Effects of an 8-week intake of lysolecithin on cognitive function and concentrations of blood choline and lysophosphatidylcholine: a randomized, double-blinded, placebo-controlled trial

Abstract

Choline is an essential nutrient for normal brain function, but its bioavailability is not as high as choline esters. Among choline esters, lysophosphatidylcholine (LPC) has unexplored potential as a choline source and cognitive enhancer in humans. This placebo-controlled, double-blinded study, involving healthy participants aged 40–74 years, aimed to assess the effects of an 8-week intake of lysolecithin containing 480 ‍mg LPC on cognitive function and plasma levels of choline and LPC. Twenty-three participants were assigned to both the placebo and lysolecithin groups, and memory was assessed as the primary outcome. Additionally, subjective mental function was assessed. Plasma levels of derivatives of reactive oxygen metabolites were also evaluated for a safety assessment. No significant between-group differences were observed in the memory or mental function score, but a post-hoc analysis yielded significant within-group increases from baseline in subjective mental acuity and calmness in the lysolecithin group. Lysolecithin intake slightly increased plasma choline and LPC18:2 concentrations over 8 weeks, but plasma levels of saturated and total LPC concentrations, associated with inflammation, and derivatives of reactive oxygen metabolites remained unchanged. No adverse events were attributed to lysolecithin supplementation. This study demonstrated lysolecithin’s good tolerability and potential as a new choline supplement.

Introduction

Choline (Ch) is an essential nutrient for neurodevelopment and cholinergic transmission. Associations between low Ch intake or diminished plasma Ch levels and low cognitive function have been reported in older adults,^(1,2) emphasizing the importance of Ch intake in maintaining and improving cognitive function. However, the amounts of Ch consumed by most Americans remain below the adequate intake values defined by the Food and Nutrition Board of the National Academy of Medicine of the USA in 1998.^(3,4) Moreover, an increasing number of individuals adopting vegetarian and vegan dietary patterns may face an elevated risk of Ch inadequacy,⁽⁵⁾ given its predominant occurrence in animal-derived food sources. Furthermore, the aging of society is contributing to a growing prevalence of cognitive deterioration among individuals. Therefore, identifying new Ch sources, particularly those from plants, enabling vegetarians and vegans to consume them, and evaluating their effects on cognitive function are crucial.

After phosphatidylcholine (PC) consumption was reported to elevate blood Ch concentration more effectively than Ch itself does, Ch esters have been explored as potential cognitive enhancers.^(6,7) However, lysophosphatidylcholine (LPC), which has a glycerol backbone esterified with a fatty acid at sn1 or sn2, has not been investigated in the context of cognitive enhancement. The limited investigation into this area is attributed to the fact that saturated LPC species, such as LPC16:0 and LPC18:0, cause the production of reactive oxygen species and inflammatory and pathogenic changes when surpassing the physiological ranges in the body.⁽⁸⁾ However, we previously demonstrated that oral administration of egg-derived LPC increased brain acetylcholine levels without elevating plasma concentrations of saturated LPC species.⁽⁹⁾ We also conducted a clinical trial in which the single-dose pharmacokinetics of soy-derived lysolecithin (LLT), a food additive mainly composed of LPC, was evaluated and showed that LLT increases blood Ch concentration, similar to glycerophosphocholine (GPC),⁽¹⁰⁾ which is a Ch ester reported to enhance memory and attention in various clinical trials.⁽¹¹⁾ However, the effects of long-term LLT supplementation on cognitive function and blood concentrations of Ch and its metabolites have not been evaluated. Therefore, here, we aimed to investigate the effects of an 8-week soy-derived LLT supplementation on memory, attention, and blood Ch concentration. In addition, other mental functions, such as mental acuity and calmness, were subjectively assessed. Moreover, changes in the plasma concentrations of LPC and derivatives of reactive oxygen metabolites (d-ROMs) were measured as part of a safety assessment of LLT supplementation.

Materials and Methods

The study was approved by the Hakata Clinic Institutional Review Board (approval number: K-39), registered in the UMIN Clinical Trials Registry (registration number UMIN000053685), and conducted at the PS Clinic (Fukuoka, Japan) in compliance with the Declaration of Helsinki.

Human volunteers

Healthy adults aged 40–74 years were recruited and included in the study if they had no health conditions hindering them from fulfilling the study requirements, as determined by a physician at the PS Clinic based on laboratory screening tests. Exclusion criteria involved the presence of sleeping problems, digestive disorders potentially affecting the absorption of test supplements, use of medications or supplements with potential effects on brain and nerve functions within a week of the study commencement, blood pressure considerably deviating from the normal range, and soy allergy. Forty-six eligible adults who provided informed consent were enrolled in the study, and 23 participants were assigned to both the placebo and lysolecithin groups. Demographic data of the study participants are presented in Table 1.

Table 1.

Demographics of the study participants

	Placebo	LLT
Number	23, including 6 females	23, including 6 females
Age (years)	55.5 ± 9.0 (41–72)	54.9 ± 9.4 (40–71)
Height (cm)	166.0 ± 9.5 (144.3–179.5)	167.0 ± 8.6 (153.5–184.8)
Weight (kg)	62.9 ± 9.7 (44.2–81.1)	62.8 ± 11.6 (45.2–90.5)

Data are presented as the mean ± SD. Numbers in parentheses indicate ranges.

Test supplements

(1) Placebo: Seven soft capsules containing 2.1 ‍g of soy oil.

(2) LLT: Seven soft capsules containing 2.1 ‍g of soy-derived LLT (SLP-PasteLyso), 240 ‍mg of LPC, and 41.2 ‍mg of a Ch moiety.

Soy oil and SLP-PasteLyso were purchased from The Nisshin OilliO Group, Ltd. (Tokyo, Japan) and Tsuji Oil Mills Co., Ltd. (Mie, Japan), respectively. Capsules were manufactured by Sankyo Co., Ltd. (Shizuoka, Japan).

The participants consumed seven capsules each after breakfast and dinner for 8 weeks (LPC 480 ‍mg/day). The dose was set by referring to a previous clinical trial in which SLP-PasteLyso containing 480 ‍mg LPC was orally administered, and no adverse events were attributed to the supplementation.⁽¹⁰⁾ The LPC purity in SLP-PasteLyso used in the present study was 11.4%. Despite the commercial availability of LLTs with higher LPC purities, SLP-PasteLyso, consisting of 55% acetone-soluble matter (fat), was chosen due to the improved absorption through the lymphatic pathway when consumed with fat.^(9,12) We confirmed that LPC is a predominant Ch source in SLP-PasteLyso, and the lipid composition of the LLT is presented in Table 2.

Table 2.

Lipid composition and Ch content in SLP-PasteLyso (LLT)

	(g/100 ‍g)
Lipid	99.4
Total fatty acid	66.9
16:0 (palmitic acid)	9.8
18:0 (stearic acid)	2.7
18:1 (oleic acid)	10.2
18:2 (linoleic acid)	37.5
18:3 (linolenic acid)	5.2
LPC	11.4
PC	2.3
Total Ch	1.96

Ch, choline; LLT, lysolecithin; LPC, lysophosphatidylcholine; PC, phosphatidylcholine.

Study design

This was a randomized, double-blinded, placebo-controlled, parallel-group study. The participants had standard meals for dinner on the day before the 0- and 8-week assessments and for breakfast on the day of the assessments. Memory assessments were conducted in the morning at both time points, and blood samples were collected at noon. After having a standard meal for lunch, participants engaged in a 15-min addition task. In addition to these assessments at 0 and 8 weeks, the participants were asked to complete a visual analog scale (VAS) to specify their subjective feelings of mental acuity, concentration, calmness, and the quality of sleep at 0, 2, 4, 6, and 8 weeks. We assumed that LPC supplementation may enhance cholinergic transmission in the brain and thereby improve mood and the quality of sleep.^(13,14)

The participants refrained from taking drugs and supplements that could affect their cognitive and mental functions during the study period. One day before the assessments, participants were prohibited from consuming alcohol or engaging in intense exercise. On assessment days, participants were required to consume only water, except for standard meals, until all assessments were completed. Blood samples were centrifuged at 1,500 ×g for 15 ‍min at 4°C, and the resulting plasma and serum were preserved at −80°C until measurement. Tubes coated with sodium heparin were used for plasma collection.

Cognitive and mental assessments

The Japanese version of the Wechsler Memory Scale-Revised (WMS-R), an internationally recognized battery for memory assessment,⁽¹⁵⁾ was used to evaluate verbal, visual, and general memory, attention/concentration, and delayed recall. The scores were adjusted for age according to the manufacturer’s instructions and analyzed as the primary outcome. Attentional function was assessed as calculation performance on the Uchida–Kraepelin test,⁽¹⁶⁾ where participants were tasked with adding two adjacent numbers and recording the last digit of their answers on a designated test form for 15 ‍min. They were instructed to calculate as fast and accurately as possible. The average number of answers per minute and the percentage of incorrect answers were calculated. Subjective feelings of mental acuity, concentration, calmness, and the quality of sleep in daily life were measured using a 100-mm VAS with anchor statements “not at all” at one end and “extremely” at the other end.

Chemical analysis

Plasma Ch and LPC levels were measured using previously reported methods.^(9,17) LPC16:0 (palmitoyl), 18:0 (stearoyl), 18:1 (oleoyl), 18:2 (linoleoyl), and 18:3 (linolenoyl) were quantified as they are the predominant LPC forms in SLP-PasteLyso. The total LPC level was calculated as the sum of the levels of these five LPC species. Additionally, the mature form of brain-derived neurotrophic factor (BDNF) in serum was determined using an ELISA kit (Biosensis Pty Ltd., Thebarton, Australia). Plasma d-ROM levels were measured at the Japan Institute for the Control of Aging, Nikken SEIL Co., Ltd. (Shizuoka, Japan).

Statistical analyses

The results of outcome measures are expressed as the mean ± SE. Statistical analyses were performed using the SAS software (ver. 9.4; SAS Institute Inc., Cary, NC). In the full analysis set, between-group differences were analyzed using the Wilcoxon rank-sum test for the percentage of incorrect answers in the Uchida–Kraepelin test and the unpaired t test for the other measures. As a post-hoc analysis, differences from baseline in VAS scores were analyzed using a two-tailed Dunnett’s test after analysis of variance for randomized block designs. Other within-group changes from baseline were analyzed using paired t test. Differences were considered statistically significant at p<0.05.

Results

Out of 46 study participants, one participant in the placebo group withdrew consent after 2 weeks, but all other participants completed the study. Although one participant in the LLT group complained of drowsiness, no abnormal changes in the laboratory test results were observed after the study, and the physician at the PS Clinic did not attribute any adverse events to LLT supplementation. Compliance with test supplement consumption exceeded 99%, and all participants were included in the analysis.

The WMS-R and Uchida–Kraepelin test scores are presented in Table 3. No significant between-group differences were observed in memory outcomes except for the general memory score at 0 week. Additionally, no significant difference was found in the average number of answers and the percentage of incorrect answers in the Uchida–Kraepelin test. VAS scores are presented in Table 4. A significant between-group difference was only observed for the calmness score at 0 week.

Table 3.

Scores of the WMS-R and Uchida–Kraepelin tests

		0 week			8 weeks
		Placebo	LLT		Placebo	LLT
WMS-R	Verbal memory	107.6 ± 3.3	100.6 ± 2.6		114.5 ± 3.3	110.9 ± 2.5
	Visual memory	106.8 ± 2.0	101.2 ± 2.2		110.5 ± 1.4	106.8 ± 1.8
	General memory	108.5 ± 2.9	100.8 ± 2.3*		115.2 ± 2.8	110.7 ± 2.3
	Attention/Concentration	107.7 ± 1.6	106.4 ± 3.3		110.8 ± 2.4	105.7 ± 2.9
	Delayed recall	105.4 ± 3.0	102.2 ± 1.8		111.3 ± 3.3	107.9 ± 2.2
Uchida–Kraepelin test	Average number of answers (/min)	52.3 ± 2.3	49.8 ± 3.8		50.9 ± 2.5	48.4 ± 3.9
	Percentage of wrong answers	1.6 ± 0.5	1.9 ± 0.7		1.7 ± 0.4	1.7 ± 0.5

Data are presented as the mean ± SE (n = 22, 23). *p<0.05 vs placebo. WMS-R, Wechsler Memory Scale-Revised.

Table 4.

Changes in VAS scores on mental acuity, concentration, calmness, and the quality of sleep

	0 week			2 weeks			4 weeks			6 weeks			8 weeks
	Placebo	LLT		Placebo	LLT		Placebo	LLT		Placebo	LLT		Placebo	LLT
Mental acuity	76.4 ± 2.6	71.6 ± 4.4		75.0 ± 3.3	75.0 ± 4.1		74.8 ± 3.9	79.8 ± 3.6		79.8 ± 3.0	79.4 ± 3.8		81.5 ± 3.4	81.6 ± 3.3
Concentration	78.9 ± 2.5	72.8 ± 4.3		77.0 ± 3.3	73.5 ± 4.2		75.8 ± 4.2	81.1 ± 3.7		82.0 ± 3.1	78.1 ± 4.1		80.9 ± 3.4	80.9 ± 3.5
Calmness	85.3 ± 2.8	73.7 ± 4.3*		85.0 ± 3.4	79.0 ± 4.1		84.4 ± 3.3	83.8 ± 3.6		87.7 ± 2.9	80.2 ± 4.3		85.5 ± 3.2	82.6 ± 3.8
Sleep	80.8 ± 3.2	76.5 ± 4.1		84.1 ± 4.2	77.7 ± 3.9		81.0 ± 4.0	75.2 ± 4.7		83.2 ± 3.4	75.7 ± 4.0		80.7 ± 3.6	77.5 ± 3.8

A 100-mm VAS with anchor statements “not at all” and “extremely” at each end was used. Data are presented as the mean ± SE (n = 22, 23). *p<0.05 vs placebo. VAS, visual analog scale.

Blood concentrations of Ch, LPC, BDNF, and d-ROMs are presented in Table 5. Changes in plasma Ch concentration from 0 to 8 weeks were significantly higher for the LLT group (0.42 ‍μM for LLT and −0.29 ‍μM for placebo, p = 0.027; Fig. 1). In addition, plasma LPC18:2 concentration was significantly elevated after 8 weeks of supplementation in both groups (p = 0.012 and 0.025 for the placebo and LLT groups, respectively). However, no significant between-group difference was observed in LPC18:2 concentration even when the change from 0 week was analyzed. Furthermore, no other significant increases from baseline or between-group differences were found in the concentrations of other LPC species, total LPC, or BDNF.

Table 5.

Blood concentrations of Ch, LPC, BDNF, and d-ROMs

		0 week			8 weeks
		Placebo	LLT		Placebo	LLT
Ch (μM)		9.7 ± 0.3	10.2 ± 0.4		9.4 ± 0.3	10.6 ± 0.5
LPC (μM)	16:0 (palmitoyl)	65.2 ± 2.1	63.2 ± 2.2		63.6 ± 2.4	63.0 ± 2.3
	18:0 (stearoyl)	19.7 ± 0.7	20.3 ± 0.9		19.5 ± 0.7	19.5 ± 0.8
	18:1 (oleoyl)	16.2 ± 0.6	16.2 ± 0.5		17.0 ± 0.6	16.7 ± 0.6
	18:2 (linoleoyl)	30.4 ± 1.2	29.9 ± 1.2		33.8 ± 1.7†	34.9 ± 2.4†
	Total	131.5 ± 3.7	129.5 ± 4.0		133.9 ± 4.8	134.0 ± 4.9
BDNF (ng/ml)		28.3 ± 1.4	27.1 ± 1.4		27.6 ± 1.4	26.2 ± 1.5
d-ROMs (U. CARR)		326.6 ± 9.4	305.4 ± 9.8		320.8 ± 10.3	299.0 ± 10.5

Plasma samples were used to determine the concentrations of Ch, LPC, and d-ROMs. LPC18:3 (linolenoyl) was below the detection limit. The total LPC concentration was calculated as the sum of the concentrations of the four detected LPC species. The serum BDNF concentration was determined. Data are presented as the mean ± SE (n = 22, 23). ^†p<0.05 vs 0 week. BDNF, brain-derived neurotrophic factor; d-ROMs, derivatives of reactive oxygen metabolites.

Fig. 1.

Changes in plasma Ch concentrations. Data are presented as the mean ± SE (n = 22, 23). *p<0.05 vs placebo.

Discussion

To prevent cognitive deterioration associated with aging, the early intake of an adequate amount of Ch seems important.⁽²⁾ From this perspective, we recruited both middle-aged and elderly individuals without imposing an inclusion criterion based on their cognitive status. In this population, the 8-week supplementation of LLT, equivalent to 480 ‍mg of LPC and 82.4 ‍mg of a Ch moiety, did not yield a significant between-group difference in memory or performance on the calculation task. In contrast, a previous study showed that a 6-week supplementation with 38.8 ‍mg of Ch in the form of egg yolk lecithin enhanced verbal memory and processing speed in individuals aged 60–80 years who either expressed concerns about or had been alerted to forgetfulness by others.⁽¹⁸⁾ Moreover, the change in plasma Ch concentration during the supplementation period was greater in a previous study (Δ1.8 ‍μM, 12.6% of the baseline) compared to that in our study (Δ0.42 ‍μM, 4.1%), despite the supplemented Ch dose being lower than that in our study.⁽¹⁸⁾ These inconsistencies may be attributed to differences in Ch form (lecithin vs LLT), the method used to determine Ch levels in test supplements, and the test populations of the two studies. Consequently, the average age-adjusted memory scores of the individuals enrolled in the present study were higher than 100 (the average for respective age groups), as shown in Table 3. Moreover, a post-hoc analysis of within-group differences revealed that the scores of verbal memory (both placebo and LLT: p<0.001), general memory (both placebo and LLT: p<0.001), and delayed recall (placebo: p = 0.004, LLT: p = 0.001) significantly increased from 0 to 8 weeks, regardless of the supplement, likely indicating a training effect. For populations with memory function below average, the efficacy of LLT should be re-evaluated. Moreover, cognitive domains other than memory should be investigated in future studies, as Ch supplementation has also been reported to improve executive function.⁽¹⁹⁾

As a result of the post-hoc analysis, we found that the visual memory score significantly improved over 8 weeks only in the LLT group. Moreover, only the LLT group showed significant within-group increases in scores of mental acuity at 8 weeks and calmness at 4 weeks (p = 0.045 and 0.040, respectively), suggesting the effects of LLT on subjective mental function and mood. The relationship between Ch and major depression has gained attention, and it has been postulated that increased Ch levels in the frontal lobe potentially improve executive function, thereby improving mood.⁽²⁰⁾ Improvements in mental acuity and calmness in the LLT group may have occurred through the same mechanism, given that oral supplementation of LLT increased Ch levels in the rat frontal cortex.⁽⁹⁾ In future studies, a higher dose of LLT should be adopted to further evaluate its potential effects on cognitive and mental functions. Meanwhile, the Ch-equivalent dose of lecithin should be evaluated to compare the bioavailability and efficacy between LLT and lecithin.

Furthermore, changes in blood trimethylamine N-oxide (TMAO) concentrations caused by these supplements warrant further investigation. TMAO, which is produced by bacterial degradation and metabolism of Ch, is associated with atherosclerosis.⁽²¹⁾ Some studies have demonstrated that PC elevates blood TMAO concentrations to a lesser extent than Ch itself and GPC at equivalent doses.^(22,23) As a portion of ingested LPC follows the metabolic pathway of PC,⁽²⁴⁾ a similar phenomenon may be observed for LPC/LLT, thereby showing the potential superiority of LLT over currently dominant Ch sources like GPC. As demonstrated in our previous work, a single administration of 480 ‍mg of LPC did not increase plasma TMAO concentration;⁽¹⁰⁾ hence, its concentration was not measured in the present study.

Notably, an 8-week LLT supplementation elevated plasma LPC18:2 concentration but did not affect the concentrations of saturated LPC species, total LPC, or d-ROMs. The increase in LPC18:2 concentration was marginal and remained within the physiological range.⁽⁸⁾ Moreover, placebo supplementation also raised plasma LPC18:2 concentration. These changes are likely due to the assimilation and reconstitution of linoleic acid (18:2), which is rich in soy derivatives (oil and LLT), as LPC. Considering that no adverse events were attributed to LLT supplementation, an 8-week supplementation of LLT at 480 ‍mg LPC per day did not raise any safety concerns.

To our knowledge, this is the first study to investigate the effects of long-term LLT consumption on cognitive function, plasma Ch, and LPC concentrations. The dosage used in the present study (480 ‍mg LPC per day) increased plasma Ch concentration but did not affect the concentration of saturated LPC, total LPC, or dROMs. Although the LLT group did not show superior memory scores (primary outcomes) compared to the placebo group, this study highlights the good tolerability of LLT and its potential as a new Ch supplement. The effects of higher doses of LLT on brain function are worthy of further investigation.

Author Contributions

RT-K: Conceptualization, data curation, formal analysis, investigation, methodology, validation, and writing of the original draft. HK: LPC measurement and validation. KH: Project administration, writing – review and editing.

Abbreviations

BDNF

brain-derived neurotrophic factor

Ch

choline

d-ROMs

derivatives of reactive oxygen metabolites

GPC

glycerophosphocholine

LLT

lysolecithin

LPC

lysophosphatidylcholine

PC

phosphatidylcholine

TMAO

trimethylamine N-oxide

VAS

visual analog scale

WMS-R

Wechsler Memory Scale-Revised

Funding

This study was conducted using the resources provided by Otsuka Pharmaceutical Co., Ltd. The funder provided support in the form of salaries to the authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

All authors are employees of Otsuka Pharmaceutical Co., Ltd.

Data Availability

The data underlying this article will be shared upon reasonable request to the corresponding author.