LABORATORIO DE URGENCIAS

Low B12 and Folate Linked to Fatigue, Lack of Motivation

                                                                                          Hiroaki Kanouchi; Ayaka Yamamoto; Akiko Kuwabara;
                                                                               Shigeo Takenaka; Eiji Nishikubo; Yasuyoshi Watanabe et al.
                                                                             * Department of Nutrition, Graduate School of Human Life and Ecology,Osaka Metropolitan University, Japan
                                                                             * Alinmin Pharmaceutical Co., Ltd., Tokyo 100-0005, Japan
                                                                             * Kobe University Graduate School of Science, Technology and Innovation, Osaka 530-0011, Japan
                                                                             * Center for Health Science Innovation, Osaka Metropolitan University,Osaka 558-8585, Japan

                                                                                                       Nutrients (March, 2026); 18(6); 941; 
                                                                                                       https://doi.org/10.3390/nu18060941

Fatigue is a prevalent and debilitating symptom that poses a significant public health burden with wide–ranging socioeconomic implications. Chronic fatigue has been linked to reduced workplace productivity, increased absenteeism, and elevated healthcare utilization, collectively contributing to substantial economic costs worldwide. In occupational settings, persistent fatigue can impair cognitive functioning, attention, and decision−making, thereby increasing the likelihood of errors and work–related accidents. Moreover, fatigue–related declines in motivation may reduce engagement in health–promoting behaviors. Identifying biological correlates or biomarkers of fatigue and motivation could offer critical insights for preventive strategies, early risk detection, and the development of targeted interventions, particularly in occupational and public health contexts.
Homocysteine (Hcy) (sulfur−containing amino acid formed during methionine metabolism). Elevated plasma Hcy levels have been associated with oxidative stress and impaired one-carbon metabolism , which may contribute to cardiovascular diseases, cognitive decline, and depression. Circulating Hcy concentrations are influenced by genetic variants, renal function, and micronutrient status, particularly folate, vitamin B12 (VB12), and vitamin B6, which act as essential cofactors in Hcy metabolism. Given its central role in one–carbon metabolism, methylation reactions, and monoamine neurotransmitter synthesis, Hcy may influence central nervous system function and contribute to fatigue-related phenotypes [. For example, Hcy accumulation can reduce the availability of S-adenosylmethionine (SAM), a universal methyl donor required for the synthesis of dopamine and serotonin, thereby impairing neuromodulation and neuroplasticity.
These neurochemical pathways are intimately involved in motivation. Motivational decline and fatigue are increasingly recognized as related constructs within central fatigue syndromes, sharing common neurobiological substrates such as dopaminergic dysfunction and impaired prefrontal cortical activity. Therefore, it is biologically plausible that elevated Hcy may not only contribute to diminishing motivational capacity but also fatigue. In addition, because Hcy reflects the integrated status of several B vitamins involved in one-carbon metabolism, Hcy may serve as a practical metabolic indicator for exploring fatigue-related outcomes in population-based studies.
Although previous studies have linked elevated Hcy levels to depression and cognitive impairment, little is known about its role in fatigue and motivational outcomes. The present study aimed to examine the associations between circulating Hcy and multiple fatigue-related measures in community-dwelling adults. Because the biological mechanisms linking Hcy to fatigue are not fully established, these analyses were considered exploratory. Multivariable models were used to examine the associations between plasma Hcy and fatigue–related outcomes while adjusting for relevant lifestyle and biochemical covariates.

MATERIALS AND METHODS

*Study Design and Participants
This study used data from RIKEN Compass to Healthy Life Research Complex Program in Kobe, Japan, from April 2018 to March 2020. Participants were recruited through advertisement posters and the program website. Eligible individuals were healthy adults aged ≥18 years residing in Kobe and Osaka areas in Japan. A total of 2618 individuals visited the Center for Health Science Innovation (CHSI), Osaka Metropolitan University, where they underwent functional measurements and blood sampling and completed questionnaires between April 2018 and March 2020 (men: 783 [ca. 30%], women: 1835 [ca. 70%]; mean age: 44 years).

*Exclusion Criteria
Participants were excluded if they had missing data for VB12, folate, Hcy, or co-enzyme form of vitamin B6, pyridoxal phosphate (PLP) concentrations (n = 1245); missing information on supplement use, daily medication, dietary variety, or fatigue assessments (n = 140); reported use of supplements or vitamin B complex (n = 437); had a diagnosis of adjustment disorder or depression (n = 6); or had biochemical values outside the mean ±3 SD for VB12, folate, Hcy, or PLP (n = 188). A total of 602 participants were included in the final analysis (men: 204; women: 398). A large proportion of exclusions was due to the absence of stored blood samples available for Hcy measurement.

*Measurement of PLP
Serum PLP concentrations were measured using high-performance liquid chromatography (HPLC) following the protocol described by Itoh et al. [22]. Briefly, serum samples were deproteinized with trichloroacetic acid, derivatized with potassium cyanide under light–shielded conditions, and analyzed using an HPLC system (Shimadzu LC–20AD series, Shimadzu, Kyoto, Japan) equipped with a Chromolith column (UM8125/004, Merck, Darmstadt, Germany).
The mobile phase consisted of 0.1 M citrate buffer (pH 3.5) containing 0.1% methanol, with a flow rate of 0.2 mL/min at 40 °C. PLP was detected by fluorescence (excitation at 418 nm, emission at 325 nm).

*Measurement of Hcy, Serum VB12 and Folate
Plasma Hcy concentrations were determined by HPLC according to Mantjoro et al. [23], with minor modifications. Briefly, serum samples were added to N–acetyl–L–cysteine, which was used as an internal standard, deproteinized with trichloroacetic acid, conjugated with 4–Fluoro–7–sulfamoylbenzofurazan (DOJINDO, Kumamoto, Japan), and analyzed using an HPLC system (Shimadzu LC-20AD series) equipped with a Chromolith column (UM8125/004). The mobile phase consisted of 0.05 mol/L potassium dihydrogen phosphate (pH 1.9) combined with 30 mL/L acetonitrile, with a flow rate of 0.2 mL/min at 40 °C. Detection was performed by fluorescence (excitation at 385 nm, emission at 515 nm).
Serum folate and VB12 concentrations were measured using commercially available kits: ACS–folic acid II kit (Bayer Medical, Osaka, Japan) and ACS–VB12 kit (Chiron Diagnostics, East Walpole, MA, USA), respectively. A fully automated ACS180 chemiluminescence analyzer (Bayer Diagnostics, Tarrytown, NY, USA) was used for the measurements. VB12 concentration was expressed as cyanocobalamin equivalents, and folic acid concentration was expressed as tetrahydrofolic acid equivalents (hereafter referred to as folate).

Fatigue Assessment
The Chalder Fatigue Scale is an 11–item self–reported questionnaire that evaluates both physical and mental fatigue. Seven items represent physical fatigue (ChaPF) and 4 represent mental fatigue (ChaMF). Each item is scored 0–3; less than usual (0), no more than usual (1), more than usual (2) and much more than usual (3).
Item ratings are summed to calculate the total score (ChaTF). The Visual Analog Scale (VAS) was used to measure the subjective intensity of both fatigue and motivation, using two separate 100 mm horizontal lines, where higher values represent greater fatigue or motivation, respectively. In addition, the CHSI Fatigue Scale, developed specifically for the Japanese population by Fukuda et al., was administered to assess multidimensional fatigue including physical, cognitive, and emotional domains. This instrument has been validated for use in both healthy individuals and those with chronic fatigue syndrome. To evaluate psychological distress, we used the Kessler Psychological Distress Scale (K6), a six–item screening tool designed to measure nonspecific psychological distress experienced over the past 30 days. Higher scores indicate greater psychological distress.

Statistical Analysis
Categorical variables are presented as numbers and percentages, and continuous variables as medians with interquartile ranges (25th–75th percentile). Descriptive statistics were computed using SPSS version 30 (IBM Corp., Armonk, NY, USA) and JMP version 14 (SAS Institute Inc., Cary, NC, USA). Trend tests for continuous variables were performed using the Jonckheere–Terpstra test in SPSS, and trend tests for categorical variables were conducted using the Cochran–Armitage test in JMP. Differences in continuous variables across Hcy tertiles were assessed using Steel’s test in JMP, with T1 as the reference group. Pearson correlation coefficients (r) and corresponding p–values for the heatmap were calculated in JMP. Multiple regression analyses were performed using JMP.
Plasma Hcy was categorized into tertiles (T1–T3). Because plasma Hcy concentrations are known to differ between men and women, all primary analyses were conducted separately by sex. We initially screened for associations between Hcy tertiles and multiple fatigue and motivation measures and identified significant relationships for ChaPF in men and VAS motivation in women. These measures were then selected for focused multivariable analyses. Multiple regression analysis was performed to assess the relationship between ChaPF and Hcy tertiles in men, and VAS motivation scores and Hcy tertiles in women, after adjusting for covariates (age, body mass index [BMI], non–optimal sleep, non–exercise habit, overwork, estimated glomerular filtration rate [eGFR], alanine aminotransferase [ALT], and dietary variety score). As a sensitivity analysis, plasma Hcy was also modeled as a continuous variable in multivariable regression models to examine potential linear associations with fatigue-related outcomes. Additionally, analyses including a sex–by–Hcy interaction term were performed using continuous Hcy in the multivariable models to evaluate potential sex differences.

RESULTS

Baseline characteristics were compared between included and excluded participants. In men, age, eGFR, overwork, BAP, d–ROMs/BAP ratio, and VAS motivation differed between the two groups. In women, ALT, d–ROMs, d–ROMs/BAP ratio, non–habitual exercise, and VAS depression also differed between the two groups.
However, ChaPF in men and VAS motivation in women—the outcomes examined in the present analyses—did not differ significantly between the included and excluded participants.

DISCUSSION

Elevated Hcy was associated with ChaPF in men and VAS motivation in women in the multivariable analyses after adjustment for sleep, work hours, exercise, BMI, dietary variety, and renal function. These associations should be interpreted as exploratory.
Fatigue is increasingly recognized as a multidimensional construct that includes physical, cognitive, and emotional components.
The Chalder Fatigue Scale primarily captures physical and mental fatigue, whereas the CHSI scale assesses broader symptom domains, including cognitive and autonomic complaints. In contrast, the VAS motivation score may be sensitive to short–term fluctuations in motivation.
Examining multiple scales, therefore, allowed us to explore whether Hcy may be differentially associated with specific dimensions of fatigue-related experiences. Fatigue primarily reflects a subjective perception of physical or mental exhaustion, whereas motivational decline reflects reduced drive to initiate or sustain goal-directed behavior.
These constructs represent distinct aspects of central fatigue syndromes.
Hcy is a key intermediate in one-carbon metabolism, requiring folate and VB12 for remethylation and vitamin B6 for transsulfuration. In both sexes, higher Hcy tertiles corresponded to lower folate and VB12, consistent with impaired clearance when these cofactors are relatively low.
Folate and VB12 were not independently associated with fatigue or motivation, suggesting that Hcy may reflect a broader metabolic state beyond micronutrient status. Mechanistically, elevated Hcy has been proposed to influence methylation processes through reductions in SAM, a universal methyl donor involved in monoamine synthesis. Such mechanisms could potentially influence dopaminergic and serotonergic signaling implicated in motivation and fatigue, although these pathways were not directly assessed in the present study.
The observed sex–specific patterns may reflect differences in Hcy metabolism and neural regulation between men and women, although these interpretations remain speculative. Plasma Hcy concentrations are generally lower in women than in men, partly due to the influence of sex differences in the prefrontal cortex and striatum may also contribute to distinct vulnerabilities to metabolic stressors.
Although Hcy has been implicated in promoting oxidative stress and excitotoxicity in previous studies, our findings did not show significant differences in the d-ROMs/BAP ratio across Hcy tertiles.
Epidemiological studies have reported positive associations between Hcy and thiobarbituric acid reactive substances (TBARS), a lipid peroxidation marker, in pathological conditions such as central retinal vein occlusion and schizophrenia. However, no studies have examined the relationship between Hcy and d-ROMs/BAP ratio, and previous reports indicate that TBARS and d-ROMs may not correlate consistently. Therefore, the absence of association in our data does not exclude a potential link between Hcy and oxidative stress.
From a clinical perspective, maintaining adequate B-vitamin status to support Hcy metabolism remains reasonable given the established links between Hcy and vascular and cognitive outcomes. In general, plasma Hcy concentrations below 15 μmol/L are often considered within the normal range; however, there is no universally accepted cutoff, and values may vary with population characteristics, assay methods, and clinical condition. For example, thresholds for defining hyperhomocysteinemia differ across studies and may be lower in neurological and cognitive outcomes. This study, the highest tertile in men (≥17.6 μmol/L) exceeded this threshold, while the highest tertile in women (≥13.9 μmol/L) approached it. These findings may suggest a possible association between Hcy levels and fatigue-related outcomes; however, the results should be interpreted cautiously given the exploratory nature of the analyses and the cross-sectional design. Hcy may reflect broader metabolic conditions that could be related to fatigue-related outcomes.
Consistent with this interpretation, folate and VB12 were strongly associated with lower Hcy concentrations, whereas their direct associations with fatigue-related outcomes were negligible, suggesting that B–vitamin status may influence fatigue primarily through its relationship with Hcy.
To further explore the relationships among B-vitamin status, Hcy, and fatigue–related outcomes, exploratory path models were constructed. These models indicated that folate and VB12 were significantly associated with Hcy tertiles, consistent with the known collinearity among these variables, whereas their direct associations with fatigue-related outcomes were not significant. This pattern suggests that B–vitamin status may influence fatigue-related outcomes primarily through its relationship with Hcy rather than through independent pathways. These findings should be interpreted cautiously.
Although these findings raise the possibility that nutritional strategies aimed at lowering Hcy may influence fatigue–related outcomes, causal relationships cannot be established from this cross-sectional study. Prospective and interventional studies are required to determine whether lowering Hcy can directly improve fatigue or motivational symptoms.
This study has several limitations that should be considered when interpreting the findings:
*First, the cross-sectional design precludes causal inference, and reverse causality cannot be excluded.
*Second, although Hcy tertiles were used to facilitate interpretation, Hcy did not show significant associations with fatigue or motivation when modeled as a continuous variable, suggesting possible threshold or non-linear effects. Therefore, tertile categorization was used to aid interpretation of potential non-linear relationships between Hcy and fatigue-related outcomes.
*Third, all fatigue and motivation measures were self-reported, which may not fully capture neurobiological correlates.
*Fourth, inflammatory processes have also been implicated in fatigue, with inflammatory cytokines such as IL-6 suggested as potential contributors to fatigue perception. However, inflammatory biomarkers were not measured in the present study.
*Fifth, residual confounding due to unmeasured factors such as stress or sex hormones remains possible.
*Sixth, participants were recruited through advertisements for a preventive health program and may therefore represent a relatively health-conscious cohort, which may limit generalizability to the broader population.
*Finally, although sex–specific associations were identified, mechanistic biomarkers—such as SAM/SAH ratios, neurotransmitter metabolites, or neuroimaging indicators—were not assessed. Because these analyses were exploratory, the findings should be interpreted cautiously and considered hypothesis-generating.
Longitudinal and mechanistic studies are needed to establish causality and clarify the biological basis of the sex differences observed..

CONCLUSIONS

In this community-based cohort, elevated Hcy was associated with ChaPF in men and VAS motivation in women, independent of lifestyle factors and measured B-vitamin status.
These findings support a model in which Hcy reflects metabolic conditions both affected by stresses and micronutrient statuses. Further longitudinal and mechanistic studies incorporating endocrine measures, oxidative stress panels, and neurobiological markers are warranted.
Future prospective and interventional studies are needed to determine whether reducing Hcy levels can influence fatigue-related outcomes.