Petra Verhoef

Effect of 3-year folic acid supplementation on cognitive function in older adults in the FACIT trial: a randomised double blind, controlled trial

BACKGROUND Low folate and raised homocysteine concentrations in blood are associated with poor cognitive performance in the general population. As part of the FACIT trial to assess the effect of folic acid on markers of atherosclerosis in men and women aged 50-70 years with raised plasma total homocysteine and normal serum vitamin B12 at screening, we report here the findings for the secondary endpoint: the effect of folic acid supplementation on cognitive performance. METHODS Our randomised, double blind, placebo controlled study took place between November, 1999, and December, 2004, in the Netherlands. We randomly assigned 818 participants 800 mug daily oral folic acid or placebo for 3 years. The effect on cognitive performance was measured as the difference between the two groups in the 3-year change in performance for memory, sensorimotor speed, complex speed, information processing speed, and word fluency. Analysis was by intention-to-treat. This trial is registered with clinicaltrials.gov with trial number NCT00110604. FINDINGS Serum folate concentrations increased by 576% (95% CI 539 to 614) and plasma total homocysteine concentrations decreased by 26% (24 to 28) in participants taking folic acid compared with those taking placebo. The 3-year change in memory (difference in Z scores 0.132, 95% CI 0.032 to 0.233), information processing speed (0.087, 0.016 to 0.158) and sensorimotor speed (0.064, -0.001 to 0.129) were significantly better in the folic acid group than in the placebo group. INTERPRETATION Folic acid supplementation for 3 years significantly improved domains of cognitive function that tend to decline with age.

A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, the 677C→T and 1298A→C variants, to MTHFR activity, homocysteine concentrations, and risk of CVD in a population of 190 vascular disease patients and 601 apparently healthy controls. The mean specific and residual MTHFR activities were significantly lower in 677CT and 677TT individuals (both P<0.001). The 1298A→C mutation alone showed no effect on MTHFR activities. However, when the 677C→T genotype was taken into account, the 1298A→C mutation also caused a significant decrease in MTHFR activities, which was observed in both the homozygous 1298CC (P<0.001) and the heterozygous 1298AC states (P=0.005). Both the 677TT as the 677CT genotypes were associated with significantly higher fasting and postload homocysteine levels than 677CC (P<0.001 and P=0.003, respectively). The 1298A→C mutation had no effect on fasting or postload homocysteine levels. Since homocysteine itself is considered to be positively associated with the risk of CVD, these findings indicate that the 1298A→C mutation cannot be considered a major risk factor for CVD.

The 677C->T mutation in the methylenetetrahydrofolate reductase gene: associations with plasma total homocysteine levels and risk of coronary atherosclerotic disease

Homozygosity for a 677C-->T mutation at the locus that codes for 5,10-methylenetetrahydrofolate reductase (MTHFR), a folate-dependent crucial enzyme in homocysteine metabolism, may render the enzyme thermolabile and less active and has been associated with increased levels of plasma total homocysteine (tHcy). We assessed whether this mutation was associated with increased risk of coronary atherosclerosis and plasma levels of tHcy and furthermore studied whether folate status would modify the associations. Data were collected from subjects with substantial coronary atherosclerosis (> or = 90% occlusion in one and > or = 40% occlusion in a second coronary artery, referred to as cases, n = 131) or virtually no coronary narrowing (referred to as coronary controls, n = 87) and from a population-based control group (n = 100), all residing in the Rotterdam area, The Netherlands. Both males and females, aged 25-65 years were studied. The frequency of homozygosity for the mutation (+/+) in cases (10.0%) did not significantly differ statistically from that observed in coronary controls (11.5%, P = 0.71), population-based controls (7.0%, P = 0.43), or combined control groups (9.1%, P = 0.80). In the overall group (as well as in the three subgroups), plasma tHcy levels, fasting and to a lesser extent after a methionine-loading test, were higher in +/+ subjects than in homozygous normal subjects (-/-), whereas heterozygous subjects (+/-) had intermediate levels (Ptrend = 0.001). The +/+ subjects with erythrocyte folate levels < 790 nmol/l (population median) had a 77%, (95% CI, 27-144%) higher geometric mean fasting tHcy (21.4, micromol/l) than those with higher erythrocyte folate (12.1 micromol/l). The odds ratio (OR) of coronary atherosclerosis for +/+ subjects, with +/- and -/- subjects as the reference group, in analyses with combined control groups, was 1.1 (95% CI, 0.5-2.4). The ORs were 2.2 (95% CI, 0.7-6.8) and 0.6 (95% CI, 0.2-1.7) among subjects with low and high folate levels, respectively. Our study indicates that homozygosity for the 677C-->T MTHFR mutation, especially in combination with low folate status, predisposes to high plasma levels of fasting tHcy. However, homozygosity for this mutation, whether or not in combination with low folate status, was not associated with increased risk of coronary artery disease.

Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias

Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.

Effects of Folic Acid Supplementation on Hearing in Older Adults: A Randomized, Controlled Trial

Context Epidemiologic evidence suggests an association between folate and homocysteine levels and hearing status. Contributions The investigators randomly assigned 728 older adults with high homocysteine levels to receive daily folic acid or placebo for 3 years. They found that hearing thresholds for low frequencies increased more slowly in participants taking folic acid, suggesting a protective effect. Cautions The trial was conducted in the Netherlands at a time when folate fortification of food was prohibited. Implications Folic acid supplementation seemed to slow the decline of low-frequency hearing in folate-deficient, older adults. The Editors Hearing loss is one of the most common chronic conditions of elderly persons, and age-related hearing loss is the most common type of hearing impairment (1). Age-related hearing loss refers to a sensorineural hearing loss due to aging and other physiologic, environmental, and pathologic processes that occur throughout the lifespan (2). Factors that vary with age, such as the increase in plasma total homocysteine concentrations, may play an etiologic role in age-related hearing loss. Folate is an important dietary determinant of plasma total homocysteine concentrations (3, 4), and folic acid supplementation can lower those levels by approximately 25% (5). Recent research has focused on the link between folate metabolism and sensorineural hearing loss (613). Two studies have examined the link between folate and age-related hearing loss. The first epidemiologic study found that high concentrations of erythrocyte folate (r = 0.37; P= 0.01) and serum folate (r = 0.36; P= 0.01) were associated with lower hearing thresholds and, thus, with better hearing in 55 women who had age-related hearing loss (7). The explanations given by the authors included the role of folate-related homocysteine lowering in cellular metabolism or in the nervous and vascular systems. However, the association of increased folate concentrations with better hearing was not confirmed in a second epidemiologic study of 91 audiologic patients with suspected age-related hearing loss (8). We investigated whether daily oral folic acid supplementation (800 g) for 3 years improved hearing thresholds compared with placebo in 728 older adults with age-related hearing loss. Methods Participants The study participants were 819 men and postmenopausal women 50 to 70 years of age from the Gelderland province in the Netherlands who were participating in the Folic Acid and Carotid Intima-media Thickness trial, a study investigating whether folic acid supplementation retards atherosclerotic progression. Additional outcomes were age-related decline in cognitive function and hearing. We recruited participants by using municipal and local blood bank registries. Assuming that high concentrations of plasma total homocysteine were risk factors, we selected participants who were expected to benefit from the homocysteine-lowering effect of folic acid. We excluded participants with plasma total homocysteine levels less than 13 mol/L (73rd percentile of those screened) or greater than 26 mol/L. We also excluded participants with elevated homocysteine concentrations that were possibly due to factors other than suboptimal folate concentrations, including serum vitamin B12 concentration less than 200 pmol/L (10th percentile of those screened [vitamin B12 concentrations >160 pmol/L indicated deficiency]), self-reported diagnosis of renal or thyroid disease, or self-reported use of medications that influence folate metabolism (14). In addition, we excluded participants with self-reported intestinal disease or terminal cancer and participants who reportedly used vitamin B supplements or medications that may influence atherosclerotic progression. In addition to using the eligibility criteria related to homocysteine metabolism, we excluded participants with hearing problems that were unlikely to be due to age-related hearing loss. Middle ear dysfunction and unilateral hearing loss are pathologic ear conditions that are not related to aging. We excluded 91 participants because of middle ear dysfunction (defined as airbone gap 15 dB on the audiogram in either ear) or unilateral hearing loss (20-dB difference in average pure-tone hearing thresholds for 0.5 kHz, 1 kHz, and 2 kHz between the right and left ear) (13). Finally, we required self-reported adherence of 80% or more during a 6-week placebo run-in period (Figure). Figure. Study flow diagram. The flowchart shows the flow of participants through the recruitment phases (November 1999 to April 2001) and the study (September 2000 to December 2004). *Participants may have been excluded for several reasons. The medical ethics committee of Wageningen University, Wageningen, the Netherlands, approved the study, and participants gave written informed consent. Randomization, Blinding, and Adherence After the initial measurement sessions, we allocated participants to folic acid (800 g/d) or placebo. When we designed the study, 800 g/d folic acid was considered a low dosage for a clinical trial. We allocated the sequence of study entry to either treatment by using permuted blocks with randomly varied block sizes of 4 and 6. Specialized personnel who were not involved in the study allocated and labeled the capsule boxes with the participants unique sequence numbers. Forty-eight participants had at least one other household member in the study. The second member of the same household received the same treatment as their previously randomly assigned partner. The capsules, produced by Swiss-Caps Benelux (Heerhugowaard, the Netherlands), were indistinguishable in appearance. Capsules were individually packaged in foil strips containing 28 capsules per strip, with the days of the week printed on the back. Participants received a 13-month supply of capsules each year. We judged adherence by counting returned capsules and reviewing a diary that registered missed capsules, which were both returned by participants every 12 weeks. At the end of the study, the proportion of participants who thought that they received the folic acid or placebo capsule did not differ significantly between the 2 groups (P= 0.26). All personnel, including the authors, were blinded to group assignment until completion of the trial and subsequent data analyses. Audiometry If present, excessive cerumen was removed from participants ears. We obtained audiometric assessments with participants seated in an acoustical booth (Audiofon G, Audiovox, Hauppauge, New York) by using an audiometer (Madsen Voyager 522, Madsen Electronics, Taastrup, Denmark) and circumaural earphones. At baseline and the final measurements, we calibrated the audiometer according to the International Organization of Standardization (ISO) standard 389 (15). The acoustical booth muted sounds up to 42 dB, but was not calibrated according to ISO standards, and was placed in a quiet room (a small, isolated, carpeted room above the college library). We performed audiometric testing by using a variation of the Hughson and Westlake (16) method: thresholds based on ascending responses using 5-dB steps up and 10-dB steps down (16). We measured pure-tone air conduction hearing thresholds at 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, 6 kHz, and 8 kHz. If the difference between the right and left ear was 50 dB or more for the air conduction hearing level, then we used contralateral masking to determine the air conduction hearing thresholds. To calculate airbone gaps for the exclusion of participants with possible middle ear dysfunction at the start of the study, we measured bone conduction hearing thresholds at 0.5 kHz, 1 kHz, 2 kHz, and 4 kHz by using contralateral masking. Other Measurements Elevated concentrations of plasma total homocysteine may be a consequence of low blood levels of B vitamins involved in one-carbon metabolism, alone or in combination with genetic polymorphisms, such as methylenetetrahydrofolate reductase (MTHFR) 677CT, or impaired renal function (14). We measured these determinants of fasting plasma total homocysteine concentrations in venous blood that was immediately processed and stored at 80C. We measured serum folate, erythrocyte folate, and serum vitamin B12 concentrations by using a chemiluminescent immunoassay (Immulite 2000, Diagnostic Products Corp., Los Angeles, California). We determined the plasma total homocysteine level with high-performance liquid chromatography (HPLC) and fluorometric detection (17). Plasma vitamin B6 concentration was also measured by HPLC (18). We determined serum creatinine and lipid levels by using the Hitachi 747 (Roche Diagnostics, Basel, Switzerland). We determined the MTHFR C677T genotype by polymerase chain reaction of DNA and restriction digestion with HinFl (19). We ascertained self-reported medical history, including current drug use and smoking habits, by questionnaire, and a research assistant reviewed the information with the participants. Education was grouped according to the highest attained level (20). We measured height and weight and calculated body mass index. We measured blood pressure by using an automated meter (Dinamap Compact Pro 100, GE Healthcare, Waukesha, Wisconsin) and used the average of 8 measurements. We used a food frequency questionnaire to measure folate intake in the previous 3 months in the elderly patients. The questionnaire asked about the intake of foods that contribute at least 80% of the average folate intake as determined in an analysis of the second Dutch Food Consumption Survey for the subpopulation of men and women 50 to 70 years of age (21). The crude Spearman correlation coefficient between serum folate concentrations and folate intake estimated by the questionnaire was 0.16 (P> 0.01), which was within the range found by other investigators (correlation coefficient range, 0.13 to 0.34). Genotyping and attained educational level were measured at the beginning of the study, and a

Effects of Betaine Intake on Plasma Homocysteine Concentrations and Consequences for Health

High plasma concentrations of homocysteine may increase risk of cardiovascular disease. Folic acid lowers plasma homocysteine by 25% maximally, because 5-methyltetrahydrofolate is a methyl donor in the remethylation of homocysteine to methionine. Betaine (trimethylglycine) is also a methyl donor in homocysteine remethylation, but effects on homocysteine have been less thoroughly investigated. Betaine in high doses (6 g/d and higher) is used as homocysteine-lowering therapy for people with hyperhomocysteinemia due to inborn errors in the homocysteine metabolism. Betaine intake from foods is estimated at 0.5-2 g/d. Betaine can also be synthesized endogenously from its precursor choline. Studies in healthy volunteers with plasma homocysteine concentrations in the normal range show that betaine supplementation lowers plasma fasting homocysteine dose-dependently to up to 20% for a dose of 6 g/d of betaine. Moreover, betaine acutely reduces the increase in homocysteine after methionine loading by up to 50%, whereas folic acid has no effect. Betaine doses in the range of dietary intake also lower homocysteine. This implies that betaine can be an important food component that attenuates homocysteine rises after meals. If homocysteine plays a causal role in the development of cardiovascular disease, a diet rich in betaine or choline might benefit cardiovascular health through its homocysteine-lowering effects. However betaine and choline may adversely affect serum lipid concentrations, which can of course increase risk of cardiovascular disease. However, whether the potential beneficial health effects of betaine and choline outweigh the possible adverse effects on serum lipids is as yet unclear.

Prospective study on dietary intakes of folate, betaine, and choline and cardiovascular disease risk in women

Objective:To investigate the association between dietary intakes of folate, betaine and choline and the risk of cardiovascular disease (CVD).Design:Prospective cohort study.Subjects:A total of 16 165 women aged 49–70 years without prior CVD. Subjects were breast cancer screening participants in the PROSPECT–EPIC cohort, which is 1 of the 2 Dutch contributions to the European Prospective Investigation into Cancer and Nutrition (EPIC).Methods:Each participant completed a validated food frequency questionnaire. Folate intake was calculated with the Dutch National Food Database. Betaine and choline intakes were calculated with the USDA database containing choline and betaine contents of common US foods. Data on coronary heart disease (CHD) events and cerebrovascular accident (CVA) events morbidity data were obtained from the Dutch Centre for Health Care Information.Results:During a median follow-up period of 97 months, 717 women were diagnosed with CVD. After adjustment, neither folate, nor betaine, nor choline intakes were associated with CVD (hazard ratios for highest versus lowest quartile were 1.23 (95% confidence interval 0.75; 2.01), 0.90 (0.69; 1.17), 1.04 (0.71; 1.53), respectively). In a subsample of the population, high folate and choline intakes were statistically significantly associated with lower homocysteine levels. High betaine intake was associated with slightly lower high-density lipoprotein (HDL)-cholesterol concentrations.Conclusion:Regular dietary intakes of folate, betaine and choline were not associated with CVD risk in post-menopausal Dutch women. However, the effect of doses of betaine and choline beyond regular dietary intake – for example, via supplementation or fortification – remains unknown.

Vitamins B6, B12, and Folate: Association with Plasma Total Homocysteine and Risk of Coronary Atherosclerosis

OBJECTIVES To investigate the association of status of vitamins B6, B12 and folate with plasma fasting total homocysteine (tHcy) and with risk of coronary atherosclerosis; and to establish whether associations between vitamins and risk of coronary atherosclerosis are mediated by tHcy. METHODS The study population consisted of 131 patients with angiography-defined severe coronary atherosclerosis and 88 referents with no or minor coronary stenosis. Previous analyses in this study population have shown that fasting tHcy is an independent risk factor for coronary atherosclerosis. In the present analyses, using multiple linear regression, we estimated differences in tHcy concentrations between subjects in the lowest and highest quartiles of concentrations of each of the vitamins, adjusting for age, gender, total:HDL cholesterol ratio, smoking habits, alcohol intake, blood pressure, serum creatinine, body mass index and the two other vitamins. We used logistic regression analysis conditional on the set of potential confounders described above to study the association between vitamin concentration and risk of coronary atherosclerosis. By comparing these estimated odds ratios (ORs) with those that were additionally adjusted for fasting tHcy, we determined whether the vitamins exerted their effects on disease risk via homocysteine metabolism. RESULTS Cases who were in the upper quartile of serum vitamin B12 and erythrocyte folate concentrations showed statistically significantly lower tHcy concentrations (-4.00 and -4.71 mumol/L, respectively) than those in the lowest quartile. Referents in the upper quartile of plasma B6 showed significantly lower tHcy concentrations (-2.36 mumol/L) than referents in the lowest quartile. Subjects in the lowest quartile of vitamin B12 concentrations had higher risk of coronary atherosclerosis (OR: 2.91; 95% CI: 1.10, 7.71) compared to those in the highest quartile. The ORs and 95% CIs for low B6 and low folate were 0.86 (95% CI: 0.33, 2.22) and 0.58 (95% CI: 0.23, 1.48), respectively. Additional adjustment for fasting tHcy weakened associations, although data indicated that low vitamin B12 concentration is a risk factor for coronary atherosclerosis, independently of tHcy. CONCLUSION The presently accepted view that vitamin B6 mainly affects tHcy after methionine loading, and not fasting tHcy, is contradicted by our findings in referents. Low vitamin B12 concentrations were associated with an increased risk of coronary atherosclerosis, partly independently of tHcy. Although low folate status was a strong determinant of elevated tHcy concentrations, it was not associated with increased risk of coronary atherosclerosis.

Effect of Homocysteine-Lowering Nutrients on Blood Lipids: Results from Four Randomised, Placebo-Controlled Studies in Healthy Humans

Background Betaine (trimethylglycine) lowers plasma homocysteine, a possible risk factor for cardiovascular disease. However, studies in renal patients and in obese individuals who are on a weight-loss diet suggest that betaine supplementation raises blood cholesterol; data in healthy individuals are lacking. Such an effect on cholesterol would counteract any favourable effect on homocysteine. We therefore investigated the effect of betaine, of its precursor choline in the form of phosphatidylcholine, and of the classical homocysteine-lowering vitamin folic acid on blood lipid concentrations in healthy humans. Methods and Findings We measured blood lipids in four placebo-controlled, randomised intervention studies that examined the effect of betaine (three studies, n = 151), folic acid (two studies, n = 75), and phosphatidylcholine (one study, n = 26) on plasma homocysteine concentrations. We combined blood lipid data from the individual studies and calculated a weighted mean change in blood lipid concentrations relative to placebo. Betaine supplementation (6 g/d) for 6 wk increased blood LDL cholesterol concentrations by 0.36 mmol/l (95% confidence interval: 0.25–0.46), and triacylglycerol concentrations by 0.14 mmol/l (0.04–0.23) relative to placebo. The ratio of total to HDL cholesterol increased by 0.23 (0.14–0.32). Concentrations of HDL cholesterol were not affected. Doses of betaine lower than 6 g/d also raised LDL cholesterol, but these changes were not statistically significant. Further, the effect of betaine on LDL cholesterol was already evident after 2 wk of intervention. Phosphatidylcholine supplementation (providing approximately 2.6 g/d of choline) for 2 wk increased triacylglycerol concentrations by 0.14 mmol/l (0.06–0.21), but did not affect cholesterol concentrations. Folic acid supplementation (0.8 mg/d) had no effect on lipid concentrations. Conclusions Betaine supplementation increased blood LDL cholesterol and triacylglycerol concentrations in healthy humans, which agrees with the limited previous data. The adverse effects on blood lipids may undo the potential benefits for cardiovascular health of betaine supplementation through homocysteine lowering. In our study phosphatidylcholine supplementation slightly increased triacylglycerol concentrations in healthy humans. Previous studies of phosphatidylcholine and blood lipids showed no clear effect. Thus the effect of phosphatidylcholine supplementation on blood lipids remains inconclusive, but is probably not large. Folic acid supplementation does not seem to affect blood lipids and therefore remains the preferred treatment for lowering of blood homocysteine concentrations.

Folate and the methylenetetrahydrofolate reductase 677C-->T mutation correlate with cognitive performance

Low folate status has been associated with cognitive decline. We investigated the association of folate status and the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism with performance on a battery of neuropsychological tests. Furthermore, we investigated whether the association of folate with cognitive performance was mediated by plasma homocysteine or risk of vascular disease. We used cross-sectional data from 818 individuals aged 50-70 years old. Low concentrations of erythrocyte folate but not serum folate were associated with poor performance on complex speed and memory tasks, independent of educational level and conventional risk factors of vascular disease. These associations were not mediated by homocysteine concentrations or carotid intima-media thickness. Subjects with the MTHFR 677TT genotype tended to perform better on cognitive tasks than CC/CT subjects, although this was significant for sensorimotor speed only (differences in Z-scores between MTHFR 677TT homozygotes and CC homozygotes -0.15, 95% CI: -0.30 to 0.00). Low concentrations of erythrocyte folate are associated with decreased cognitive performance, possibly through a homocysteine-independent mechanism such as DNA infidelity and mitochondrial decay.