM, Appelmans KE, Renshaw PF, Wurtman RJ, Cohen BM.
Differential effect of CDP-choline on brain cytosolic choline levels in
younger and older subjects as measured by proton magnetic resonance
spectroscopy. Psychopharmacology (Berl) 1996;127(2):88–94.
39. Zamroziewicz MK, Zwilling CE, Barbey AK. Inferior prefrontal cortex
mediates the relationship between phosphatidylcholine and executive
functions in healthy, older adults. Front Aging Neurosci 2016;8:
226.
40. Chaleckis R, Murakami I, Takada J, Kondoh H, Yanagida M. Individual
variability in human blood metabolites identies age-related differences.
Proc Natl Acad Sci 2016;113(16):4252–9.
41. Romero A, Grau T, Sacristán A, Ortiz JA. CDP-choline: 6-month
study on toxicity in dogs. Arzneimittelforschung 1983b;33(7A):
1038–42.
42. Romero A, Grau T, Sacristán A, Ortiz JA. Study of subacute
toxicity of CDP-choline after 30 days of oral administration to rats.
Arzneimittelforschung 1983a;33(7A):1035–8.
43. Schauss AG, Somfai-Relle S, Financsek I, Glavits R, Parent SC, Endres
JR, Varga T, Szücs Z, Clewell A. Single- and repeated-dose oral toxicity
studies of citicoline free-base (choline cytidine 5-pyrophosphate) in
Sprague-Dawley rats. Int J Toxicol 2009;28(6):479–87.
44. Alvarez XA, Mouzo R, Pichel V, Pérez P, Laredo M, Fernández-
Novoa L, Corzo L, Zas R, Alcaraz M, Secades JJ, et al. Double-
blind placebo-controlled study with citicoline in APOE genotyped
Alzheimer’s disease patients. Effects on cognitive performance, brain
bioelectrical activity and cerebral perfusion. Methods Find Exp Clin
Pharmacol 1999;21(9):633–44.
45. Clark WM, Williams BJ, Selzer KA, Zweier RM, Sabounjian LA,
Gammans RE. A randomized efcacy trial of citicoline in patients with
acute ischemic stroke. Stroke 1999;30(12):2592–7.
46. Masel MC, Peek MK.Ethnic differences in cognitive function over time.
Ann Epidemiol 2009;19(11):778–83.
47. Schwartz BS, Glass TA, Bolla KI, Stewart WF, Glass G, Rasmussen M,
Bressler J, Shi W, Bandeen-Roche K. Disparities in cognitive functioning
by race/ethnicity in the Baltimore Memory Study. Environ Health
Perspect 2004;112(3):314–20.
2160 Nakazaki et al.
