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1 From the Rowett Research Institute, Aberdeen, United Kingdom (SJD, ARC, and KB); the Clinical Research Center, Department of Mental Health, University of Aberdeen, Royal Cornhill Hospital, Aberdeen, United Kingdom (LJW and SL); and the Department of Psychology, University of Edinburgh (IJD).
2 Supported by the United Kingdom Biotechnology and Biological Sciences Research Council, the Scottish Executive Environment and Rural Affairs Department, and the World Cancer Research Fund. LJW holds a career development award from the Wellcome Trust. 3 Reprints not available. Address correspondence to SJ Duthie, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, United Kingdom. E-mail sd{at}rri.sari.ac.uk.
ABSTRACT
Background: Old age is associated with reduced cognitive performance. Nutritional factors may contribute to this association.
Objective: We tested associations between cognitive performance and plasma vitamin B-12, folate, and homocysteine concentrations in the elderly.
Design: We studied survivors of the Scottish Mental Surveys of 1932 (Aberdeen 1921 Birth Cohort, or ABC21) and 1947 (Aberdeen 1936 Birth Cohort, or ABC36), which surveyed childhood intelligence quotient. We measured folate, vitamin B-12, and homocysteine concentrations in fasting blood samples and cognitive performance by the Mini Mental State Examination (MMSE), National Adult Reading Test (NART), Raven's Progressive Matrices (RPM), Auditory Verbal Learning Test (AVLT), digit symbol (DS) subtest, and block design (BD) subtest.
Results: Homocysteine was higher in the ABC21 than in the ABC36 (P < 0.0001). There were positive correlations between folate and vitamin B-12 and negative correlations between homocysteine and both folate and vitamin B-12. MMSE, RPM, AVLT, DS, and BD scores were higher in the ABC36. In the ABC21, folate, vitamin B-12, and MMSE score were positively correlated and homocysteine was negatively correlated with RPM, DS, and BD scores. Folic acid was positively correlated with AVLT and DS scores. In the ABC36, folate was positively correlated with BD score. After adjustment for childhood intelligence quotient, partial correlations were strengthened between vitamin B-12 and NART score and between homocysteine and RPM score but weakened between red blood cell folate and DS score.
Conclusions: B vitamins and homocysteine are associated with cognitive variation in old age. In the ABC21 but not the ABC36, homocysteine accounted for 7–8% of the variance in cognitive performance. This may prove relevant to the design of neuroprotective studies in late life.
Key Words: Folate • vitamin B-12 • homocysteine • cognition • elderly • community-based survey • Scottish Mental Survey
INTRODUCTION
Age-related cognitive variation ranges from benign memory loss to progressive dementia (1). Investigations of cognitive variation suggest that nutritional factors are important for cognitive function in late life and that specific dietary deficiencies may be relevant to the retention of mental abilities, although the findings are inconsistent. Consumption of particular food components (antioxidants, marine oils, and fat-soluble vitamins) is probably relevant to cognitive function across the life span. Both maternal and infant nutrition have a profound effect on brain development and function. Folic acid has a critical role in neural tube closure in the neonate; maternal folate deficiency during pregnancy is associated with neural tube defects in the newborn (2). Several B vitamins, including folate, vitamin B-12, and vitamin B-6, are essential to the maintenance of normal nervous system function in adults (1). Low folate and vitamin B-12 intakes and blood concentrations are associated with neuropsychiatric disorders (3), and intervention with B vitamin supplements may reduce the severity of symptoms (3).
Age-related changes in absorption, metabolic pathways, and physiologic systems may result in older persons obtaining insufficient dietary folate and vitamin B-12, which may cause excess homocysteine to accumulate (4,5). High plasma homocysteine concentrations are associated with impaired memory and lower total life satisfaction in healthy elderly (6,7). Although plasma homocysteine is also reported to be elevated in Alzheimer disease (8), few studies have measured the effect of homocysteine on cognitive variation in older persons.
We identified a unique sample of elderly individuals who took part at the age of 11 y in the Scottish Mental Ability Surveys of 1932 or 1947 (SMS32 and SMS47). Survey archives are preserved by the Scottish Council for Research in Education and contain 157998 individual results on an intelligence quotient (IQ)–type test. We recruited local survivors of the SMS32 and SMS47 cohorts into a longitudinal observational study on brain aging and health. Our program aims to determine sources of individual differences in brain aging. Previous work established childhood IQ as the single most important predictor of cognitive function in late life (9,10). Here, we examine the potential contributions of blood vitamin B-12, folate, and homocysteine concentrations to individual differences in life cognitive variance after taking childhood IQ into account.
SUBJECTS AND METHODS
Subject recruitment, study design, and cognitive assessment
The Scottish Council for Research in Education gave us access to their unique archive of childhood IQ records. The council conducted 2 national surveys of the intelligence of Scottish schoolchildren. All children born in Scotland in 1921 and at school on 1 June 1932 (n = 87498) or born in 1936 and at school on 4 June 1947 (n = 70500) were given a group-administered test of general psychometric intelligence. The test used, the Moray House Test, was published as the "verbal test" (9). It has 71 numbered items, 75 items in total, and a maximum possible score of 76 points. The mean test score of the total Scottish population born in 1921 was 34.5. In a representative subsample of 1000 randomly selected children (500 boys and 500 girls), scores on the Moray House Test correlated with those on the individually administered Stanford Binet Test (r = 0.8), supporting its criterion validity.
In 1997–1999, with the permission of the Grampian Ethics Committee, we traced Aberdeen survivors of the 2 surveys (Aberdeen 1921 Birth Cohort, or ABC21, and Aberdeen 1936 Birth Cohort, or ABC36) in local health registers and invited a subsample to take part in a study of brain aging, nutrition, and health. This report concerns the first 199 ABC21 and 148 ABC36 subjects who agreed to take part. Men and women were recruited in approximately equal numbers. All subjects were living independently in the local community. Each cohort was invited to take part in 2 further assessments: the first in 1998–1999 (wave 1) and the second in 1999–2000 (wave 2) 15 mo later. Fasting blood samples were obtained during wave 2.
Current cognitive status was assessed during wave 1 and wave 2 with the use of 6 standardized tests: the Mini Mental State Examination (MMSE), the National Adult Reading Test (NART), Raven's Progressive Matrices (RPM), the Auditory Verbal Learning Test (AVLT), and the digit symbol (DS) and block design (BD) subtests of the revised Wechsler Adult Intelligence Scale. The MMSE is a useful screening tool for dementia; scores <24 are suggestive of mild dementia and scores <20 are indicative of dementia (11). The NART consists of a set of 50 phonologically irregular words and relies on the relative preservation of verbal abilities. It is used as a valid estimate of premorbid mental ability (12). The RPM assesses nonverbal intelligence and consists of 60 simple-to-complex abstract pattern-recognition problems that are solved by conceptualizing spatial or design or numerical relations within patterns (13). The AVLT (14) is a measure of verbal learning and memory. The DS subtest is a measure of cognitive performance involving the substitution of symbols for numbers according to a code. It is often referred to as a test of speed of information processing (15). The BD subtest is a constructional test that measures visuospatial organization (15). All subjects underwent cognitive examinations in a standardized setting within the Clinical Research Center. This examination formed part of a research assessment that included measures of general health and functional independence not reported here (16).
Preparation of plasma and erythrocytes for measurement of folate, vitamin B-12, and homocysteine
Blood samples (10 mL) were collected by venipuncture from the subjects at home after they had fasted overnight. The samples were collected in EDTA-treated evacuated tubes, stored at 4°C for transport, and processed within 4 h of collection.
Blood was spun at 2400 x g for 15 min at 4°C. The plasma was portioned into 1.5-mL plastic tubes, snap frozen in liquid nitrogen, and stored at -80°C until analyzed. The lymphocyte-containing buffy coat was removed as waste. Erythrocytes, reconstituted to initial blood volume with sterile phosphate-buffered saline (pH 7.4, 4°C) after plasma separation, were portioned into 1.5-mL plastic tubes, snap frozen, and stored at -80°C. Erythrocyte protein was determined by using bovine serum albumin as a standard (17).
Plasma (200 µL) and erythrocyte (100 µL) folate and vitamin B-12 were measured by using a commercially available kit (Simultrac Radioassay Kit vitamin B-12 [57Co]/folic acid [125I]; ICN Flow, Irvine, United Kingdom). Plasma homocysteine (200 µL) was measured by reversed-phase HPLC with a DS30 Hcy Homocysteine Assay Kit and a DS30 analyzer (Drew Scientific, Barrow-in-Furness, United Kingdom). Samples were reanalyzed if the CV between duplicates was >6%.
Statistical methods
The biomarker data were log transformed to approximate normality. SPSS (9th ed; SPSS Inc, Chicago) was used for all analyses. A priori, we recognized that statistical outliers would potentially confound tests of association and that these were most likely to occur among individuals who took vitamin supplements regularly. Thus, individuals were excluded if their blood vitamin B-12 concentration was >664 pmol/L (>900 ng/L) or their blood folate concentration was >59 nmol/L (>26 µg/L).
Cognitive test scores (except those on the MMSE) approximated normality. Comparisons between birth cohorts were made by multivariate analysis of variance (MANOVA) with and without adjustment for the effect of sex. Pearson's correlations were examined to test relations between transformed biomarkers and cognitive test data. Correlations between MMSE scores and biomarkers were reestimated by using Spearman's distribution-free method. Moray House Test scores were transformed from raw data to IQ-type scores (
RESULTS
A total of 186 ABC21and 148 ABC36 subjects were recruited. Data from 3 subjects (all ABC21) were removed before analysis because blood vitamin B-12 (2 subjects) or folate (1 subject) concentrations were greater than the preset limits for exclusion.
Mean plasma homocysteine, plasma vitamin B-12, and plasma and red blood cell folate concentrations of the ABC21 and ABC36 subjects are presented in Table 1. MANOVA detected an overall difference in blood biomarker concentrations between cohorts (Hotelling's trace = 0.11, P < 0.001). Univariate analysis of variance (ANOVA) located the source of this difference as greater plasma homocysteine concentrations in the 1921 birth cohort. No significant differences were detected between cohorts in plasma or red blood cell concentrations of folic acid or in plasma concentrations of vitamin B-12.
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TABLE 1 . Plasma and red blood cell biomarkers in 2 Aberdeen birth cohorts (ABC21 and ABC36) tested in 1999–20001
Significant inverse relations were observed between homocysteine and both plasma and red blood cell folate concentrations and plasma vitamin B-12 in both cohorts (Table 2). In both cohorts, positive correlations were found between blood concentrations of B vitamins.
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TABLE 2 . Correlations between biomarkers and psychological test scores in 2 Aberdeen birth cohorts (ABC21 and ABC36) tested in 1999–20001
MANOVA showed an overall difference between cohorts in cognitive test performance (P < 0.001; Table 3). Univariate ANOVA showed that the ABC36 cohort performed better than the ABC21 cohort on the AVLT, BD subtest, DS subtest, MMSE, and RPM. There was no significant difference between cohorts in scores on the NART. After sex was entered into the model, overall cohort differences persisted in nutritional and cognitive variables (P < 0.001). These were identified in the ABC21 cohort as greater homocysteine concentrations, lower vitamin B-12 concentrations, and lower scores on the MMSE, RPM, AVLT, BD subtest, and DS subtest.
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TABLE 3 . Psychological test scores in 2 Aberdeen birth cohorts (ABC21 and ABC36) tested in 1932–1947 and 1999–20001
Correlations between blood biomarker concentrations and cognitive test scores are also shown in Table 2. Note that the number of subjects varies in the table because not all subjects completed all cognitive tests. All effect sizes were small to medium. A positive relation was seen in the ABC21 cohort between both plasma folate and plasma vitamin B-12 and MMSE score (r = 0.20 and 0.24, respectively). Plasma folate was positively associated with NART score (r = 0.19). There was a negative relation between RPM score and plasma homocysteine (r = –0.22). Folate was positively associated with AVLT (r = 0.19) and DS subtest (r = 0.19) scores. Homocysteine was negatively associated with DS (r = –0.25) and BD (r = –0.27) subtest scores. In the ABC36 cohort, plasma folate was positively associated with BD subtest score (r = 0.24).
The correlations between blood biomarker concentrations and cognitive test scores after adjustment for the contribution of childhood IQ to cognitive scores in late life are shown in Table 4. These adjusted associations estimate the contribution of individual biomarkers to lifelong change in cognitive function. Partial correlations in both cohorts strengthened the associations between plasma vitamin B-12 and folate and MMSE and NART scores, respectively, but weakened those between red blood cell folate and the DS subtest score.
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TABLE 4 . Partial correlations between biomarkers and psychological test scores in 2 Aberdeen birth cohorts (ABC21 and ABC36) after control for childhood intelligence quotient1
DISCUSSION
The main finding of this study is that plasma homocysteine was higher in the ABC21 cohort than in the ABC36 cohort. Additionally, after adjustment for childhood IQ, homocysteine remained positively associated with RPM (r = –0.24), BD (r = –0.29), and DS (r = –0.29) scores in the ABC21. Previously (9), we showed that childhood IQ accounts for 50% of the variance in late-life cognitive ability. The present study adds to these earlier findings and shows that plasma homocysteine concentrations account for an additional 7–8% of this variance. These findings may prove relevant to susceptibility to late-onset Alzheimer disease. Total plasma homocysteine concentrations are higher in patients with dementia attributable to Alzheimer disease (8) and concentrations >15 µmol/L are associated with lower total life satisfaction, reasoning, memory recognition, and spatial copying in nondemented elderly (7). Similarly, homocysteine is strongly related to spatial copying performance (6) and significantly inversely associated with test scores on the cognitive subscale of the Cambridge Dementia Inventory (18).
Others have suggested that plasma homocysteine is not associated with performance on the MMSE (18,19). The findings presented here are not directly comparable with these earlier reports because the MMSE provides a global measure of cognitive performance, whereas the present study reports test scores in specific cognitive domains. In the ABC36 cohort, cognitive test scores were not associated with homocysteine concentrations. This is consistent with the findings of Hernanz et al (20), who observed cellular B vitamin concentrations to be similar but homocysteine concentrations to be higher in subjects aged 75–90 y than in those aged 65–75 y. However, note that renal function was not measured in our study and that renal insufficiency in the older birth cohort cannot be excluded.
B vitamins and homocysteine affect brain function and the mechanisms that explain these effects may also prove relevant to the associations reported here. Both folate and vitamin B-12 are important in the remethylation of homocysteine to methionine, which is subsequently adenosylated to S-adenosylmethionine. S-Adenosylmethionine is the primary methyl donor in most biochemical reactions, including the synthesis of monoaminergic neurotransmitters. In addition, it is required in the methylation of membrane phospholipids essential for receptor coupling and in protein and nucleic acid synthesis (21). B vitamin deficiencies may alter cellular methylation reactions and normal brain function by lowering S-adenosylmethionine concentrations. Supplementation with S-adenosylmethionine improves cognitive performance (22). Moreover, S-adenosylmethionine itself may have antidepressant properties (23).
B vitamin deficiency also increases plasma homocysteine concentrations. Homocysteine can undergo autoxidation to various metabolites and reactive oxygen species that are directly toxic to the endothelium and that negatively alter the dilatory properties of the vasculature (24). Elevated homocysteine concentrations are associated with extracranial carotid artery stenosis in the elderly (25) and may affect brain function as a result of the excitatory effects of homocysteine (26). One study of human neuronal cells reported that homocysteine metabolites such as homocysteic acid and cysteine sulphinic acid may act as endogenous agonists of N-methyl-D-aspartate receptors, overstimulating glutamate receptor activation and ultimately inducing neuronal injury and death in vitro (21,26). Homocysteine was also found to be associated with elevated cyclin E (a marker of cell division) in the hippocampus of patients with Alzheimer disease (27). Homocysteine may act mitogenically, stimulating neurons to proliferate and increasing atrophy (27).
These data may have implications for those designing neuroprotective nutritional strategies aimed at the delay of cognitive decline in late life and the onset of Alzheimer disease. We detected differences between birth cohorts for which homocysteine, a potential neurotoxin, accounted for 7–8% of the variation in cognitive function in the older cohort. This was not found in a cohort 15 y younger, suggesting that interventions started at this younger age aimed at homocysteine reduction may have benefits. Confidence in the likely success of a homocysteine-lowering strategy is encouraged by strong circumstantial evidence that raised concentrations of plasma homocysteine are linked to the later development of Alzheimer disease (28).
In this community-based study of healthy elderly, concentrations of folate and vitamin B-12 were positively associated with cognitive ability after control for childhood IQ. Plasma homocysteine was significantly higher in the ABC21 cohort than in the ABC36 cohort. Cognitive function was inversely related to plasma homocysteine concentrations, but only in the ABC21 cohort. This could represent folate or vitamin B-12 deficiencies in the tissues of older subjects that may in turn affect brain function. Indeed, folate concentrations were not significantly different between groups, suggesting either that blood vitamin concentrations do not accurately represent cellular or tissue status or that homocysteine and folate may act on cognitive performance independently.
ACKNOWLEDGMENTS
Helen Lemmon and Helen Fox recruited all subjects, collected the data, and managed the study database. We thank the local family doctors and volunteers, without whom this study would not have been possible.
REFERENCES