Nutrition and Aging. Janet Bryan, Eva Calvaresi 3 and Donna Hughes

Transcription

1 Nutrition and Aging Short-Term Folate, Vitamin B-12 or Vitamin B-6 Supplementation Slightly Affects Memory Performance But Not Mood in Women of Various Ages 1,2 Janet Bryan, Eva Calvaresi 3 and Donna Hughes Commonwealth Scientific and Industrial Research Organisation, Health Sciences and Nutrition, Kintore Avenue, Adelaide, 5000 Australia ABSTRACT Based on research demonstrating associations between folate, B-12 and B-6 vitamins and cognition and mood, we investigated the effects of short-term supplementation in 211 healthy younger, middle-aged and older women who took either 750 g of folate, 15 g of vitamin B-12, 75 mg of vitamin B-6 or a placebo daily for 35 d. In addition, we examined associations between dietary intake of these vitamins and cognition and mood. Usual dietary intake status was estimated using a retrospective, self-report, quantified food frequency questionnaire. Participants completed alternate forms of standardized tests of cognitive processing resources, memory, executive function, verbal ability and self-report mood measures before and after supplementation. Supplementation had a significant positive effect on some measures of memory performance only, and no effect on mood. Dietary intake status was associated with speed of processing, recall and recognition and verbal ability. J. Nutr. 132: , KEY WORDS: folate vitamin B-12 vitamin B-6 mood cognition humans The association between nutrition and cognitive function and mood is a topic of public and scientific interest (1,2) and recent research has focused on the effects of the B vitamins folate, B-12 and B-6 on cognitive aging (3,4). These vitamins are of particular interest because mild to moderate subclinical deficiencies, particularly of folate, are believed to be relatively common in the general population and in older adults and women in particular (5,6). This work is based on the assumption that the modification of nutrition may be important to the maintenance of cognitive function and mood across the lifespan (7). Individual micronutrients may be especially important for cognitive performance and mood because the effective functioning of the central nervous system (CNS) 4 depends in part on an adequate and constant nutrient supply (1,8,9). Research to date indicates two interlinked neurochemical mechanisms by which folate, with vitamins B-12 and B-6 as catalyzing 1 Presented, in part in preliminary form at the XXVII International Congress of Psychology, Stokholm, Sweden, July 23 28, 2000 (Bryan, J., Calvaresi, E. & Hughes, D. The effects of B vitamin supplementation and dietary intake on cognition and mood in women), at the 33rd Annual Convention of the Australian Industry of Food Science and Technology, Brisbane, Australia, August 20 23, 2000 (Bryan, J., Calvaresi, E. & Hughes, D. Nutrition, cognitive performance and mood: folate, vitamins B-12 and B-6.) and at the National Conference of the Australian Association of Gerontology, Adelaide, Australia (Bryan, J., Calvaresi, E. & Hughes, D. Folate, vitamin B-12 and vitamin B-6 supplementation and cognitive performance. Proceedings: Aust. J. Aging 19.4: suppl. 10.). 2 Financial support was received from the Australian Association of Gerontology, R.M. Gibson Award, To whom correspondence should be addressed. eva.calvaresi@csiro.au. 4 Abbreviations used: CESD, Center for Epidemiological Studies-Depression Scale; CNS, central nervous system; FFQ; food frequency questionnaire; POMS, Profile of Mood States Questionnaire; RAVLT, Rey Auditory-Verbal Learning Test; RDI, Recommended Daily Intake; SAM, S-adenosylmethionine; TCS, Technical Consultancy Services. cofactors, influences cognitive performance and mood via its role in methylation in the CNS (3,10,11). The first mechanism, the hypomethylation hypothesis, posits that these B vitamins may have a direct and possibly acute effect by inhibiting methylation reactions throughout the CNS involving proteins, membrane phospholipids, DNA, the metabolism of neurotransmitters such as the monoamines (e.g., dopamine, norepinephrine and serotonin), and melatonin, all of which are crucial to neurological and psychological status (2,11 13). The second mechanism, the homocysteine hypothesis, proposes that there is an indirect and possibly longer-term effect of folate, and vitamin B-12 or B-6, on the brain via the cerebrovasculature (10,14 17), and that these B vitamins may function to preserve the integrity of the CNS via their role in the prevention of vascular disease, which is crucial to cognitive function (2,10,18 21). Evidence from cross-sectional (22 28), longitudinal (29,30) and intervention studies (31 35) have demonstrated associations among folate, vitamin B-12 and vitamin B-6 intake and/or status and many aspects of cognition, in particular, memory performance (5). However, most of the evidence for the link between the B vitamins and cognitive performance is based on studies utilizing older participants, particularly those with clinical deficiencies, while very few have investigated the links between the B vitamins and cognition among younger adults or across a broader age range. Moreover, many studies have used measures of cognitive impairment such as the Mini Mental State Examination (36) as outcome measures of cognitive performance. Because nutritional effects are likely to be subtle, these tests of impairment may not be sensitive enough, when used in unimpaired populations, to capture sufficient variability in performance scores to facilitate the detection of subtle effects /02 $ American Society for Nutritional Sciences. Manuscript received 4 September Initial review completed 27 November Revision accepted 5 March

2 1346 BRYAN ET AL. There is a substantial body of literature linking the B vitamins with mood. In a comprehensive review of the literature, Young and Ghadirian (37) found that the most consistent finding from all the studies surveyed was the high incidence of folate deficiency in individuals with mood disorders compared with individuals with various other psychological disorders or normal controls (38,39). The level of folate deficiency has also been related to the severity and duration of depression (25,40) and the response to antidepressant treatment (41,42), leading to the use of folate or related compounds, such as S-adenosylmethionine (SAM), in the treatment of depressive disorders, either as an adjunct to standard antidepressant pharmacotherapy or even as a single agent in patients with low or subclinical levels of folate (11,37,43,44). Moreover, Fontanari and his colleagues (45) found that the administration of SAM not only confirmed its efficacy as an antidepressant in their work with older adults, but also resulted in significant improvements in measures of cognitive impairment, providing evidence for a link between folate, mood, cognitive performance and the hypomethylation hypothesis (9,46,47). The relationship between vitamin B-12 and depression is less clearly supported because research into vitamin B-12 and mood has not been extensive. Although vitamin B-12 deficiency in the absence of anemia is believed to be unusual (48), an association between vitamin B-12 deficiency and depressive symptomatology is frequently cited (25,40,49,50) and the benefits of supplementation have been evidenced (46). The few studies investigating vitamin B-6 have found neither associations with depression (51) nor any benefits of supplementation (32). Although studies to date suggest that folate may play a role in mood, they have utilized mainly older adults or clinical populations (11,12), and the effect of supplementation in those with only mild depressive symptomatology, or on mood in healthy populations across the lifespan, remains to be investigated. We investigated the effects of folate, B-12 and B-6 vitamin supplementation and dietary intake on cognition and mood in healthy women from three different age groups: younger, middle-aged, and older, using a randomized, double-blind, placebo-controlled design. In contrast to previous research, we used a comprehensive range of standardized neuropsychological outcome measures, sensitive enough to capture variability and subtle effects on performance in the cognitive functions believed to be vulnerable to the influence of nutritional factors known to affect the integrity and functioning of the CNS, such as, speed of information processing, memory (including working memory) and executive functions, as well verbal ability and measures of depressive symptomatology and mood states. MATERIALS AND METHODS Participants and background measures. Participants were recruited by a letter of invitation sent to a random sample of females from three age bands whose names were selected from the Adelaide Metropolitan Electorates of the Australian Electoral Rolls. The Australian Electoral Rolls contain 98% of the adult voting population because voting is compulsory in Australia. The letters of invitation stated that eligibility for participation was restricted to healthy women who did not smoke, who were not pregnant or lactating, not taking oral contraceptives or hormone replacement therapy, and not taking any medication likely to affect mental performance or mood. English as a first language, or proficiency in English, was also a requirement, because the cognitive performance measures and mood questionnaires were language rich. Women only were used for practical reasons of recruitment and accessibility and to control for gender effects. Prospective participants were asked to discontinue taking all B vitamins 2 wk before commencement of the study. Females (221) volunteered to participate and the final sample of participants for whom pre- and post-treatment data were available consisted of 211 women. Fifty-six formed a younger group aged between 20 and 30 y, 80 formed a middle-aged group aged between 45 and 55 y, and 75 formed an older age group aged between 65 and 92 y. The study was approved by the Human Experimentation Ethics Committee of the CSIRO Division of Health Sciences and Nutrition and the Social and Behavioral Research Ethics Committee of the Flinders University of South Australia. Design. A randomized, double-blind, placebo-controlled experiment was used to assess the effect of folate, vitamin B-12 and vitamin B-6 supplementation on cognitive performance and mood. A mixed factorial design was used, in which there were two between-subjects factors: treatment condition (four levels: folate, vitamin B-12 or vitamin B-6 supplementation and placebo control) and age group (three levels: younger, middle-aged and older); and one withinsubjects factor: time of testing (pre- and post-treatment). Participants were randomly allocated to one of four supplement treatment conditions: folate, vitamin B-12, vitamin B-6 or placebo as they entered the study. The dependent variables were the cognitive performance and mood measures. Materials. The vitamin supplements, in capsule form, were purchased from Technical Consultancy Services (TCS) of New South Wales Australia. Daily dosages were set at 75% of the tolerable daily upper limit (Food and Nutrition Board of the Institute of Medicine in the United States of America) for each of the supplements, i.e., 750 g of folate, 15 g of vitamin B-12, and 75 mg of vitamin B-6. These dosages were used because they have been found to be effective for other important physiological effects (13) and might, therefore, be large enough to elicit any subtle effects on cognition and mood. Participants took one capsule daily of folate, vitamin B-12, vitamin B-6 or placebo for 5 wk. Capsules for the placebo contained microcrystalline cellulose, calcium phosphate, soy polysaccharide and magnesium. The capsules for each treatment group were identical in color and shape and each container of capsules was numbered. TCS provided a sealed list identifying which supplement corresponded to each numbered container. These were not referred to until completion of the study; thus, both experimenter and participants were unaware of the treatment conditions. Participants were asked to return any unused capsules in their containers at the post-treatment session, enabling a capsule count to assess compliance. Apparent compliance for ingesting the total number of capsules was very high. The percentage of each treatment group who reported taking (i.e., 95%) of the capsules was: folate, 96%; vitamin B-12, 93%; vitamin B-6, 94% and placebo, 88%. Measures. Standardized measures of cognition and mood were administered before and after treatment. A self-completed, quantified food frequency questionnaire (FFQ), which provides an estimate of usual dietary and nutrient intake, was completed before the first testing session. Alternate forms of cognitive performance tests were counterbalanced across participants. Cognitive measures Speed of processing. Speed of information processing was assessed using three measures: the Boxes Test (52), a measure of sensory-motor speed; and the Digit Symbol-Coding and Symbol Search subtests of the Wechsler Adult Intelligence Scale-III (53), both measures of perceptual speed. For the Boxes Test, participants were presented with a printed sheet of 100 boxes, each with one side missing. They were required to complete as many boxes as possible by drawing in the missing side. The score represents the number of boxes completed correctly in 30 s. Alternate form reliability was good ( 0.92). For Digit-Symbol-Coding, participants were required to complete 133 digit symbol substitutions on a printed sheet. The number completed in 120 s and the time taken to complete the substitutions was recorded. Alternate form reliability was good ( 0.95 and 0.97, respectively). For Symbol Search, participants were required to scan two columns of symbols and to indicate whether the symbols of one column appeared among the symbols of the other.

3 MEMORY PERFORMANCE AND MOOD IN WOMEN OF VARIOUS AGES 1347 Scores represent the number identified correctly in 60 s. Alternate form reliability was good ( 0.83). Working memory. Two measures of working memory were administered: Digit Span-Backwards and Letter-Number Sequencing (WAIS-III) (53). For Digit Span-Backwards, strings of numbers (from two to eight numbers, with two trials of each string length) were read to participants at the rate of one per second. Immediately after presentation, participants were required to repeat the number strings in reverse order. One point was awarded for each number string repeated correctly. Alternate form reliability was moderate ( 0.70). For Letter-Number Sequencing, strings of numbers and letters (from two to eight items, with three trials of each string) were read to participants at the rate of one per second. Immediately after presentation, participants were required to repeat the items with numbers first in numerical order and letters second in alphabetical order. One point was awarded for each string repeated correctly. Alternate form reliability was moderate ( 0.68). Memory. Three measures of memory were used in this study. The Rey Auditory-Verbal Learning Test (RAVLT) (54) and two measures of incidental recall: recall of symbols from Digit-Symbol-Coding (WAIS-III) (53) and Activity recall. The measures of incidental recall were administered at post-treatment only. For the RAVLT, 15 nouns (list A) were read to participants (over five trials) at the rate of one word per second. Immediately after hearing the list at each of the five trials, participants were required to repeat any words recalled. Scores for trials 1 to 5 were summed to produce a measure of immediate recall. The alternate form reliability was moderate ( 0.74). After a sixth trial consisting of 15 different words (list B), participants were required to again recall the words presented in list A and then again after an interval of 20 min. Scores from these two trials produced measures of delayed recall. The alternate form reliability was moderate ( 0.67 and 0.66, respectively). After the final delayed recall trial, participants were presented with a printed sheet with 50 words containing the words from list A and B among 20 distracter words. Participants were required to identify the words from lists A and B and also to specify the list they came from. One point was awarded for each word correctly recalled or identified. This produced two measures of recognition memory, one for list A and one for list B. Alternate form reliabilities were fair to moderate ( 0.65 and 0.70, respectively). For Symbol recall, participants were required to recall as many symbol-digit pairs from the Digit Symbol-Coding test immediately after completing the test. For activity recall, participants were required to name or describe each of the 13 cognitive tasks they had completed, scoring one point for each activity recalled. Executive function. Executive function is conceptualized as a higher order cognitive function that controls and integrates other cognitive activities involved in planning and implementing strategies for performance, monitoring performance and using feedback to adjust future responding (55). Neuropsychological tests sensitive to frontal lobe function were used as measures of executive function (55 57). Six measures were administered: the Stroop Test (58); the Self-Ordered Pointing Task (59); Uses for Common Objects (60); the Trail Making Test (61); and Verbal Fluency, comprising Initial Letter Fluency (62) and Excluded Letter Fluency (63). For the Stroop Test, participants were presented with a printed sheet of color names printed in incongruent colored ink. First, participants were required to read the words, and second, to name the color of the ink that the words were printed in. Scores on this task represent a measure of interference that was calculated as a ratio of the time taken to name the colors compared with the time taken to read the words, such that lower scores reflect less interference and consequently better performance. Alternate form reliability was good ( 0.86). For the Self-Ordered Pointing Task, participants were presented with 3 trials of 16 pages, each printed with 16 different visual patterns in a different random order on each page. Participants were required to point to one different pattern on each page, aiming to point to each of the 16 different patterns once, such that by page 16 each pattern had been identified only once. The repetition of a pattern represented an error and scores represented the total errors over the three trials. The time taken to complete each trial was also recorded. Test-retest reliability across the three trials was fair ( 0.49). The Uses for Common Objects Test required participants to produce as many different uses as possible for a common object in two 90-s trials (brick and newspaper or bottle and paper clip). Responses were rated independently by two scorers and inter-rater reliabilities ranged from 0.92 to 0.97 for the four objects. Alternate form reliability was moderate ( 0.75). The Trail Making Test had two parts. In part A, participants were presented with a sheet on which the numbers 1 25 were printed in circles in a random pattern. Participants were required to join the numbers in numerical order as quickly as possible. Scores represent the time (in seconds) taken to complete the task. Alternate form reliability was fair ( 0.45). In part B participants were presented with a sheet on which the numbers 1 13 and the letters A to L were printed in circles in a random pattern. Participants were required to join the numbers and letters in alternate numeric and alphabetic sequence. Scores represent the time (in seconds) taken to complete the task. Alternate form reliability was good ( 0.86). The Verbal Fluency task also consisted of two parts: initial letter fluency and excluded letter fluency. For initial letter fluency, participants were required to generate as many words as possible according to an initial letter. Participants completed two, 60-s trials (F and C or L and S). The number of words correctly generated over the two trials was summed to provide the total initial letter fluency score. Alternate form reliability was moderate ( 0.75). For excluded letter fluency participants were required to generate as many words as possible not containing a specified letter. Participants completed two 60-s trials (E and R or A and T). The number of words correctly generated over the two trials was summed to provide the total excluded letter fluency score. Alternate form reliability was fair ( 0.57). Verbal ability. Two measures of verbal ability were used: Vocabulary (WAIS-III) (53) and Spot-the-Word (64). For Vocabulary, participants were asked to define 15 words (e.g., generate and encumber) and were awarded a score of 0, 1, or 2 depending on the quality of the definition. Alternate form reliability was good ( 0.83). For Spot-the-Word, participants were presented with two printed sheets each containing 60 real word/nonword pairs. The nonwords were designed to look like a real word (e.g., harrick). Participants were required to identify the real word in each pair. Contrary to standard instructions, participants were instructed not to guess. Scores on this task were calculated as the number of words correctly identified minus the number of errors to correct for guessing (65). Alternate form reliability was good ( 0.89). Mood measures. Current mood state was assessed using two self-report questionnaires that were completed before the cognitive testing sessions: The Center for Epidemiological Studies-Depression Scale (CESD) (66) and The Profile of Mood States Questionnaire (POMS) (67). Participants were required to rate the frequency with which they experience 20 depressive symptoms on a 4-point scale ranging from 1 (rarely or none of the time) to 4 (most or all of the time). Scores range from 20 to 80 with higher scores indicating greater frequency of depressive mood. The POMS assesses six aspects of mood: tension-anxiety; depression-dejection; anger-hostility; vigor-activity; fatigue-inertia; and confusion bewilderment, reflected in 65 adjectives describing mood states (e.g., tired and anxious). Participants were required to report the frequency with which they had experienced the 65 mood over the last week on a 5-point scale ranging from 1 (not at all) to 5 (extremely). Dietary intake. Dietary intake was assessed using a self-completed, quantified, FFQ based on Baghurst and Record (68). This form of the FFQ is regularly updated and has been used extensively with Australian population samples and national dietary surveys (1988, 1993 and 1998). It has been shown to have a high repeatability and consistency with other dietary intake measurement techniques and has demonstrated good reliability compared with urinary and protein measures (69,70). The FFQ takes the form of a 20-page booklet including a list of over 180 common food and beverage items and questions relating to food preparation and dietary habits. Participants were required to indicate how often each food and beverage was usually consumed per month, week or day. Average daily consumption was based on participants reports of how often a specified serving size of each food or beverage item was consumed. All this information, together with the nutrient composition of the food item per unit

4 1348 BRYAN ET AL. weight taken from Australian and British food tables (71,72), allowed participants daily nutrient intakes, including the B vitamins, to be calculated using the Frequency Questionnaire Analysis dietary analysis program (68). Procedure. After initial contact and scrutiny of eligibility for participation, individuals were sent the two mood questionnaires and an FFQ to be completed the day before or on the day of the testing sessions. Both pre- and post-treatment testing sessions, 5 wk apart, took h, with a 15- to 20-min break midway through the session. Participants were tested individually in quiet and well-lit interview rooms. The order of events was as follows. During the testing sessions, information on demographic and background information was collected. Participants then completed the cognitive tests in the following order: Boxes, Stroop, Trails A and B, Digit Symbol- Coding, Digit Span-Backwards, Self-Ordered Pointing Task (session 2 only), and the Rey Auditory-Verbal Learning Test. This was followed by a 15- to 20-min break. After the break, participants completed Letter-Number Sequencing, Verbal Fluency, Symbol Search, Uses for Objects, Vocabulary, Spot the Word and activity recall (session 2 only). Statistical methods. Values are presented as means SD unless stated otherwise. All data were analyzed using SPSS, Version 10 (73). Before hypothesis testing the distributions for all variables were examined for violations to assumptions of normality. Tests for skewness and kurtosis revealed five instances of departure from normality: Digit-Symbol recall, recognition memory for RAVLT List A, Trail Making Test parts A and B, WAIS vocabulary and CESD total mood score. Data for these variables were transformed using logarithmic (base10) transformations (74). Subsequent analyses on transformed and untransformed scores produced comparable results. Therefore, untransformed scores were used in all analyses. To assess the effects of usual dietary intake before supplementation on cognitive performance and mood, and to determine whether there were any threshold effects, dietary intake ranges for each B vitamin were divided into quartiles with the first quartile reflecting the lowest intake and the fourth reflecting the highest. Table 1 presents a summary of the dietary intake ranges and percentages of participants whose dietary intakes were below the Australian Recommended Daily Intakes (RDI) (75), and those who were below 70% of the Australian RDI, which is considered more indicative of nutrient inadequacy (76). Table 2 presents a summary of dietary intake quartile ranges. Differences in cognitive performance and mood before supplementation between intake quartiles were examined using two-way ANCOVA. The between subjects factors were intake quartile and age group. Years of education was used as a covariate to control for the possible influence of education on dietary habits. Post hoc comparisons using Tukey s Honestly Significant Difference procedure were conducted to determine significant differences between groups for interaction effects. A value of P 0.05 was set as the criterion of significant difference. To assess the effect of supplementation on cognitive performance and mood measures administered at both pre- and postsupplementation, (treatment condition age group time of testing) repeated measures ANOVA was performed. Univariate ANOVA was performed for measures administered at postsupplementation only (Digit-Symbol symbol and symbol/position recall, Activity recall and Self-Ordered Pointing Task). Post hoc comparisons, using Tukey s HSD procedure, were conducted to determine significant differences between groups for interaction effects, and a value of P 0.05 was set as the criterion of significant difference. Demographic and other background information variables were examined as potential covariates. (Table 3 presents a summary of the means and standard deviations.) The four treatment groups did not differ on background measures, although, as expected, there were some age group differences. The younger and middle-aged groups experienced more years of formal education than the older age group [F (2,199) 17.64, P 0.05]. The older age group experienced more medical conditions than the younger and middle-aged groups [F (2,199) 40.49, P 0.05] and took more medications than the younger and middle-aged groups [F (2,199) 61.09, P 0.05]. The two measures of verbal ability (WAIS Vocabulary and Spot the Word) were used as indicators of premorbid intelligence and the younger age group performed more poorly than did both the older and middle-aged groups [F (2,199) 13.91, P 0.05]. Because the background measures covaried with the dependent variables of cognition and mood as well as with the independent variable age, they were not considered useful as covariates because they would remove any main effects (74). Furthermore, because there were no significant differences among treatment groups in the variables considered as potential covariates, the decision was made to conduct all ANOVA without covariates. RESULTS Effects of usual dietary intake on cognitive performance and mood Folate. The effects of usual dietary intake on cognitive performance and mood before supplementation was assessed using two-way ANCOVA with years of education as a covariate. Folate intake affected Boxes performance across age groups [F (3,196) 3.23, P 0.05], with those in the 2nd intake quartile ( ) completing significantly more boxes than those in the 3rd ( ) and 4th ( ). There was an age group folate intake interaction for recall performance of List B for the RAVLT [F (6,196) 2.41, P 0.05; HSD 2.07, q 4.62, MSw 3.33], with younger age group participants in the 4th ( ) intake quartile recognizing more words than did those in the 1st ( ) and 3rd ( ) intake quartiles. Excluded letter fluency performance also differed between intake quar- TABLE 1 Dietary intake of B vitamins as assessed by food frequency questionnaire, and proportion of participants below the Australian Recommended Daily Intake (RDI) and below 70% of the Australian RDI Folate TUL g RDI 200 g Vitamin B-12 TUL 20 g RDI 2 g Vitamin B-6 TUL 100 mg RDI mg Age group, y n Range, % 0.7 g % RDI 2 RDI 3 Range, g % RDI % 0.7 RDI Range, mg % RDI % 0.7 RDI Younger (20 30) Middle (45 55) Older (65 92) Total Sample Tolerable upper limit. 2 Percentage of participants below the Australian RDI. 3 Percentage of participants below 70% of the Australian RDI.

5 MEMORY PERFORMANCE AND MOOD IN WOMEN OF VARIOUS AGES 1349 TABLE 2 Dietary intake of folate, vitamins B-12 and B-6 by quartile for participants as assessed by food frequency questionnaire (FFQ)1,2 Age group n Folate, g Vitamin B-12, g Vitamin B-6, mg Younger (20 30 y) 56 1st quartile nd quartile rd quartile th quartile Middle (45 55 y) 80 1st quartile nd quartile rd quartile th quartile Older (65 92 y) 75 1st quartile nd quartile rd quartile th quartile Values are dietary intake ranges and means SD, n 7 21 (younger), (middle) and (older). 2 The 1st quartile reflects the lowest intake and the 4th the highest. tiles [F (3,196) 3.12, P 0.05], with those in the 2nd intake quartile ( ) performing better than did those in the 3rd ( ). There was also a significant interaction of age group intake quartile [F (6,196) 2.18, P 0.05], for the younger age group participants in the 4th intake quartile generating more words according to the excluded letter criteria than the other intake quartiles. There were no effects of folate intake on mood measures. Vitamin B-12. There was a significant effect of vitamin B-12 intake that interacted with age group for recognition memory performance of list B for the RAVLT [F (6,196) 2.13, P 0.05; HSD 3.49, q 4.62, MSw 9.65], with those in the 3rd ( ) intake quartile performing better than those in the 1st ( ) intake quartile for younger adults only. There were no effects of vitamin B-12 intake on the other cognitive or mood measures. Vitamin B-6. Vitamin B-6 intake interacted with age group on the number of words recalled from list B for the RAVLT [F (6,196) 3.10, P 0.05; HSD 2.07, q 4.62, MSw 3.29], with younger age group participants in the 4th ( ) intake quartile recalling more words than those in the 1st ( ), 2nd ( ) and 3rd ( ). There was an effect of vitamin B-6 intake that interacted with age group for the short delay recall of the RAVLT [F (6,196) 2.47, P 0.05; HSD 2.94, q 4.62, MSw 6.76], with older age group participants in the 2nd ( ) intake quartile recalling more words than those in the 1st ( ). Vitamin B-6 intake interacted with age group for the long delay recall of the RAVLT [F (6,196) 2.56, P 0.05; HSD 3.13, q 4.62, MSw 7.65], with younger age group participants in the 4th ( ) intake quartile of the recalling more words than did those in the 2nd ( ) intake quartile. There were no effects of vitamin B-6 intake on the other cognitive measures or mood measures. Effects of short-term supplementation on cognitive performance and mood. Tables 4 7 present a summary of the F values for main effects of age group, treatment group and time of testing, interaction effects of time age, age treatment, time treatment and time age treat- TABLE 3 Demographic and background measures for participants as assessed by self-report questionnaire1,2 Younger Middle-age Older n Age, y Education,3 y Health4 (self-rated) Number of medical conditions Number of medications Number of supplements Verbal ability: WAIS vocab Spot the Word Values are means SD. 2 No significant differences between B vitamin treatment groups. 3 The older age group experienced fewer years of formal education. 4 Health rated 1 (poor) to 5 (excellent). 5 The older age group had a greater number of medical conditions and 6 took a greater number of medications than did the younger and middle age groups (P 0.001). 7 The younger age group performed more poorly in tests of verbal ability than did the middle and older age groups (P 0.001).

6 1350 BRYAN ET AL. ment, and descriptive statistics for age group and time of treatment effects. The effects of interest from the ANOVA are time treatment interactions. These would indicate differential effects of supplementation on cognition and mood outcomes from pre- to post-treatment. There were significant main effects of age for measures of cognitive performance. Exceptions to this were age effects for Spot the Word and Vocabulary, which showed increases in performance with increasing age. There were also age effects for many of the mood measures. The POMS differed between age groups (Table 7) with the older age group scoring significantly lower than the younger and middle age groups indicating a more positive overall mood. Similarly, for the POMS subscales of anger/hostility, fatigue/inertia and confusion/bewilderment, the older age group s scores were significantly more positive than those of the younger and middle age groups. For the POMS subscales of depression/ dejection and tension/anxiety, the older age group s scores were significantly more positive than those of the younger age group. There were also main effects of time for many of the cognitive performance and mood measures in which performance and mood was more generally more positive at time 2 than time 1, reflecting practice and placebo effects. There were few treatment interaction effects. (See Table 8 for descriptive statistics for interaction effects.) There was a trend for a significant time age treatment interaction (P 0.06) on immediate recall for RAVLT. The trend was greater improvement in number of words recalled by younger age participants in the folate and vitamin B-12 treatment groups and the middle age participants in the vitamin B-6 treatment group. There was a significant time age treatment interaction effect on the RAVLT memory task for the number of words correctly recognized. Post hoc comparisons (HSD 1.28, q 5.01, MSw 3.47) revealed that older age participants in the folate treatment group identified significantly more words than did those in the placebo group. There was a significant time treatment interaction effect TABLE 4 Cognitive processing resources of women in three age groups before and after supplementation with B vitamins as assessed by measures of speed of processing and working memory1 Cognitive processing resource measures Age group2 Time3 Folate Vitamin B-12 Vitamin B-6 Placebo Speed of processing Boxes Y Pre Post M Pre Post O Pre Post Effects of age (F 55.75, P 0.001), time (F , P 0.001), and time age (F 3.72, P 0.05) Digit-Symbol Coding (120 s) Y Pre Post M Pre Post O Pre Post Effects of age (F 76.64, P 0.001), and time (F , P 0.001) Symbol Search Y Pre Post M Pre Post O Pre Post Effects of age (F 89.78, P 0.001), and time (F 13.31, P 0.001) Working memory Digit Span Backwards Y Pre Post M Pre Post O Pre Post Effects of age (F 6.04, P 0.01) and time (F 19.96, P 0.001) Letter-Number Sequencing Y Pre Post M Pre Post O Pre Post Effects of age (F 18.65, P 0.001) and time (F 10.08, P 0.01) 1 Values are means SD, n Younger (Y), middle (M), and older (O) age groups. 3 Pre- and post-treatment.

7 MEMORY PERFORMANCE AND MOOD IN WOMEN OF VARIOUS AGES 1351 TABLE 5 Memory of women in three age groups before and after supplementation with B vitamins as assessed by measures of free recall and incidental recall1 Memory measures Age group2 Time3 Folate Vitamin B-12 Vitamin B-6 Placebo Free recall Immediate recall: RAVLT 1 54 Y Pre Post M Pre Post O Pre Post Effects of age (F 42.27, P 0.001), time (F 35.66, P 0.001) and time age treatment (F 2.05, trend P 0.06) RAVLT B Y Pre Post M Pre Post O Pre Post Effects of age (F 38.71, P 0.001) Delayed recall: RAVLT 6 Y Pre Post M Pre Post O Pre Post Effects of age (F 29.39, P 0.001) and time (F 27.34, P 0.001) RAVLT 7 Y Pre Post M Pre Post O Pre Post Effects of age (F 33.17, P 0.001) and time (F 14.05, P 0.001) Recognition: RAVLT list A Y Pre Post M Pre Post O Pre Post Effect of age (F 23.87, P 0.001) RAVLT list B Y Pre Post M Pre Post O Pre Post Effects of age (F 23.11, P 0.001), time (F 3.03, P 0.05), and time age treatment (F 2.38, P 0.05) Incidental recall5 Digit Symbol symbol Y M O Effect of age (F 6.84, P 0.01) Symbol/position Y M O Effects of age (F 11.94, P 0.001), and age treatment (F 2.82, P 0.05) Activity Y M O Effect of age (F 16.48, P 0.001) 1 Values are means SD, n Younger (Y), middle (M), and older (O) age groups. 3 Pre- and post-treatment. 4 Trend only (P 0.06). 5 Administered at post-treatment only.

8 1352 BRYAN ET AL. of the Verbal Fluency task for the number of words correctly generated in 60 s according to an initial letter. Post hoc comparisons (HSD 2.26, q MSw 14.68) revealed that participants in the vitamin B-6 and placebo groups generated significantly more words than did those in the folate and vitamin B-12 treatment groups. DISCUSSION We have sought to establish whether supplementation with folate, vitamin B-12 or vitamin B-6 influences cognitive performance and mood among healthy women across the lifespan. We reasoned that short-term supplementation, consistent TABLE 6 Executive function and verbal ability of women in three age groups assessed before and after supplementation with B vitamins1 Executive function and verbal ability measures Age group Time Folate Vitamin B-12 Vitamin B-6 Placebo Executive Function Stroop Y Pre Post M Pre Post O Pre Post Effect of age (F 8.66, P 0.001) and time (F 15.49, P 0.001) Self-Ordered Pointing Task2 Y M O Effect of age (F 49.73, P 0.001) Uses for Objects Y Pre Post M Pre Post O Pre Post Effect of age (F 13.92, P 0.001) Trail Making Test: Test A Y Pre Post M Pre Post O Pre Post Effect of age (F 40.55, P 0.001) Test B Y Pre Post M Pre Post O Pre Post Effects of age (F 44.46, P 0.001) and time (F 20.40, P 0.001) Verbal Fluency: Initial letter Y Pre Post M Pre Post O Pre Post Effects of age (F 4.35, P 0.05), time (F 15.81, P 0.001), and time treatment (F 2.97, P 0.05) Excluded letter Y Pre Post M Pre Post O Pre Post Effect of age (F 8.53, P 0.001) Verbal Ability Vocabulary Y Pre Post M Pre Post O Pre Post Effect of age (F 3.47, P 0.05)

9 MEMORY PERFORMANCE AND MOOD IN WOMEN OF VARIOUS AGES 1353 TABLE 6 (Continued) Executive function and verbal ability of women in three age groups assessed before and after supplementation with B vitamins1 Executive function and verbal ability measures Age group2 Time Folate Vitamin B-12 Vitamin B-6 Placebo Spot the Word Y Pre Post M Pre Post O Pre Post Effect of age (F 17.46, P 0.001) 1 Values are means SD, n Younger (Y), middle (M), and older (O) age groups. 3 Pre- and post-treatment. with the direct or acute effects posited by the hypomethylation hypothesis, might have a subtle influence on the CNS, and, therefore, on measures of fluid function believed to be vulnerable to the impact of nutritional factors (2,8,9,29). Furthermore, we have also examined whether intake status of the three vitamins is associated with any aspects of cognitive performance and mood. Previous research has demonstrated associations between B-vitamin intake and cognitive performance and affective symptoms, although findings have been inconsistent. However, most studies have utilized older aged or clinical samples and very few placebo-controlled intervention studies have been conducted. To our knowledge this is the first study to investigate supplementation and intake status in women across the lifespan using a comprehensive battery of standardized neuropsychological outcome measures and mood. We found age effects for measures of fluid function, which support the robust findings of age-related cognitive decline on measures of cognition reflecting fluid functions (77). Age effects in favor of middle and older age women were found on the tests of verbal ability in keeping with previous findings that crystallized cognitive abilities, reflected in tasks of verbal ability, are well-maintained in older age (78). Mood measures also differed between age groups with older women reporting more positive moods than younger and middle-age women, consistent with research demonstrating age-related improvements in psychological well-being in women (79). We also found effects of time of testing (pre- vs. posttreatment) reflecting practice or placebo effects. When cognitive tasks are given repeatedly, improvement in performance is expected; hence, the need for placebo control groups in intervention studies. Improvements in mood were also evident at post- compared with pre-treatment. This finding suggests that simply being in the study influenced participants to report mood as being more positive at post-treatment. However, there was little evidence of general positive effects of short-term B vitamin supplementation. Contrary to expectations, there were very few time treatment interactions. It was expected that if women and older adults were at risk of even subclinical levels of deficiency in the B vitamins, particularly folate (5,6), cognitive performance and mood would be enhanced by supplementation, especially in older women. Hence, we had expected that those in the supplemented groups would show a greater improvement in cognitive performance and mood scores compared with those in the placebo groups from pre- to post-treatment, and that these effects might also interact with age. Such time supplement age effects were evident on two measures of memory: immediate recall and delayed recognition of words. There was also a time by supplement group interaction for verbal fluency performance. However, for this test, those in both the vitamin B-6 and placebo groups performed better post-treatment compared with pretreatment, relative to the other supplement groups. These findings suggest there were few general positive effects of short-term B vitamin supplementation on cognitive performance, with effects being evident for some aspects of memory performance only. Contrary to expectations, there were no effects of supplementation on depressive symptoms or mood states. We also investigated the effects of estimated usual dietary intake on cognition and mood before supplementation. A higher percentage of younger women had usual dietary intakes of folate and vitamin B-12 that were below 70% of the RDI compared with middle-aged and older women, although 9% of the middle-aged women had intakes of folate that were below 70% of the RDI. This suggests that younger women may be particularly at risk of nutrient inadequacy for folate. Contrary to expectations, very few of the older women had intakes of the B vitamins that were below the RDI. Dietary intake ranges were split into quartiles to examine threshold effects of intake on cognition and mood. Some aspects of cognitive performance differed between intake quartiles. Folate intake was associated with speed of processing, memory recall and verbal fluency. However, contrary to previous research (22,26), effects were generally nonlinear except for younger women in whom higher intake was associated with better performance. Linear effects were also observed for younger women for whom vitamins B-12 and B-6 intake was associated with memory performance, with higher intake associated with better recognition memory for vitamin B-12 and better immediate and delayed memory for vitamin B-6. However, the effects of dietary intake of B vitamins on cognitive performance were variable and, generally nonlinear, except for younger women. These results may reflect the lack of a doseresponse relationship, or it could be argued that because middle and older age women had better intakes of all three vitamins, any possible effects may have been masked. In summary there were few effects of short term B vitamin supplementation on cognitive performance and none on mood. Effects of supplementation were seen mainly for memory performance. The general lack of effects could be due to the short duration of supplementation. It was believed that supplementation for 5 wk would be sufficient to produce the metabolic outcomes (80) likely to have an effect on the cognitive measures considered to reflect fluid abilities, which are more vulnerable to CNS integrity, and, therefore, more likely to demonstrate acute effects as proposed by the hypo-