The major finding from the present study is the lack of significant associations in covariate-adjusted models between choline measures (and other biomarkers of one-carbon metabolism) and various cognitive outcomes in the 5-year-old children in the Seychelles Child Development Nutrition Cohort. We did not find a significant association between the plasma concentration of free choline, or its related metabolites, and children’s intelligence assessed by the Kaufman Brief Intelligence Test, a test which measures both verbal and non-verbal intelligence and provides an indication of visual perception, cognitive ability and receptive vocabulary. The Woodcock–Johnson Test of Achievement, which assesses the overall level of scholastic achievement, was also non-significantly associated with choline concentration. In addition, we included measures of neurodevelopment that may be more specifically related to choline. For example, the Finger Tapping Test, which provides a measure of fine motor speed, may be relevant in light of reports that postnatal dietary choline supplementation improves gross motor locomotion in mice(35)
. The PLS and Child Behaviour Checklist provide a more specific assessment of visuospatial and auditory memory and social or emotional responses, domains which may be particularly sensitive to choline(36,37)
, potentially owing to the influence of ACh on hippocampal plasticity(20)
. Nevertheless, we did not find any consistent relationships between the endpoints measured and the plasma concentration of choline, DMG, methionine or tHcy. However, plasma betaine concentration was positively associated with the PLS-total language score. A previous intervention study in healthy elderly adults reported a positive effect of betaine on memory performance, which the authors speculated was explained by the greater availability of choline metabolites for synthesis of ACh and structural phospholipids, such as phosphatidylcholine and sphingomyelin(38)
. Such a mechanism is also plausible in children, particularly as the early years are a time of rapid brain growth, with the peak time for development between 5 months gestation and 4 years of age(39)
. Albeit scientifically plausible, the present finding that the PLS-total language score was significantly associated with betaine requires confirmation in future research, as it may be owing to chance.
To our knowledge, the present study is the first to examine the relationship between the plasma concentration of choline and other biomarkers of one-carbon metabolism and concomitant cognitive performance in children. Animal studies, however, have consistently shown a cause-and-effect relationship between postnatal choline intake (and other biomarkers of one-carbon metabolism) and subsequent performance in offspring(7)
, with beneficial effects reported to be long term and evident in adulthood and old age(13)
. The majority of research investigating choline intake and performance has been undertaken in animal models, specifically in rodents(7)
. The growth and development of the rat and human brain progress at different rates, making direct extrapolation of animal data to human subjects difficult. However, similar to the rat, it has been reported that a large proportion (80 %) of the human brain growth spurt is postnatal(40)
. This observation, together with the evidence that neural plasticity in response to learning continues throughout childhood, would suggest that requirements for brain-related nutrients, including choline, would be important in early childhood(19)
. The essentiality of nutrients will be related to the timing of their delivery, compared with the critical periods during brain development. However, unlike the prenatal period, the windows of exposure for brain development during the pre-school years are relatively broad(41)
, while, furthermore, the capacity for plasticity within the human brain may allow it to compensate for fluctuations in nutritional status.
Choline circulates in a bound form, mostly in the form of phosphatidylcholine, or in a free form initially reported as a likely mechanism supplying choline to the brain(42)
. Although an accepted and often used biomarker of choline status, plasma free choline represents only a fraction of the total choline pool(43)
and may be a poor marker of ACh synthesis and status in the brain(44)
. Indeed, another study that reported a positive association between betaine status and memory also observed no association with plasma free choline concentration(38)
. Our lack of association may also be attributed to the high plasma choline concentration (status) of the children in the Seychelles. A previous study only observed a relationship between plasma choline concentration and leucocyte ACh concentration in children who had low choline status(45)
; no relationship was evident in children who had plasma concentrations of choline, betaine and DMG, similar to those reported in the present analysis. The lack of significant associations may also be owing to the small sample size of the present study.
The present study has a number of strengths. The cohort of 300 mothers initially recruited to the present study represented one-fifth of total annual deliveries in the Seychelles and 75% of all women booking at antenatal clinics during the enrolment period, and was therefore considered to be a representative sample of the population(46)
. In addition, the children were sampled and tested early in childhood within a period of significant brain development, and the test battery used specific and reproducible measures of pre-school neurological function. Extensive data on additional factors that influence child development were also collected and controlled for in the present analysis. Furthermore, choline, betaine, DMG and tHcy were assessed using a sensitive method based on liquid chromatography-MS/MS(28)
, and plasma free choline concentrations were in keeping with values reported previously in US children(17,45)
. However, the study also has limitations. The cohort size was not adequate to examine interactions (between choline and the other biomarkers of one-carbon metabolism on neurodevelopmental endpoints) and other unmeasured covariates, such as folate and vitamin B12
, which may have been significant. In addition, the critical period, if any, for human postnatal development with respect to choline may not correspond to 5 years of age. As non-fasting samples were collected from the children, plasma choline concentrations may have been sensitive to recent dietary intakes.
In summary, we found few significant associations between the concentration of free choline (within the normal physiological range for children), or its metabolites, and cognitive outcome in children at 5 years of age. However, the experimental evidence suggesting that choline status is positively associated with cognitive outcome in animals supplemented with choline in the postnatal period suggests that this issue needs further investigation.