Association between egg-derived phosphatidylcholine intake and trimethylamine N-oxide in healthy middle-aged and older adults

Abstract

Background

Egg-derived phosphatidylcholine may support the maintenance of cognitive function. However, its metabolite trimethylamine N-oxide is considered a potential cardiovascular risk factor. The present study aimed to assess, for the first time, the effects of long-term intake of egg-derived phosphatidylcholine on serum trimethylamine N-oxide concentrations in healthy Japanese middle-aged and older adults.

Methods

A secondary analysis was conducted using preserved serum samples obtained from a previous randomized controlled trial (UMIN000041554). The study population, comprising thirty-two healthy middle-aged and older Japanese adults, was categorized into four groups based on their level of phosphatidylcholine intake: placebo (0 g/day), low-dose (0.3 g/day), medium-dose (1.0 g/day), and high-dose (3.0 g/day). Serum trimethylamine N-oxide concentrations were measured before and after the 12-week intervention.

Results

In the high-dose phosphatidylcholine group, serum trimethylamine N-oxide concentration increased significantly at 12 weeks (Week 12) compared with Baseline (Week 0) (P = 0.028). In contrast, no significant between-group differences were observed in serum trimethylamine N-oxide concentrations or in the magnitude of change from Baseline to Week 12, including comparisons with placebo. Across all groups, mean trimethylamine N-oxide values remained substantially below the threshold associated with high clinical risk.

Conclusions

Twelve weeks of egg-derived phosphatidylcholine supplementation did not result in serum trimethylamine N-oxide concentrations approaching clinically concerning levels under the specific conditions tested. These findings suggest that egg-derived phosphatidylcholine may serve as a dietary source of choline without inducing a substantial elevation in trimethylamine N-oxide in healthy middle-aged and older adults. Confirmation of these results in studies with larger sample sizes is warranted.

Background

Acting as the fundamental building block for the neurotransmitter acetylcholine, choline is recognized as a vital vitamin-like nutrient. Proper neural maturation and the preservation of mental acuity across the lifespan depend on sufficient levels of this nutrient. To reflect its biological necessity, health authorities in North America, Europe, Australia, and China have established specific Dietary Reference Intakes. Nevertheless, intake levels in several populations continue to fall short of these recommendations [1]. Eggs represent a primary dietary source of choline among frequently consumed foods. A large proportion of the choline content in eggs is present in the form of phosphatidylcholine (PC), a principal component of biological membranes [1, 2].

Multiple studies have reported that egg-derived PC may help maintain or enhance aspects of cognitive performance, such as verbal memory, in healthy older adults [3–6]. Current scientific data regarding the relationship between egg ingestion and cognitive health continue to yield conflicting results. For example, a recent study observed no significant association in older adults between egg intake and changes in cognition over a 4-year follow-up [7]. Similarly, a recent systematic review concluded that current evidence supporting the cognitive benefits of egg consumption remains inconclusive [8]. In addition, egg phospholipids containing PC may provide several additional health benefits, including improvements in lipid metabolism and anti-inflammatory activity [9].

Nonetheless, concerns have been raised regarding the safety profile of dietary choline intake. Previous studies of feeding behavior have demonstrated that consumption of PC or whole eggs can lead to acute postprandial increases in plasma trimethylamine N-oxide (TMAO) concentrations [10, 11]. A portion of ingested choline is converted by gut microbiota into trimethylamine, which is subsequently oxidized in the liver to form TMAO [10]. In several studies, elevated serum TMAO concentrations have been associated with increased risk of cardiovascular disease (CVD), including atherosclerosis [10, 12–14]. As a result, the influence of egg consumption on circulating TMAO concentrations has garnered increasing attention in the field of nutrition science [15].

Previous investigations, including a meta-analysis of randomized controlled trials, have shown that consuming up to three eggs per day does not significantly alter serum TMAO concentrations in healthy adults [16]. Furthermore, recent work has demonstrated that various egg-derived components, including lipid fractions such as PC, egg yolk protein hydrolysates [17], and egg white peptides [18], can improve cognitive function in rodents. Overall, this evidence has increased the interest of investigators in the functional properties of eggs.

While the utility of egg-derived PC has become increasingly clear, its long-term effects on safety, particularly through changes in circulating TMAO concentration, remain poorly defined. It was hypothesized that continuous intake of egg-derived PC would not substantially elevate serum TMAO levels in a Japanese population, given the distinct dietary habits and differing gut microbiota composition compared with Western populations. To the best of the authors’ knowledge, no study thus far has examined the mechanism by which prolonged PC intake affects serum TMAO concentrations in a Japanese population. Therefore, the present study aimed to evaluate the dose-dependent effects of continuous intake of egg-derived PC for 12 weeks on the TMAO serum concentrations in healthy middle-aged and older adults.

Methods

Study design

The current investigation involved a retrospective analysis of stored serum specimens collected during an earlier randomized, double-blind, placebo-controlled clinical trial. This earlier study examined the efficacy of egg-derived PC on cognitive function in healthy middle-aged and older Japanese adults (University Hospital Medical Information Network [UMIN] Clinical Trials Registry: UMIN000041554). The Ethics Review Committee of Kobuna Orthopedic Clinic (Approval No: MK-2402–02) reviewed and approved the study protocol adhering to the ethical standards established by the Declaration of Helsinki. Written informed consent for the secondary utilization of serum samples was obtained from every individual. The present study was also registered with the UMIN Clinical Trials Registry (Registration No. UMIN000053605). The results are reported in accordance with the CONSORT guidelines.

Participants

The present study involved 32 healthy adults (19 men and 13 women) aged 60–79 years who participated in the previous study (UMIN000041554). These individuals received full information regarding the purpose and procedures of this ancillary study and provided written consent for their participation. The primary reasons for exclusion were insufficient serum sample volume for analysis or determination of ineligibility by the principal investigator.

Intervention

Randomization was utilized to allocate participants into four groups: placebo (i.e., PC 0 g/day; 0 mg/day as egg yolk choline [EYC]; n = 9), low-dose PC (i.e., PC 0.3 g/day; 40.5 mg/day as EYC; n = 8), medium-dose PC (i.e., PC 1.0 g/day; 135 mg/day as EYC; n = 8), and high-dose PC (i.e., PC 3.0 g/day; 405 mg/day as EYC; n = 7). Participants consumed their assigned test foods for 12 weeks.

Test foods

The preparation and composition of the test foods used in this study have been previously described in detail [5]. Briefly, baked sweets containing 300, 1,000, and 3,000 mg of EYC (expressed as PC equivalents) were provided to the intervention groups. These amounts corresponded to 40.5, 135, and 405 mg of free choline, respectively, calculated on the basis of the molecular weights of free choline (104.17 g/mol) and egg-derived PC (772 g/mol). In addition, the placebo group received baked sweets containing egg yolk oil without EYC (Kewpie Corporation, Tokyo, Japan). To maintain blinding, all test foods were prepared to be indistinguishable in taste and appearance. Participants were instructed to consume two pieces of baked sweets once daily, specifically between lunch and dinner (total intake: 48 g/day).

Measurements

Serum samples were collected before intake (Baseline; Week 0) and after 12 weeks of intake (Week 12), frozen at − 80 °C, and analyzed. The primary outcome was serum TMAO concentration, while the secondary outcome was the association with plasma choline concentration, as measured in a previous study [5] (Registration No. UMIN000041554). LSI Medience Corporation (Tokyo, Japan) performed the measurement of serum TMAO concentrations using liquid chromatography–tandem mass spectrometry and specific TMAO clinical risk classifications based on company reference values (high risk levels: ≥ 2.0 μg/mL).

Statistical analyses

The Shapiro–Wilk test was performed to evaluate data normality. Intragroup comparisons between Baseline and Week 12 involved the application of paired t-tests for data with a normal distribution, while the Wilcoxon signed-rank test was employed for data that were not normally distributed. Comparisons among the four groups at Baseline and Week 12 were subsequently conducted. Changes over time were quantified using one-way analysis of variance followed by Tukey’s post hoc tests when assumptions of normality and homogeneity of variance (evaluated by Levene’s test) were satisfied. When these assumptions were not met, the Kruskal–Wallis test was applied, followed by pairwise comparisons with Bonferroni adjustment. To evaluate potential associations between serum trimethylamine N-oxide levels and other clinical laboratory parameters, Spearman’s rank correlation coefficient was employed. For all analyses, a two-sided P value < 0.05 was considered to indicate statistical significance.

Results

Participant flow and baseline characteristics

Figure 1 illustrates the participant flow throughout the investigation.

Fig. 1.

Study participant flow diagram. n, number of participants; PC, phosphatidylcholine

Initial demographics and clinical profiles of the 32 subjects analyzed are documented in Table 1.

Table 1.

Baseline characteristics of study participants

Variable	Placebo (n = 9)			Low-dose PC (n = 8)			Medium-dose PC (n = 8)			High-dose PC (n = 7)			Total (n = 32)			P-value
Age (years)	68.7	±	4.3	68.5	±	4.3	67.8	±	3.5	69.9	±	4.4	68.7	±	4.0	0.803	a
Sex (Male/Female)	6/3 (66.7%/33.3%)			4/4 (50.0%/50.0%)			5/3 (62.5%/37.5%)			4/3 (57.1%/42.9%)			19/13 (59.4%/40.6%)			0.911	b
Weight (kg)	63.6	±	11.2	60.0	±	14.1	60.7	±	9.2	60.2	±	13.9	61.2	±	11.6	0.918	a
BMI (kg/m2)	23.7	±	2.2	23.1	±	3.1	22.6	±	2.6	22.2	±	3.6	23.0	±	2.8	0.739	a
Systolic blood pressure (mmHg)	131.6	±	12.5	145.0	±	22.1	133	±	12.2	133	±	15.3	136	±	16.1	0.304	a
Diastolic blood pressure (mmHg)	76.8	±	10.4	85.1	±	11.8	81.9	±	7.5	70.3	±	7.6	78.7	±	10.7	0.031	a
Heart rate (bpm)	71.4	±	10.8	74.0	±	18.6	69.9	±	8.3	58.9	±	12.9	68.9	±	13.7	0.154	a
Average egg intake (count/day)	0.54	±	0.32	0.75	±	0.40	0.48	±	0.29	0.70	±	0.44	0.61	±	0.36	0.406	a

Data are presented as mean ± standard deviation (SD) or number (%). P-values were calculated using one-way analysis of variance (ANOVA)

PC Phosphatidylcholine, BMI Body mass index, SD Standard deviation, ANOVA Analysis of variance

^*P < 0.05

^afor continuous variables and the chi-squared test

^bfor categorical variables. Group definitions: Low-dose PC (0.3 g/day), Medium-dose PC (1.0 g/day), High-dose PC (3.0 g/day)

In addition, lipid and glucose metabolism parameters are shown in Table 2, while renal and hepatic function parameters are shown in Table 3.

Table 2.

Baseline lipid and glucose metabolism parameters

Variable	Placebo (n = 9)			Low-dose PC (n = 8)			Medium-dose PC (n = 8)			High-dose PC (n = 7)			Total (n = 32)			P-value
Total cholesterol (mg/dL)	219	±	29	229	±	20	213	±	32	229	±	41	222	±	30	0.653	ª
HDL-C (mg/dL)	60	±	13	68	±	26	71	±	12	68	±	19	67	±	18	0.599	ᵇ
LDL-C (mg/dL)	137	±	31	139	±	34	114	±	32	143	±	31	133	±	33	0.292	ª
Triglycerides (mg/dL)	107	±	71	127	±	65	140	±	145	80	±	19	114	±	87	0.227	ᵇ
Fasting blood glucose (mg/dL)	89	±	8	88	±	6	91	±	10	90	±	8	90	±	8	0.948	ª
HbA1c (%)	5.3	±	0.3	5.5	±	0.2	5.5	±	0.2	5.6	±	0.2	5.5	±	0.2	0.078	ª

Data are presented as mean ± SD. P-values were calculated using one-way ANOVA

PC Phosphatidylcholine, HDL-C High-density lipoprotein cholesterol, LDL-C Low-density lipoprotein cholesterol, HbA1c Hemoglobin A1c, SD Standard deviation, ANOVA Analysis of variance

^aor the Kruskal–Wallis test

^bamong the four groups. Group definitions: Low-dose PC (0.3 g/day), Medium-dose PC (1.0 g/day), High-dose PC (3.0 g/day)

Table 3.

Baseline renal and hepatic function parameters

Variable	Placebo (n = 9)			Low-dose PC (n = 8)			Medium-dose PC (n = 8)			High-dose PC (n = 7)			Total (n = 32)			P-value
Renal function
BUN (mg/dL)	15	±	4	14	±	2	15	±	3	15	±	5	15	±	3	0.997	a
Creatinine (mg/dL)	0.80	±	0.13	0.76	±	0.14	0.81	±	0.14	0.84	±	0.12	0.80	±	0.13	0.752	a
Uric acid (mg/dL)	5.2	±	0.7	5.7	±	1.5	5.6	±	1.1	5.1	±	1.1	5.4	±	1.1	0.637	a
Hepatic function
AST (U/L)	19	±	4	23	±	3	19	±	7	24	±	6	21	±	5	0.116	b
ALT (U/L)	16	±	4	19	±	5	17	±	7	16	±	4	17	±	5	0.556	b
LDH (U/L)	179	±	28	200	±	22	169	±	37	229	±	122	193	±	63	0.233	b
γ-GTP (U/L)	22	±	9	47	±	47	45	±	47	27	±	8	35	±	34	0.362	b
ALP (U/L)	72	±	12	86	±	18	74	±	12	78	±	16	77	±	15	0.491	b
TBil (mg/dL)	0.84	±	0.26	0.90	±	0.54	0.73	±	0.24	0.76	±	0.27	0.81	±	0.34	0.778	b

Data are presented as mean ± SD. P-values were calculated using one-way ANOVA

PC Phosphatidylcholine, BUN Blood urea nitrogen, AST Aspartate aminotransferase, ALT Alanine aminotransferase, LDH Lactate dehydrogenase, γ-GTP gamma-glutamyl transpeptidase, ALP Alkaline phosphatase, SD Standard deviation, ANOVA Analysis of variance

^aor the Kruskal–Wallis test

^bamong the four groups. Group definitions: Low-dose PC (0.3 g/day), Medium-dose PC (1.0 g/day), High-dose PC (3.0 g/day)

The mean age of the participants was 68.7 ± 4.0 years, and all groups were comparable in terms of participant characteristics. Although a statistically significant difference among groups was observed for diastolic blood pressure at Baseline (P = 0.031), the mean values for all groups remained within the clinically normal range. No statistically significant differences at Baseline were observed for other physical measurements, markers of lipid and glucose metabolism, or markers of renal and hepatic function.

Effect of PC supplementation on serum TMAO concentration

Changes in serum TMAO concentration for each group are presented in Table 4.

Table 4.

Effect of egg-derived PC intake on serum TMAO concentration

Group	Baseline (μg/mL)			Week 12 (μg/mL)			ΔWeek 12 (μg/mL)			P-value (within-group)†
Placebo (n = 9)	0.38	±	0.26	0.30	±	0.15	–0.08	±	0.32	0.779
Low-dose PC (n = 8)	0.35	±	0.33	0.36	±	0.13	0.01	±	0.24	0.528
Medium-dose PC (n = 8)	0.45	±	0.23	0.85	±	1.05	0.40	±	1.18	0.484
High-dose PC (n = 7)	0.28	±	0.17	0.80	±	0.64	0.52	±	0.68	0.028	*
P-value (between-groups)§	0.288			0.055			0.099

Data are presented as mean ± SD. ^†P-values for within-group comparisons (Baseline; Week 0 vs. Week 12) were calculated using the Wilcoxon signed-rank test. ^§P-values for between-group comparisons were calculated using the Kruskal–Wallis test. ^*P < 0.05 vs. Baseline (Week 0). ΔWeek 12 represents the change from Baseline (Week 0) to Week 12. PC, phosphatidylcholine; TMAO, trimethylamine N-oxide. Group definitions: Placebo (vehicle control); Low-dose PC (0.3 g/day); Medium-dose PC (1.0 g/day); High-dose PC (3.0 g/day); SD Standard deviation

In the high-dose PC group, serum TMAO concentration increased significantly at Week 12 relative to Baseline. In contrast, no significant changes in TMAO concentration at Week 12 relative to Baseline were observed in the placebo, low-dose PC, or medium-dose PC groups. In addition, no statistically significant differences were detected among the four groups for serum TMAO concentration at Week 12 (P = 0.055) or for change from Baseline (P = 0.099).

Correlation analyses

The results of the correlation analysis between serum TMAO and plasma choline concentrations are presented in Table 5.

Table 5.

Correlation between serum TMAO and plasma choline concentration

Group	Baseline		Week 12		ΔWeek 12
	Spearman's r	P-value	Spearman's r	P-value	Spearman's r	P-value
Placebo (n = 9)	–0.268	0.486	–0.519	0.152	–0.350	0.356
Low-dose PC (n = 8)	–0.214	0.610	0.228	0.588	–0.263	0.528
Medium-dose PC (n = 8)	0.214	0.610	–0.595	0.120	–0.071	0.867
High-dose PC (n = 7)	–0.216	0.641	0.536	0.215	–0.357	0.432
Total (n = 32)	–0.082	0.655	0.071	0.701	0.014	0.938

Values represent Spearman's rank correlation coefficients (r) between serum TMAO and plasma choline concentrations

PC Phosphatidylcholine, TMAO Trimethylamine N-oxide

ΔWeek 12 represents the change from Baseline (Week 0) to Week 12

No significant correlations were detected between these two markers at Baseline, at Week 12, or in their respective changes. The results of the correlation analysis examining relationships between serum TMAO concentration and other clinical laboratory parameters are provided in Tables 6 and 7.

Table 6.

Correlation between serum TMAO concentration and clinical parameters at Baseline

Variable (at Baseline; Week 0)	Placebo (n = 9)	Low-dose PC (n = 8)	Medium-dose PC (n = 8)		High-dose PC (n = 7)		Total (n = 32)
Lipids
Total cholesterol (mg/dL)	–0.167	–0.167	0.190		0.072		–0.068
HDL-C (mg/dL)	0.021	0.299	–0.310		0.131		0.088
LDL-C (mg/dL)	0.075	–0.31	–0.156		0.180		–0.135
Triglycerides (mg/dL)	–0.326	–0.071	0.524		–0.108		–0.031
Glucose metabolism
Fasting blood glucose (mg/dL)	–0.326	0.071	0.262		0.306		0.017
HbA1c (%)	–0.145	–0.253	0.361		–0.667		–0.175
Renal function
BUN (mg/dL)	0.403	0.048	0.381		0.342		0.354	*
Creatinine (mg/dL)	0.343	–0.122	0.571		0.252		0.215
Uric acid (mg/dL)	0.561	–0.095	0.333		0.288		0.268
Hepatic function
AST (U/L)	–0.378	0.530	0.826	*	0.111		0.016
ALT (U/L)	–0.325	–0.024	0.855	**	0.823	*	0.205
LDH (U/L)	0.109	–0.431	0.695		–0.252		–0.160
γ-GTP (U/L)	–0.349	0.071	0.905	**	0.118		0.062
ALP (U/L)	–0.378	–0.074	–0.619		–0.418		–0.319
TBil (mg/dL)	0.073	0.659	0.512		0.173		0.347

Values represent Spearman's rank correlation coefficients (r) between serum TMAO concentrations and other clinical parameters at Baseline (Week 0)

Group definitions: Placebo (vehicle control); Low-dose PC (0.3 g/day); Medium-dose PC (1.0 g/day); High-dose PC (3.0 g/day)

PC Phosphatidylcholine, TMAO Trimethylamine N-oxide, LDL-C Low-density lipoprotein cholesterol, HDL-C High-density lipoprotein cholesterol, HbA1c Hemoglobin A1c, AST Aspartate aminotransferase, ALT Alanine aminotransferase, LDH Lactate dehydrogenase, γ-GTP gamma-glutamyl transpeptidase, ALP Alkaline phosphatase, Tbil Total bilirubin, BUN Blood urea nitrogen

* P < 0.05

** P < 0.01

Table 7.

Correlation between changes in serum TMAO concentration and changes in clinical parameters

Variable (at ΔWeek 12)	Placebo (n = 9)	Low-dose PC (n = 8)	Medium-dose PC (n = 8)	High-dose PC (n = 7)	Total (n = 32)
Lipids
Total cholesterol (mg/dL)	0.276	0.036	0.299	0.821*	0.441	*
HDL-C (mg/dL)	0.427	0.367	0.619	0.429	0.501	**
LDL-C (mg/dL)	0.300	–0.133	0.455	0.893**	0.414	*
Triglycerides (mg/dL)	–0.167	0.431	–0.287	–0.429	–0.234
Glucose metabolism
Fasting blood glucose (mg/dL)	–0.339	0.335	0.216	–0.321	–0.123
HbA1c (%)	0.138	0.253	–0.195	–0.291	–0.168
Renal function
BUN (mg/dL)	0.200	–0.132	0.060	0.000	0.100
Creatinine (mg/dL)	–0.075	–0.537	–0.181	0.286	–0.166
Uric acid (mg/dL)	0.117	–0.253	0.356	0.309	0.139
Hepatic function
AST (U/L)	–0.008	0.594	–0.012	0.360	0.069
ALT (U/L)	–0.160	0.404	–0.619	–0.143	–0.128
LDH (U/L)	–0.494	0.120	0.323	0.643	0.093
γ-GTP (U/L)	–0.322	0.564	0.551	–0.107	0.227
ALP (U/L)	–0.458	–0.236	0.120	–0.643	–0.222
TBil (mg/dL)	–0.017	0.370	0.393	0.598	0.358	*

Values represent Spearman's rank correlation coefficients (r) between the change in serum TMAO concentrations and the change in other clinical parameters. ΔWeek 12 represents the change from Baseline (Week 0) to Week 12

Group definitions: Placebo (vehicle control); Low-dose PC (0.3 g/day); Medium-dose PC (1.0 g/day); High-dose PC (3.0 g/day)

PC Phosphatidylcholine, TMAO Trimethylamine N-oxide, LDL-C Low-density lipoprotein cholesterol, HDL-C High-density lipoprotein cholesterol, HbA1c Hemoglobin A1c, AST Aspartate aminotransferase, ALT Alanine aminotransferase, γ-GTP gamma-glutamyl transpeptidase, ALP Alkaline phosphatase, BUN Blood urea nitrogen

* P < 0.05

** P < 0.01

At Baseline, significant positive correlations were identified between TMAO concentration and hepatic function markers (aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase) in the medium-dose PC group. In the high-dose PC group, TMAO concentration was positively correlated with alanine aminotransferase. In the total study population, a significant positive correlation was observed between TMAO and blood urea nitrogen. At Week 12, sporadic correlations were observed between TMAO and several clinical parameters. Regarding the magnitude of change, significant positive correlations were identified between changes in TMAO and changes in lipid markers (total cholesterol, high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C]) in the total population. Notably, in the high-dose PC group, strong positive correlations were observed between changes in TMAO and total cholesterol (r = 0.821) as well as LDL-C (r = 0.893), whereas HDL-C demonstrated a relatively high correlation coefficient (r = 0.429).

Discussion

This is the first study examining the effects of continuous intake of egg-derived PC for 12 weeks on serum TMAO concentration in healthy Japanese middle-aged and older adults. Collectively, our results indicated that serum TMAO concentration increased significantly from Baseline in the group receiving the highest dose of PC (3.0 g/day). However, even in this high-dose group, the mean value at Week 12 was 0.80 ± 0.64 μg/mL, remaining markedly below the established threshold for high clinical risk of CVD (≥ 2.0 μg/mL). Moreover, no statistically significant differences were observed among the four groups. These findings suggest that the rise in TMAO concentration following intake of egg-derived PC is limited and unlikely to cause an immediate increase in clinical risk. Regarding the dosage, the lowest dose of PC used in this study (300 mg/day) contained approximately 40.5 mg of choline. Since a typical large egg contains approximately 125–150 mg of choline [1], this dose corresponds to roughly one-third of the choline present in a single egg. In contrast, the high-dose group (3,000 mg of PC) represents the equivalent of consuming approximately three eggs.

Recent studies have indicated that the causal relationship between elevated TMAO concentration and CVD risk may be complex [14, 15, 19, 20]. Furthermore, a study showed that TMAO may be a marker of renal function rather than a direct causal toxin for CVD [21]. Consistent with this view, a significant positive correlation was observed between serum TMAO concentration and blood urea nitrogen at Baseline (r = 0.354, P < 0.05; Table 6). Importantly, no significant correlation was identified between changes in TMAO concentration and alterations in renal function markers (Table 7). This lack of association supports the safety of the intervention, suggesting that PC intake did not induce TMAO elevation through renal impairment.

For instance, research revealed no rise in the levels of inflammatory markers or oxidized LDL in populations in whom TMAO increased after egg consumption [11]. In addition, large-scale epidemiological studies in Japanese populations found no significant association between dietary choline intake and CVD mortality risk [21]. Considering the typical choline intake levels in the general Japanese diet [21], these findings align with our observation that a limited increase in TMAO does not directly lead to increased CVD risk.

Recent systematic reviews have shown that the association between egg consumption and increased TMAO concentration remains unclear [15, 22]. Furthermore, several studies have reported no effect or noted that background factors may strongly influence outcomes [19]. In addition, a recent meta-analysis limited to randomized controlled trials clearly demonstrated that egg consumption does not have a statistically significant impact on serum TMAO concentration [16]. Taken together, these findings further reinforce our conclusion that elevation of TMAO concentration due to intake of egg-derived PC remains limited.

In the total population analysis, significant positive correlations were observed between changes in TMAO concentration and alterations in lipid markers (total cholesterol, HDL-C, LDL-C) (Table 7). Furthermore, in the high-dose PC group, strong positive correlations were identified between the levels of TMAO and total cholesterol (r = 0.821), as well as LDL-C (r = 0.893), while HDL-C also showed a positive trend (r = 0.429). While the potential for a chance correlation (Type I error) due to the small sample size cannot be excluded, this observation also suggests that PC intake influences both TMAO production and lipid metabolism through a common pathway. Overall, this hypothesis warrants further investigation.

In this study, a statistically significant difference in diastolic blood pressure across groups was observed at Baseline (Table 1), although the mean values in all groups remained within the clinically normal range. The concentration of TMAO is mainly influenced by renal function and the intestinal environment [10, 19, 22]. Therefore, it is unlikely that this difference in blood pressure at Baseline had a substantial impact on the observed changes in TMAO concentration, which served as the primary endpoint of this study.

Furthermore, the concentration of TMAO is strongly affected by various confounding factors, including individual differences in gut microbiota composition and the activity of hepatic metabolic enzymes [8, 13]. In this study, one participant in the medium-dose PC group and another in the high-dose PC group exhibited values exceeding the threshold for high clinical risk (2.0 μg/mL) at Week 12. Miller et al. [11] and Lombardo et al. [23] proposed that these elevations may be a temporary effect produced by meals consumed immediately before measurement. Indeed, dietary records confirmed that one participant had eaten squid (a rich source of TMAO) the day before the measurement, while the other had consumed six Silkie chicken eggs on the same day. To further examine whether the statistically significant increase recorded in the high-dose group (n = 7) (P = 0.028) was driven by this single outlier, a sensitivity analysis was conducted using a dataset that excluded this participant (n = 6). The data showed that the difference remained statistically significant (P = 0.046). Importantly, the mean value of the high-dose group showing this increase (0.80 ± 0.64 μg/mL) remained substantially below the threshold for high clinical risk (2.0 μg/mL). Therefore, this increase is considered statistical rather than indicative of an immediate rise in clinical risk. Furthermore, prior research has shown that TMAO production can be modulated via changes in the intestinal environment, including probiotic intake [24], highlighting the influence of background factors when interpreting TMAO concentration.

In conclusion, considering the diverse health benefits of egg-derived PC [9] and its significance during development [25], the findings of this study support the utility of eggs as part of a balanced diet. Moreover, these results alleviate concerns regarding TMAO levels.

Strength and limitation

The strengths of this study include its randomized, double-blind, placebo-controlled design, which allowed for a rigorous evaluation of the dose-dependent effects of egg-derived PC. Additionally, this was the first study to investigate the long-term impact of egg-derived PC on TMAO levels specifically in a Japanese population, providing valuable insights into ethnic and dietary differences in the metabolism of choline.

However, this study also has several limitations. Firstly, the investigation had a small sample size as this was a secondary analysis, which may have limited the statistical power to detect minor changes. Secondly, although participants were instructed to maintain their habitual diet and were stratified by egg intake at Baseline, they utilized self-reported diaries to record overall daily dietary intake during the study. Due to the lack of strict monitoring, this approach may have introduced confounding variables. Thirdly, blood samples were collected after a fasting period of at least 4 h rather than under strict overnight fasting conditions. While this approach minimizes the immediate influence of recent food intake, it remains a limitation of the study protocol compared with overnight fasting. Finally, this study examined the effects of egg-derived PC intake; when individuals consume whole eggs, other lipids or components within the egg yolk may affect choline metabolism or gut microbiota composition, and the resulting TMAO production dynamics may differ from those observed in this investigation.

Conclusions

In healthy middle-aged and older adults, continuous intake of up to 3.0 g/day of egg-derived PC for 12 weeks did not lead to serum TMAO concentrations approaching clinically concerning levels. In addition, no distinct increase was detected relative to the levels recorded in the placebo group. These findings reduce concerns that PC intake immediately elevates cardiovascular risk through TMAO and offer scientific support for its use as a dietary component for maintaining cognitive function. These results suggest that egg-derived PC can be utilized as a safe and effective dietary strategy to support cognitive health in older adults without raising immediate concerns regarding cardiovascular risk associated with TMAO.

Abbreviations

CVD

Cardiovascular disease

EYC

Egg yolk choline

HDL-C

High-density lipoprotein cholesterol

LDL-C

Low-density lipoprotein cholesterol

PC

Phosphatidylcholine

TMAO

Trimethylamine N-oxide

UMIN

University Hospital Medical Information Network

Authors’ contributions

W.W., Y.T., M.K., and R.M. conceived and designed the study. W.W., Y.T., M.K., R.M., and T.H. acquired the data. W.W. and Y.T. analyzed and interpreted the data. W.W. drafted the manuscript. N.Y. and M.S. critically reviewed and revised the manuscript for important intellectual content. R.M. supervised the project. All authors have read and approved the final version of this manuscript.

Funding

The Kewpie Corporation funded this study.

Data availability

All data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The Ethics Review Committee of Kobuna Orthopedic Clinic approved the study protocol (Approval No: MK-2402–02). All experiments were performed in accordance with the ethical principles of the Declaration of Helsinki. Finally, all participants provided written informed consent for the secondary use of their serum samples.

Consent for publication

Not applicable.

Competing interests

W.W., Y.T., M.K., and R.M. are employees of Kewpie Corporation, which funded the study. T.H. is an employee of LSI Medience Corporation. N.Y. and M.S. declare that they have no competing interests.