Serum Folate and B12 Levels in Association With Cognitive Impairment Among Seniors

Abstract

Objective: To summarize existing evidence on the effect of serum folate and vitamin B12 levels on cognitive impairment among elders via a meta-analysis, also including unpublished data from a cross-sectional study of seniors ( > 65 years) residing in Velestino, Greece. Method: Serum measurements and Mini-Mental State Examination (MMSE) assessments were available for 593 Velestinians. In addition, 12 studies availing data on folate blood levels (N = 9,747) and 9 on B12 (N = 8,122) were identified following a search algorithm; pooled effect estimates were derived. Results: Cognitive impairment (MMSE < 24) among Velestenians was associated with lower education level in both genders; decreased social activity, depressive symptoms and low folate levels in males; older age in females. Meta-analyses showed an adverse effect of low-folate levels on cognition (OR: 1.66, 95% CI: 1.40-1.96); B12 was nonsignificantly associated (OR: 1.11, 95% CI: 0.88-1.40). Discussion: Low folate levels are associated with cognitive impairment of seniors; underlying pathophysiological mechanisms should be further explored.

Keywords

folate B12 cognitive impairment meta-analysis aged

Introduction

Cognitive impairment refers to changes in cognition ranging from normal ageing to dementia (Lonie et al., 2010; Voisin, Touchon, & Vellas, 2003). During the past decades, considerable attempts at discovering biological markers related to cognitive impairment have been made but results remain somewhat controversial. In particular, ever since the mid-1990s, lower levels of B-complex vitamins among impaired patients have been reported in several studies (Bernard, Nakonezny, & Kashner, 1998; Malouf & Areosa Sastre, 2003; Malouf, Grimley, & Areosa, 2003; Ortega et al., 1996). The evidence for a role of these specific vitamins on the pathogenesis of cognitive impairment comes primarily from reported cases of severe vitamin deficiencies (such as pernicious anemia) and defects in genes that encode for enzymes of one-carbon metabolism. The strong association between the B-complex vitamins and impaired nerve cell homeostasis, probably through mechanisms of homocysteine-induced increased neurotoxicity, vasotoxicity, and inefficient s-adenosylmethionine methylation (Kim, Cho, & Kwon, 2008; Pizzolo et al., 2011), but the topic is still considered an open debate, despite the plausible biochemical mechanisms.

The effect of folate and folic acid, two different forms of B9, tetrahydrofolate, a folate derivative and active form of B9, as well as B12 on cognition has been addressed in various observational studies (mostly cross-sectional designs) in which the levels of vitamins were assessed either in serum or food and/or supplement intake, yielding mostly inconclusive results (M. C. Morris, 2012). A number of trials has been implemented to test the effect of vitamin supplementation in cognitive function, as the B-complex vitamins have been considered good candidates for biological interventions, due to their physiological actions and properties. However, the results of recent randomized controlled trials (RCTs) have shown contradictive evidence and it remains to be established whether supplementation with B-vitamins can reduce the risk of cognitive impairment in later life (Ford & Almeida, 2012; Walker et al., 2012). Evidently, some authors suggest following an approach aiming at the rehabilitation of the deficiency with B-complex vitamin supplementation while others deny this strategy. Therefore, taking a step back and systematically assessing the association between folate and/or B12 and cognitive impairment in observational studies is important in this context.

Identifying cognitive decline predictors undeniably constitutes a challenge in public health in that it would allow interventions which could protect or delay the occurrence of cognitive disorders in elderly people (Perquin et al., 2012). Apart from B-complex vitamins, numerous other congenital and lifestyle factors seem to be implicated in the pathophysiology of cognitive impairment including age, gender, education, family support (Dimopoulos et al., 2006; Fountoulakis, Tsolaki, Chantzi, & Kazis, 2000; James, Wilson, Barnes, & Bennett, 2011; Sachdev et al., 2011), and, evidently from longitudinal studies, participation in social activity (James et al., 2011). It is obvious that cognitively impaired individuals suffer difficulties in preserving their productivity and protecting themselves from all-cause morbidity and therefore, in this context, such type of research is of prime interest amidst the financial crisis.

Our team conducted a community-based study in the mountainous town of Velestino, Central Greece, aiming to explore the role of inherent, environmental, and social factors in preserving cognition among the elderly, with special emphasis on the role of blood measurements of the two aforementioned B-complex vitamins. Furthermore, we conducted a meta-analysis in order to synthesize existing evidence and quantify the association of the two B-complex vitamins, if any. The rationale would be to offer a definite answer on the association between the low folate and/or B12 levels and cognitive impairment; thereafter proper long-term nutrition intervention studies could be designed.

Method

The “VELESTINO” Study

Study’s Sample

Data collection was conducted in the municipality of Velestino, a town in central Greece of about 4,000 inhabitants, during the period January 2005-April 2006. According to the town registry, 845 individuals were recorded to be 65 years of age or older on January 1, 2005; out of them, 150 had moved in the past to other residential areas of Greece and did not qualify as permanent residents, whereas another 13 individuals were residing in Velestino, though not included in the town registry; lastly, 26 died during the study period and before being interviewed, leading to a total of 682 individuals, potentially available for participation in the study. Six of the eligible study subjects were physically or mentally disabled and therefore, unable to communicate and respond to the questionnaire of this investigation, whereas 83 were not willing to participate. The remaining 593 seniors provided informed consent for participation. The study protocol was reviewed and approved by the Ethics Committee of the Athens University Medical School.

Demographic, Social Activity and Family Support Information

During the study period, two physicians (AA, KPK), who had served in the local health center, interviewed the participants using a standardized, precoded questionnaire that covered various sociodemographic, anthropometric, and lifestyle characteristics, as well as medical history. Thereafter, a “social activity” proxy was created by combining the reported variables “level of autonomy” (3 categories of self-help ability) plus “activity” (mean time per day spent outside the home, in 3 categories) as elderly, especially males in rural Greece, use to spend hours socializing in café stores (the “social activity” was categorized as normal and subnormal). Similarly, a family support scale was created, comprising the variables “marital status” (currently married or not), “cohabitation status” (3 categories of cohabitation: with close family member; with distant family member or friend or living in a nursing home; living alone) and the number of alive offspring (0; 1-2; more).

Body Mass Index (BMI) was calculated as body weight divided by height squared, and classified in four levels according to established criteria (< 25, 25-29.9, 30-34.9, and ≥ 35kg/m²); smoking was classified in two categories (ever smoker vs. never smoker); and alcohol consumption in three gender specific increments (a) Zero glasses/day, (b) recommended: 1 glass/day for women and 2 glasses/day for men, and (c) > recommended, namely consuming more than the respective recommended consumption limits; Neafsey & Collins, 2011). Information was also retrieved concerning folate (n = 14) and B12 (n = 13) supplements.

Proxies for Cognitive Function and Depression

The validated Greek version of the Mini-Mental State Examination (MMSE) questionnaire (Folstein, Folstein, & McHugh, 1975) was applied and used as a proxy of cognitive impairment. The validated Greek version of the 15-question Geriatric Depression Scale (GDS-15; Sheikh & Yesavage, 1986) was used for the evaluation of depressive symptoms. Relevant authorization for the use of the questionnaires has been acquired. According to the validation protocol, MMSE score < 24 indicates cognitive impairment (Fountoulakis et al., 2000); similarly, GDS score > 6 indicates existence of depressive symptoms (Fountoulakis, Tsolaki, Chantzi, & Kazis, 1999).

Biochemical Measurements

Fasting morning blood samples have also been collected and determinations were available for serum folate (n = 404), B12 (n = 387) and macrocytosis with or without anemia (yes: Mean Corpuscular Volume, MCV > 98fl vs. no: MCV ≤ 98fl) (n = 443). Folate and B12 levels were assessed in tertiles. The cutoff point of the lowest folate tertile was < 4.4ng/ml and of the highest ≥ 7.3ng/ml among males, whereas among females: < 5.5ng/ml and ≥ 8.8ng/ml, respectively. Similarly, the cutoff points for B12 were < 255.6pg/ml and ≥ 371.8pg/ml for males, whereas for females: < 242.1pg/ml and ≥ 366.2pg/ml, respectively.

Statistical Analyses

Complete sociodemographic, anthropometric, lifestyle, medical history information, and MMSE assessments were available for 593 (87.7%) Velestinian seniors. Initially, the proportion of cognitively impaired (MMSE < 24) individuals by study variables were derived and p values were calculated separately for each gender given the gender differences in the MMSE score and several other variables. Sensitivity analyses of the basic characteristics and the MMSE score between subjects availing or not availing blood measurements for the indicated vitamins and macrocytosis were also performed and no significant differences were detected.

A multiple logistic regression core model was thereafter developed with cognitive impairment (MMSE < 24) as the dependent variable and sociodemographic/lifestyle characteristics, BMI and depressive symptoms as independent variables, and the respective adjusted odds ratios (OR) and 95% confidence intervals (95% CI) were estimated. Consequently, serum folate and B12 levels were alternatively introduced as categorical (1st vs 3rd and 2nd vs 3rd tertile) variables (Models 1 and 2, respectively) or simultaneously (Model 3). The possible independent effect of macrocytosis was also explored by introducing the variable in the core model (Model 4). In addition to the above and following a similar methodology, a multiple logistic regression core model was developed for subjects with less than 6 years of education. The rationale for the latter derived from the fact that the MMSE scores are somewhat inconsistent for subjects with inadequate education. All statistical analyses were carried out using SAS software, Version 9 of the SAS system for Windows 9 (SAS Institute Inc., Cary, NC, USA).

Meta-analysis

Search Strategy and Data Extraction

Publications of interest were identified by a search of Medline bibliographical database, without language restrictions using the mesh terms: (“folate” OR “folic acid” OR “cobalamin” OR “B12”) AND (“cognition” OR “cognitive impairment” OR “MMSE”) AND (“age*” OR “seniors” OR “elder*”). Thereafter, reference lists were systematically searched for further relevant articles by the two reviewers (TM, AAK), who independently also abstracted the relevant information; lastly, corresponding authors were contacted for missing data. Excluded articles comprised: case reports, secondary literature reviews, analyses of unpublished data, those with overlapping or wider age range populations in which results for elderly were not exclusively presented, those comprising exclusively cognitively impaired subjects, and those solely exploring the effect of supplementation on impaired subjects.

Assessment of quality of observational studies is considered more difficult than of RCTs. In fact, there is a lack of a single obvious candidate tool for assessing quality of observational epidemiological studies and appraising observational studies with scales designed for RCTs may not be appropriate (Sanderson, Tatt, & Higgins, 2007). The “Newcastle-Ottawa Scale,” an instrument recommended by the Cochrane Non-Randomized Studies Methods Working Group, is not either designed for cross-sectional studies (Stang, 2010). Therefore, it has not been considered appropriate to arbitrarily assign a quality score to the studies included in this meta-analysis, which comprised mostly cross-sectional studies.

Statistical Analyses

ORs and CIs for cognitive impairment were extracted from each individual study, if readily available or calculated from original data on the basis of 2×2 tables [biochemical level (high or low) in cognitive impaired and nonimpaired participants]. Separate meta-analyses were run by gender and for both genders, whereas additional analyses were also conducted separately for cross-sectional and cohort studies. For homogeneity purposes the ORs and CIs of the current study represent the comparison of the 1st versus 2nd and 3rd folate or B12 tertile.

Between-study heterogeneity and between-study inconsistency were assessed by using Cochran Q statistic and by estimating I², respectively. In case of no significant heterogeneity, the fixed effects model was chosen, while random effects model was used for heterogeneous data. Evidence of publication bias was determined using Egger’s formal statistical test and by visual inspection of the funnel plot. OR calculations, pooling of individual studies through fixed or random effects models and assessment of publication bias were performed using the Comprehensive Meta-analysis v2.2, (Biostat, Englewood, NJ, USA).

Results

The “VELESTINO” Study

Table 1 shows participants’ characteristics by MMSE score class. Results in this table serve mostly descriptive purposes and cannot be directly interpretable due to correlation among variables.

Table 1.

Distribution of the 593 Study Participants by Sociodemographic Characteristics, Lifestyle Variables, GDS, Serum Folate and B12 Levels, Macrocytosis, and MMSE Score.

	Men (n = 252)					Women (n = 341)
	MMSE < 24		MMSE ≥ 24			MMSE < 24		MMSE ≥ 24
	N	%	N	%	p value	N	%	N	%	p value
Age (years)					0.04					0.0001
65-69	16	12.9	29	22.7		36	16.0	52	44.8
70-74	29	23.4	36	28.1		69	30.7	34	29.3
75-79	38	30.6	26	20.3		58	25.8	18	15.5
80+	41	33.1	37	28.9		62	27.5	12	10.4
Education (years)					0.0001					0.0001
< 6	93	75.0	54	42.2		173	76.9	68	58.6
6-8	28	22.6	52	40.6		49	21.8	41	35.4
9+	3	2.4	22	17.2		3	1.3	7	6.0
Social activity					0.003					0.35
Subnormal	34	27.4	16	12.5		111	49.3	51	44.0
Normal	90	72.6	112	87.5		114	50.7	65	56.0
Family support					0.64					0.03
Low	6	4.8	7	5.5		47	20.9	22	19.0
Medium	16	12.9	11	8.6		74	32.9	20	17.2
High	102	82.3	110	85.9		104	46.2	74	63.8
BMI (kg/m²)					0.47					0.63
< 25	26	21.0	23	18.0		27	12.0	11	9.5
25-29.9	62	50.0	58	45.3		66	29.3	36	31.0
30-34.9	27	21.8	42	32.8		82	36.5	41	35.4
35+	9	7.2	5	3.9		50	22.2	28	24.1
Smoking					0.65					0.19^d
Never	46	37.1	44	34.4		223	99.1	112	96.6
Ever	78	62.9	84	65.6		2	0.9	4	3.4
Alcohol					0.06					0.93
No drinking	23	18.5	13	10.2		117	52.0	59	50.9
Up to recommended limit	78	63.0	84	65.6		99	44.0	53	45.7
> Recommended limit	23	18.5	31	24.2		9	4.0	4	3.4
GDS score					0.0009					0.26
0-6	65	52.4	93	72.7		81	36.0	49	42.2
7+	59	47.6	35	27.3		144	64.0	67	57.8
Folate^a					0.16					0.49
1st tertile	31	35.2	25	29.4		64	37.2	25	30.1
2nd tertile	32	36.4	26	30.6		53	30.8	31	37.4
3rd tertile	25	28.4	34	40.0		55	32.0	27	32.5
B12^b					0.85					0.58
1st tertile	30	34.9	26	32.1		59	35.6	23	28.4
2nd tertile	27	31.4	28	34.6		52	31.3	32	39.5
3rd tertile	29	33.7	27	33.3		55	33.1	26	32.1
Macrocytosis^c					0.06					0.28^d
No	93	96.9	85	90.4		184	97.9	86	95.6
Yes	3	3.1	9	9.6		4	2.1	4	4.4

Note. GDS = Geriatric Depression Scale. MMSE = Mini-Mental State Examination. BMI = Body Mass Index. ^aAvailable: 173 males, 255 females. ^b167 males, 247 female. ^c190 males, 278 females. ^dp value derived from Fisher’s Exact test.

Multiple logistic regression derived adjusted ORs and 95% CIs by gender are presented in Table 2. Higher education seems to exert a statistically significant positive effect in preserving cognition in both genders (males: OR: 0.28, 95% CI: 0.17-0.45; females: OR: 0.44, 95% CI: 0.28-0.71), whereas among females, older age was significantly associated with cognitive impairment (OR: 1.96, 95% CI: 1.52-2.54). Among males, normal social activity seems to impact positively in preserving cognition (OR: 0.38, 95% CI: 0.18-0.82), in contrast to the negative effect imparted by depressive symptomatology (OR: 2.20, 95% CI: 1.21-4.02). Regarding the main variables of interest, low serum folate levels imparted a sizeable 2.4-fold negative effect (95% CI: 1.03-5.50) among males, whereas no statistically significant association was evident for B12 or macrocytosis among either gender. Results did not materially change in sensitivity subanalyses restricted to subjects with < 6 years of education or to individuals not supplemented by folate and/or B12 for either gender (data not shown).

Table 2.

Logistic Regression Derived Odds Ratio (OR) and 95% Confidence Intervals (95% CI) for Cognitive Impairment by Sociodemographic Characteristics, Lifestyle Variables, GDS, Serum Folate and B12, Macrocytosis and Gender.

		Men					Women
Variable	Category and increment	OR	95% CI			p value	OR	95% CI			p value
Core model
Age	5 years more	1.03	0.78	—	1.36	0.82	1.96	1.52	—	2.54	0.0001
Education	1 level more	0.28	0.17	—	0.45	0.0001	0.44	0.28	—	0.71	0.0006
Social activity	Normal vs. subnormal	0.38	0.18	—	0.82	0.01	1.00	0.60	—	1.66	1.00
Family support	1 level more	1.23	0.71	—	2.12	0.46	0.88	0.63	—	1.23	0.46
BMI	1 category more	0.96	0.67	—	1.37	0.82	1.14	0.87	—	1.50	0.33
Smoking	ever vs never	0.69	0.38	—	1.23	0.21	0.40	0.06	—	2.73	0.35
Alcohol	1 level more	0.89	0.55	—	1.44	0.63	1.25	0.81	—	1.93	0.31
GDS score	7+ vs. 0-6	2.20	1.21	—	4.02	0.01	0.98	0.59	—	1.64	0.95
Additional, alternatively introduced variables to the core model
Model 1^a
Folate	1st vs. 3rd tertile	2.38	1.03	—	5.50	0.04	1.03	0.49	—	2.17	0.93
	2nd vs. 3rd tertile	2.04	0.90	—	4.63	0.09	0.83	0.40	—	1.72	0.62
Model 2^b
B12	1st vs. 3rd tertile	0.93	0.41	—	2.09	0.85	0.96	0.45	—	2.03	0.91
	2nd vs. 3rd tertile	1.06	0.46	—	2.42	0.90	0.84	0.41	—	1.72	0.63
Model 3^c
Folate	1st vs. 3rd tertile	2.79	1.17	—	6.68	0.02	1.01	0.48	—	2.15	0.98
	2nd vs. 3rd tertile	2.14	0.92	—	5.01	0.08	0.83	0.39	—	1.73	0.61
B12	1st vs. 3rd tertile	0.88	0.38	—	2.05	0.77	0.94	0.44	—	2.00	0.87
	2nd vs. 3rd tertile	1.04	0.44	—	2.43	0.93	0.83	0.40	—	1.71	0.61
Model 4^d
Macrocytosis	yes vs. no	0.30	0.06	—	1.46	0.14	0.31	0.07	—	1.39	0.12

Note. OR = Odds Ratios. CI = Confidence Intervals. BMI = Body Mass Index. GDS = Geriatric Depression Scale. ^aAvailable for 173 males and 255 females. ^bavailable for 167 males and 247 females. ^cavailable for 164 males and 246 females. ^davailable for 190 males and 278 females.

Meta-analysis

Eligible Studies

As a result of the search strategy, 428 studies were deemed irrelevant, 2 studies (Clarke et al., 2008; Selhub, Morris, Jacques, & Rosenberg, 2009) were excluded on account of overlapping populations, whereas results from 5 studies (Ariogul, Cankurtaran, Dagli, Khalil, & Yavuz, 2005; Feng, Ng, Chuah, Niti, & Kua, 2006; Ravaglia et al., 2003; Ravaglia et al., 2000; Skarupski et al., 2010) could not be included due to missing data. Data was missing, in part, because there was no response from some corresponding authors following an initial and two reminder emails. Eventually, after adding the unpublished current VELESTINO study, 13 studies (Argyriadou, Vlachonikolis, Melisopoulou, Katachanakis, & Lionis, 2001; Clarke et al., 2007; Ebly, Schaefer, Campbell, & Hogan, 1998; Hin et al., 2006; Lindeman et al., 2000; Marengoni et al., 2004; Mooijaart et al., 2005; M. S. Morris, Jacques, Rosenberg, & Selhub, 2007; Ortega et al., 1996; Quadri et al., 2004; Ramos et al., 2005; Stewart, Asonganyi, & Sherwood, 2002) were included in this meta-analysis availing serum folate levels for 9,747 individuals and 10 articles with serum B12 data for 8,122 individuals (Figure 1).

Figure 1.

Flow chart of search strategy leading to included studies.

Characteristics of Eligible Studies

As shown in Tables 3 and 4, nine studies originate from Europe and four from North America. Ten out of the 13 studies used a cross-sectional design and only three were cohort studies in which baseline measurements were assessed. Original or modified MMSE was used for the assessment of cognitive impairment in all but two studies (M. S. Morris et al., 2007; Quadri et al., 2004). In nine studies the whole population was over 65 years old, while in three cases the starting age was 60 years old and in one occasion 55; the results remained essentially unchanged in sensitivity analysis, excluding the four studies with less than 65 year age inclusion criteria.

Table 3.

Low Folate Levels and Cognitive Impairment Meta-Analysis: Characteristics of Included Studies.

				Population
Study (year)	Design	Cognitive impairment definition	Low folate definition	Age Range	Area	N	Gender	Unadjusted OR (95% CI, p value) Adjusted OR (95% CI, p value)	Adjustment factors
Ortega et al (1996)	Cross-sectional	MMSE-35 ≤ 27	< 14nmol/l	65-89	Community of Madrid, Spain	166	Both	0.71 (0.40-1.27, 0.25) NA	No
Ebly et al (1998)	Cross-sectional	3MSE ≤ 77	Lowest quartile (≤ 10nmol/l)	≥ 65	Canada	1409	Both	1.71 (1.31-2.22, 0.0001) NA	No
Lindeman et al (2000)	Cross-sectional	MMSE ≤ 24	Lowest tertile (< 7.3nmol/l)	≥ 65	Bernalillo County, New Mexico, USA	380	Females	1.67 (0.98-2.86, 0.06) NA	No
			Lowest tertile (< 6.35nmol/l)			433	Males	2.39 (1.44-3.97, 0.001) NA
			Lowest tertiles for each gender			813	Both	2.01 (1.39-2.91, 0.001) NA
Argyriadou et al (2001)	Cross-sectional	Greek validated MMSE ≤ 24	< 4.85ng/ml (< 11nmol/l)	≥ 65	Northern Greece	291	Females	1.90 (1.07-3.39, 0.03) NA	No
						245	Males	2.35 (1.24-4.45, 0.01) NA
						536	Both	2.09 (1.36-3.21, 0.001) NA
Stewart et al (2002)	Cross-sectional	MMSE ≤ 25	Two lowest quartiles (≤ 4.5μg/l, ≤ 10.2nmol/l)	55-75	African-Caribbean in South London, UK	238	Both	1.16 (0.66-2.05, 0.60) NA	No
Marengoni et al (2004)	Cohort	Italian validated MMSE ≤ 23	≤ 11nmol/l	≥ 65	Division of Internal Medicine, Brescia, Italy	68	Females	2.40 (0.83-6.88, 0.11) NA	No
						12	Males	5.73 (0.23-142.55, 0.29) NA
						80	Both	2.67 (0.98-7.25, 0.055) NA
Quadri et al (2004)	Cross-sectional	CDR 0.5	< 13.5nmol/l	≥ 60	Memory Clinic, Mendrisio, Switzerland	139	Females	1.65 (0.84-3.27, 0.15) NA	No
						85	Males	2.90 (1.12-7.52, 0.03) NA
						224	Both	2.04 (1.18-3.54, 0.01) NA
Mooijaart et al (2005)	Cross-sectional	MMSE ≤ 24	≤ 11nmol/l	≥ 85	Leiden, Netherlands	371	Females	2.19 (1.42-3.37, 0.0001)	No
						188	Males	1.87 (1.01-3.46, 0.045)
						559	Both	1.98 (1.40-2.80, 0.0001)
Ramos et al (2005)	Cohort	3MSE ≤ 78	RBC folate ≤ 160ng/ml	≥ 60	Latinos, Sacramento, USA	848	Females	2.72 (0.60-12.24, 0.19) NA	No
						626	Males	0.82 (0.04-17.2, 0.90) NA
						1474	Both	1.91 (0.48-7.68, 0.36) NA
Hin et al (2006)	Cross-sectional	MMSE ≤ 21	Lowest quartile (mean level 11nmol/l)	≥ 75	Banbury, UK	1000	Both	NA 3.38 (1.31-8.71, 0.01)	Age, sex, smoking
Morris et al (2007)	Cross-sectional	Wechsler Adult Intelligence Scale III ≤ 33	≤11nmol/l	≥ 60	USA	763	Females	2.15 (1.48-3.12, 0.0001) NA	No
						538	Males	1.61 (1.08-2.39, 0.02) NA
						1301	Both	1.90 (1.45-2.49, 0.0001) NA
Clarke et al (2007)	Cohort	MMSE ≤ 23	Lowest tertile	≥ 65	Oxford, UK	1549	Both	1.31 (0.98-1.75, 0.07) NA	No
Current study	Cross-sectional	Greek validated MMS ≤ E 23	Lowest tertile (< 12.46nmol/l)	≥ 65	Velestino, Greece	255	Females	NA 1.14 (0.60-2.15, 0.69)	Age, education, social activity, family support, BMI, smoking, alcohol consumption, depressive symptoms
			Lowest tertile (< 9.97nmol/l)			173	Males	NA 1.64 (0.80-3.32, 0.17)
			Lowest tertiles for each gender			428	Both	NA 1.23 (0.78-1.93, 0.38)

Note. CI = Confidence Intervals. 3MSE = Modified Mini-Mental State Examination. CDR = Clinical Dementia Rating scale. MMSE = Mini-Mental State Examination. NA = Not Applicable. RBC folate = Red Blood Cell folate.

Table 4.

Low B12 Levels and Cognitive Impairment Meta-Analysis: Characteristics of Included Studies.

				Population
Study (year)	Design	Cognitive impairment definition	Low B12 definition	Age Range	Area	N	Unadjusted OR (95% CI, p value) Adjusted OR (95% CI, p value)	Gender	Adjustment factors
Lindeman et al. (2000)	Cross-sectional	MMSE ≤ 24	Lowest tertile (< 364pmol/l)	≥ 65	Bernalillo County, New Mexico, USA	381	Females	0.68 (0.38-1.22, 0.19) NA	No
			Lowest tertile (< 350.1pmol/l)			434	Males	1.47 (0.88-2.46, 0.14) NA
			Lowest tertiles for each gender			815	Both	1.03 (0.70-1.51, 0.88) NA
Argyriadou et al. (2001)	Cross-sectional	Greek validated MMSE ≤ 24	< 271pg/ml (< 200pmol/l)	≥ 65	Northern Greece	291	Females	1.07 (0.64-1.79, 0.81) NA	No
						245	Males	1.55 (0.85-2.83, 0.15) NA
						536	Both	1.26 (0.85-1.86, 0.25) NA
Marengoni et al. (2004)	Cohort	Italian validated MMSE ≤ 23	≤ 200pmol/l	≥ 65	Division of Internal Medicine, Brescia, Italy	199	Females	1.25 (0.63-2.49, 0.53) NA	No
						36	Males	0.40 (0.07-2.28, 0.30) NA
						235	Both	0.97 (0.52-1.79, 0.91) NA
Quadri et al. (2004)	Cross-sectional	CDR 0.5	< 234pmol/l	≥ 60	Memory Clinic, Mendrisio, Switzerland	139	Females	0.72 (0.36-1.44, 0.35) NA	No
						85	Males	0.82 (0.32-2.10, 0.68) NA
						224	Both	0.76 (0.43-1.31, 0.32) NA
Mooijaart et al. (2005)	Cross-sectional	MMSE ≤ 24	≤ 200pmol/l	≥ 85	Leiden, Netherlands	371	Females	0.71 (0.44-1.15, 0.17) NA	No
						188	Males	0.51 (0.26-1.00, 0.05) NA
						559	Both	0.62 (0.42-0.92, 0.02) NA
Ramos et al. (2005)	Cohort	3MSE ≤ 78	≤ 271pg/ml (≤ 200pmol/l)	≥ 60	Latinos, Sacramento, USA	883	Females	0.95 (0.59-1.52, 0.82) NA	No
						635	Males	1.30 (0.81-2.09, 0.27) NA
						1518	Both	1.09 (0.78-1.52, 0.61) NA
Hin et al. (2006)	Cross-sectional	MMSE ≤ 21	Lowest quartile (mean level 125pmol/l)	≥ 75	Banbury, UK	1000	Both	NA 2.17 (1.11-4.26, 0.02)	Age, sex, smoking
Morris et al. (2007)	Cross-sectional	Wechsler Adult Intelligence Scale III ≤ 33	< 200pmol/l	≥ 60	USA	764	Females	1.89 (1.32-2.71, 0.0001) NA	No
						538	Males	1.92 (1.26-2.91, 0.002) NA
						1302	Both	1.90 (1.45-2.49, 0.0001) NA
Clarke et al. (2007)	Cohort	MMSE ≤ 23	Lowest tertile	≥ 65	Oxford, UK	1549	Both	1.08 (0.81-1.45, 0.60) NA	No
Current study	Cross-sectional	Greek validated MMSE ≤ 23	Lowest tertile (< 178.7pmol/l)	≥ 65	Velestino, Greece	247	Females	NA 1.04 (0.54-2.03, 0.90)	Age, education, social activity, family support, BMI, smoking, alcohol consumption, depressive symptoms
			Lowest tertile (< 188.6pmol/l)			167	Males	NA 0.90 (0.45-1.83, 0.77)
			Lowest tertiles for each gender			414	Both	NA 1.00 (0.63-1.59, 0.99)

Note. OR = Odds Ratios. 3MSE = Modified Mini-Mental State Examination. CDR = Clinical Dementia Rating scale. MMSE = Mini-Mental State Examination. NA = Not Applicable.

Folate Status and Cognitive Impairment

A statistically significant association between low folate status and cognitive impairment in both genders combined, was observed (OR: 1.66, 95% CI: 1.40-1.96, random effects model), as well as in gender-specific analyses (males: OR: 1.95, 95% CI: 1.55-2.46, fixed effects model; females: OR: 1.90, 95% CI: 1.55-2.32, fixed effects model) (Figure 2). The results were of the same magnitude in both cross-sectional and cohort studies (both genders combined, cross-sectional studies: OR: 1.69, 95% CI: 1.40-2.04, random effects model; cohort studies: OR: 1.40, 95% CI: 1.06-1.84, fixed effects model) or in the gender-specific analyses (data not shown). Likewise, no essential changes were noted in further meta-analyses pertaining to groups of studies according to variable MMSE cutoffs and/or different folate cutoff levels.

Figure 2.

Forest plot of the meta-analyses on low folate levels and cognitive impairment.

B12 Status and Cognitive Impairment

By contrast, the positive association of low B12 status with cognitive impairment was far from reaching statistical significance either for both genders combined (OR: 1.11, 95 % CI: 0.88-1.40, random effects model) or in gender specific analyses (males: OR: 1.15, 95 % CI: 0.83-1.60; females: OR: 1.01, 95 % CI: 0.75-1.35, random effects model in both instances; Figure 3). Likewise, study design did not seem to modify the results (both genders combined, cross-sectional studies: OR: 1.13, 95 % CI: 0.80-1.60, random effects model; cohort studies: OR: 1.07, 95 % CI: 0.87-1.32, fixed effects model), nor did alternative analyses by variable MMSE cutoffs or B12 cutoff levels (data not shown).

Figure 3.

Forest plot of the meta-analyses on low B12 levels and cognitive impairment.

Discussion

The present study investigated the effect of serum folate and vitamin B12 levels on cognitive function. First, these associations were examined in a cross-sectional study with unpublished data pertaining to seniors residing in Velestino, Greece. Secondly, we synthesized the existing evidence and quantified the aforementioned associations via a meta-analysis.

The “VELESTINO” study

In the unpublished VELESTINO study, low folate levels are associated with abnormal cognitive status only in males, whereas, serum levels of vitamin B12 do not seem to be associated with cognitive function in any gender.

Sociocultural factors affecting dietary habits and variations of the duration of coexisting macrocytic state may implicate levels of B-complex vitamins. Indeed, low B-complex vitamin levels were found even among individuals under vitamin supplementation. Apart from inadequate dietary intake, poor nutritional status or age-associated decline in the intestinal absorption of B-complex vitamins may all play a part in the measured low concentrations (Hin et al., 2006).

Alcohol consumption, which affects folate metabolism, has also been accounted for and a nonstatistically significant association was found in both genders. A possible explanation could be that heavy drinking, a known risk factor for cognitive impairment, is a rather rare condition overall in this country. A recent meta-analysis pertaining exclusively to the role of alcohol reported that moderate drinking reduces the risk for Alzheimer’s disease and other forms of dementia among men (Neafsey & Collins, 2011). Findings are not conclusive, however, as other studies showed that moderate drinking may actually decrease the risk of cognitive decline or the rate of progression to dementia among older women, while these apparent benefits were not clearly seen in older men (Neafsey & Collins, 2011; Stampfer, Kang, Chen, Cherry, & Grodstein, 2005; Stott et al., 2008). On the other hand, some studies in Turkish (Ariogul et al., 2005) and Chinese (Huang, Dong, Zhang, Wu, & Liu, 2009) populations, showed that there were no significant differences among women.

The effect of other lifestyle variables, such as smoking, has been taken into account; an inverse association of borderline statistical significance was found among males (smokers: 65%) but not among females (smokers: 2%). Control for folate deficiency, a known factor confounding the association (Mannino, Mulinare, Ford, & Schwartz, 2003) has been undertaken in the study; nevertheless, it is not easy to disentangle whether this borderline association might imply a potential neuroprotective action of nicotine during aging—as it is rather the case for Parkinson’s disease (Ferrea & Winterer, 2009)—or simply reflect reverse causation. To this end, we reran the analysis by introducing the “current smoking” variable (yes: ~20%) instead of the “ever smoking” and a nonsignificant positive association emerged (OR = 1.34, p value = .42) with no essential changes of the remaining findings.

The paucity of individuals in the “lean” category (7 men and 2 women) in our study did not allow drawing any meaningful results; essentially null results were yielded with regards to the role of body structure, despite the fact that lean individuals in other studies were found to be at higher risk of cognitive decline and probable dementia (Brubacher et al., 2004; Kerwin et al., 2011).

By contrast, consistent with existing literature, a robust positive relation of GDS score with MMSE was found only among men (Lindeman et al., 2000; Ortega et al., 1996). To this end, it is worth noting imperfections of the tools assessing depressive symptoms in presence of severe cognitive impairment (MMSE < 15; Yesavage, 1986). In fact, initial cognitive impairment can possibly result in increased depressive symptoms, neurological processes can give rise to both cognitive impairment and depression, and depressive symptoms can lead to cognitive impairment (possibly through a glucocorticoid cascade; Hall, O’Bryant, Johnson, & Barber, 2011; Hinkelmann et al., 2009; Kobayashi & Kato, 2011; Marques, Silverman, & Sternberg, 2009). Thus, specific clusters of gender and disease status—namely depression only, depression and cognitive impairment, and cognitive impairment alone, among each gender—should be explored.

Despite the fact that depression symptomatology was actually slightly higher among women in the VELESTINO study (38% vs. 36%), there is a null association of GDS with MMSE among females, in contrast to the sizeable association found for men. Given the complex underlying interactions, one can only stipulate on the origin of this presumably contradictory finding. It may well be that extrinsic modifiable environmental factors exert their impact in shaping the gender differentials in either of these two pathological entities.

The country specific proxy of social activity seems to exert an independent effect also only among males, pointing to risk factors other than biological and seems to be part of a constellation of extrinsic factors associated with preservation of cognition. As a matter of fact, gender-specific leisure time patterns apply in this population, with men socializing in coffee shops and staying out of home for longer periods, whereas women seem to spend time indoors with housekeeping activities alone or cohabiting with other family members. The protective effect of increased social activity on cognition found among Velestinian men has been previously pointed out in other investigations using similar measures (James et al., 2011; Sachdev et al., 2011). These facts, along with the notably higher prevalence of cognitive decline among women, evident in a number of studies including VELESTINO, are in agreement with suggestions to customize relevant interventions for distinct subtypes in a gender-specific manner (Sachdev et al., 2011).

In addition, low education seems to be another independent risk factor for cognitive impairment, as also reported in studies conducted in both urban and rural regions of Greece (Argyriadou et al., 2001; Dimopoulos et al., 2006); nevertheless, despite the use of the validated in the Greek setting MMSE test, strong links between MMSE scores and education have been reported (Crum et al., 1993). Consequently, the interpretation of the relationship between education and MMSE-measured cognitive function must be performed with caution.

Finally, deterioration of cognition with advancement of age, in line with our findings, has been consistently reported since the early 1990s (Crum et al., 1993); It should be pointed out though that effect measurements in some studies do not reach statistically significant levels (Argyriadou et al., 2001; Mooijaart et al., 2005).

The strength of the population based VELESTINO study is that it assures high quality data derived from interviews by two physicians who were familiar with the population under investigation, their overall cognitive performance and mental health status, rather than being based merely on the assessment through the tests. In addition, gender-specific analyses were carried out with detailed mutual control for a series of biological, environmental, and social factors, which might have been associated with cognition. Potential weaknesses of the cross-sectional study include inherent limitations of the tools used, lack of homocysteine, and apolipoprotein E genotype determinations (Farlow, 2010), as well as lack of assessment for related psychological disorders, such as anxiety and stress also through biological measurements. Moreover, as cognition was only measured at one time point, it was not possible to evaluate the time-trend of cognitive decline related to aging, or the consequences of the long-term exposure to low levels of B-complex vitamins.

Meta-analysis

The current meta-analysis comprising both cross-sectional and cohort study designs on published plus an unpublished dataset from Greece, availing valid information on serum folate levels of about 10,000 subjects and B12 levels of about 8,000 subjects, shows a sizeable (OR: 1.66) and statistically significant inverse association of low folate levels on cognitive function of both gender seniors assessed through MMSE, whereas the association with B12 (OR: 1.11) was far from being statistically significant.

It is not clear whether folate deficiency may eventually exert an adverse effect on cortical neurons directly or by increasing homocysteine concentration, especially in a state of chronic low folate levels, which have been associated with white matter hyperintensities volume or with hippocampal volume shrinking (Feng et al., 2011). Homocysteine is also reported to have a neurotoxic action independent of its vascular effects by overstimulation of N-methyl-D-aspartate receptors (Lipton et al., 1997). Besides, studies using animal models demonstrate that folate deficiency may promote neuronal degeneration (Kruman et al., 2002).

B12 deficiency was not practically associated with cognitive impairment in any of the two genders in the current meta-analysis. In line with the literature, B12 is considered to account for only a small fraction of the reversible causes of dementia in older people (Malouf et al., 2003). Studies have shown that decrease in brain volume is greater among older community-dwelling adults with lower vitamin B12 and holotrasncobalamin levels, and thus the vitamin’s active form can be a potentially important modifiable risk factor for cognitive decline in the elderly. However, larger studies on brain atrophy, using sensitive vitamin B12 status markers, might be needed to support these conclusions and reveal underlying mechanisms (Vogiatzoglou et al., 2008). Control for possible confounding variables other than sex is reported in only three out of the 13 meta-analyzed studies related to the effect of folic acid and the 10 of B12.

Vitamin B12 and folate have long been studied in the literature in relation to neurological function. Low levels of these B-complex vitamins have been proposed as a modifiable risk factor for cognitive impairment, thus implying a specific direction of causation between the two variables. Nonetheless, it is worth mentioning that impaired cognitive status itself could theoretically result in the individual adopting different dietary habits and therefore affect the measured levels of these vitamins. Moreover, low measured levels of B-complex vitamins could possibly reflect vitamin malabsorption due to advanced age, or inefficient enzymatic activity attributed to aging. In such a context, it is likely that the association between B-complex vitamins and cognitive status could in fact be bidirectional.

Results of the current meta-analysis indicating a positive effect of blood serum folate levels, derive from a total of approximately 10,000 individuals, who reside mostly in Europe and North America and are similar for both genders, irrespective of the study design used. The results are more speculative with regards to a tentative association with B12. Most of the studies’ cross-sectional designs, though, may challenge the clarity as to whether any deficiencies in folate or B12 are the cause or the result of processes that lead to cognitive decline. However, intensive efforts were made to elaborate on inherent heterogeneity issues, such as unavailability of common confounding factors in the constituent studies and to contact the corresponding authors for provision of unconfounded and gender-specific estimates to the extent possible; yet the MMSE or modified versions used for assessing cognitive impairment are somewhat biased in the case of illiterate populations and when individuals suffer severe depression, which might also go unnoticed by the nonpsychiatric professionals who usually interview the study subjects.

In conclusion, this large meta-analysis confirms an adverse effect of low serum folate levels on cognitive function among both genders of elderly individuals. The underlying pathophysiological mechanisms should be further explored along with the role of B12, homocysteine, and genetic predisposition (Sachdev et al., 2011; Stephan et al., 2011). Apart from biological factors, however, the VELESTINO study points out the need to further explore the potential protective role of extrinsic factors, such as social environment, in preserving cognition and increasing quality of life among seniors.

Footnotes

Acknowledgements

The contribution of the Velestino Municipality in the planning and data collection phase is acknowledged as well as the contributions of Drs Robert D Lindeman, Stella Argyriadou, Pierluigi Quadri, Alessandra Marengoni, Anton JM de Craen, Martha S Morris, and Robert Clarke for providing original data related to their publications.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported in part by the National and Kapodistrian University of Athens and a grant of the A. S. Onassis Public Benefit Foundation on the health of Greek elderly to Prof E. Petridou.

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