ISSN 0017-8748 doi: 10.1111/head.12490 Published by Wiley Periodicals, Inc.
Headache © 2015 American Headache Society
Research Submission Effects of Dietary Folate Intake on Migraine Disability and Frequency Saras Menon, PhD; Rodney A. Lea, PhD; Sarah Ingle, MSc; Michelle Sutherland, BSc; Shirley Wee, PhD; Larisa M. Haupt, PhD; Michelle Palmer, PhD; Lyn R. Griffiths, PhD
Background.—Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The methylenetetrahydrofolate reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid (FA), vitamin B6, and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However, the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyze the association of dietary folate intake in the form of dietary folate equivalent, FA, and total food folate (TFF) on migraine frequency, severity, and disability. Methods.—A cohort of 141 adult females of Caucasian descent with MA was genotyped for the MTHFR C677T variant using restriction enzyme digestion. Dietary folate information was collected from all participants and analyzed using the “FoodWorks” 2009 package. Folate consumption was compared with migraine frequency, severity, and disability using linear regression. Results.—A significant inverse relation was observed between dietary folate equivalent (R2 = 0.201, B = −0.002, P = .045, 95% confidence interval [CI] [−0.004, −0.001]) and FA (R2 = 0.255, B = −0.005, P = .036, 95% CI [−0.009, −0.002]) consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, migraine frequency was significantly linked to FA consumption (R2 = 0.106, B = −0.004, P = .029, 95% CI [−0.007, −0.004]). Conclusions.—The results from this study indicate that folate intake in the form of FA may influence migraine frequency in female MA sufferers. Key words: migraine with aura, methylenetetrahydrofolate reductase C677T, folic acid, dietary folate equivalent, homocysteine Abbreviations: CBS cystathionin beta synthase, CVD cardiocerebrovascular disease, DFE dietary folate equivalent, FA folic acid, MA migraine with aura, MIDAS migraine disability assessment score, MO migraine without aura, MTHFR methylenetetrahydrofolate reductase, MTRR methionine synthase reductase, TFF total food folate (Headache 2015;55:301-309)
From the Genomics Research Centre, Institute for Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia (S. Menon, R.A. Lea, M. Sutherland, L.M. Haupt, and L.R. Griffiths); Griffith Health Institute, Griffith University, Gold Coast, QLD, Australia (S. Ingle, S. Wee, and M. Palmer). Address all correspondence to L.R. Griffiths, Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland University of Technology, Musk Ave, Kelvin Grove, QLD 4059, Australia, email: [email protected] Accepted for publication October 9, 2014. Conflict of Interest: None. Financial Support: This study was supported by funding from the Nutricia Research Foundation project grant and the Department of Innovation, Industry, Science and Research Funding agreement: International Science Linkages grant. R.A. Lea was supported partially by a Corbett Research Fellowship.
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302 Migraine is an extremely debilitating and highly prevalent neurovascular disorder presenting multiple symptoms.1 Migraine affects a significant proportion of the Australian population, thus placing a large economic burden on the economy.2 The International Headache Society has subdivided migraine into 2 broad categories: migraine with aura (MA) and migraine without aura (MO).3 MA has been linked to the methylenetetrahydrofolate reductase (MTHFR) gene mutation C677T.4,5 Individuals with the MTHFR C677T variant have shown diminished capacity to remethylate homocysteine to methionine, consequently inducing the increase in circulating plasma homocysteine levels.4-7 Elevated homocysteine levels have been shown to be an independent risk factor for cardiovascular disease as well as being associated with endothelial dysfunction.8,9 Although alteration in vascular function and cerebral blood flow has been identified in MA sufferers, the exact role vascular dysfunction plays in migraine pathogenesis is yet to be unravelled.10,11 The B group vitamins are crucial coenzymes in the maintenance of homocysteine homeostasis via the methylation cycle and deficiencies in these vitamins have been implicated in hyperhomocysteinemia.12 Folic acid (FA), B6, and B12 supplementation have been previously shown to reduce migraine disability, frequency, and severity in MA sufferers.6,13,14 However, the effects of dietary B vitamin consumption on migraine severity, frequency, and disability are yet to be determined. The aims of the current study were to analyze the consumption of dietary folate among a cohort of adult females with MA and to determine if there is an association among the levels of folate consumption, MTHFR genotype, migraine frequency, severity, and disability prevalence.
METHODS Study Design and Participant Group.—This study involved the secondary analysis of data collected from a randomized, double-blind, placebo-controlled clinical trial on the effects of vitamin B supplementation and MTHFR and methionine synthase reductase (MTRR) genotype on migraine disability, severity, and frequency.14 The clinical trial investigating the effects of vitamin B supplementation and
February 2015 MTHFR and MTRR genotype of migraine disability, severity, and frequency was registered with the Australian New Zealand Clinical Trial Registry and had previously obtained ethical approval from the Griffith University Ethics Committee for experimentation on human subjects. The analysis of the current study was included in the approval. The intervention period for this trial was 6 months with serum folate and plasma homocysteine levels recorded at baseline and at the end of the trial. MTHFR genotype was determined, and migraine disability and frequency were also recorded. Participants of the clinical trial were asked to complete diet diaries, which were used to analyze the composition of dietary folate intake (total food folate [TFF], FA, and dietary folate equivalents [DFE]). Pretrial data (baseline measurement) from the participants were used for the dietary analysis. The baseline folate intake of the participants was compared with the baseline measurements of plasma homocysteine, serum folate, MTHFR C677T variant, migraine severity, frequency, and disability. Patient Group.—In total, 245 Caucasian females with MA of European ancestry between the ages of 18 and 65 years were recruited from Australia and were randomly assigned either to the placebo group or to the vitamin-treated group. A detailed inclusion criterion for participant recruitment for the migraine trial is described in the study by Menon et al.14 Participants were included if they had suffered from migraine for more than 5 years and had a current diagnosis of MA (>90% of their migraine were associated with aura), and a 1-year history of severe, long-lasting attacks of MA episodes with severe headaches lasting 4-72 hours and had a family history of migraine. Participants were excluded if they were pregnant or suffered from a clinically recognized cardiovascular or neuropsychiatric condition (eg, depression and anxiety) as reported by the participant at the time of the screening process to identify their eligibility for the trial. All participants were instructed to cease all vitamin supplementation 2 weeks prior to commencement of the trial.14 Three participants dropped out before the commencement of the trial and the remaining 242 participants received baseline assessment and commenced the trial. There were 119
Headache participants in the vitamin-treated group and 123 participants in the placebo group. Of these, 44 participants were lost to follow-up because of lack of compliance, and 162 participants completed the trial (76 vitamin and 86 placebo). Diet diaries were successfully completed for 141 participants. The remaining participants either did not complete the diet diary or the diary was not successfully retrieved from the participants after the trial ended. Baseline Assessment and Genotyping.—Total fasting plasma homocysteine and serum folate concentrations were taken at baseline and at the conclusion of the 6-month trial intervention. The plasma homocysteine (μmol/L), serum folate (nmol/L), serum B6 (nmol/L), and serum B12 (pmol/L) were measured in an accredited laboratory. Genotype assessment was performed by extracting DNA from the participants’ blood and analyzed using standard techniques described in Lea et al.5 Participants were assessed for migraine disability using the Migraine Disability Assessment Score (MIDAS) Instrument, which provides a measure of productive days lost to migraine headache in the previous 3 months. Questions 6 and 7 of the MIDAS instrument were on migraine frequency and head pain severity, respectively. Participants were asked to complete a MIDAS prior to starting the trial, 3 months after starting the trial, and at the end of the 6-month intervention of the trial. Dietary Assessment Tool.—The dietary assessment tool used in this trial was designed to focus on foods that were rich sources of folate, >200 μg/100 g according to the Nutrient Tables for Use in Australia 2006 Composition Table and was created to document the subject’s daily food consumption, with an emphasis on folate consumption. A day consisted of a breakfast, lunch, dinner, and snack section. Participants were instructed to indicate what they ate at each corresponding meal time and to write down “other food” items that they had consumed that were not on the list of the foods given in the diet diary provided. Subjects were advised that one serve of food was equal to 100 g and were instructed to indicate if fruits and vegetable consumed were cooked (fried, boiled, baked) or raw. Participants were advised to complete one diary before commencing
303 the trial and one diary for every fortnight of the trial duration. Diet Diary Analysis.—Analysis of estimated dietary folate intake was performed using the nutrient analysis software FoodWorks Professional (Version 6, 2009, Xyris Software, Brisbane, QLD, Australia). The TFF, FA, and DFE content of each diet diary completed by each participant was analyzed. In instances where participants did not specify cooking techniques, an unspecified option (Not Specified – NFS) was always selected. If serve sizes were not specified, a “Standard” serve size was selected as specified by FoodWorks. In instances where FoodWorks did not have a specific food item in its database, the closest possible option was selected. All assumptions were made to be as consistent as possible. Diet diary data were excluded if folate intake was not reported within 2 weeks of relevant biomarker measurements. Assessment of Folate Intake.—Total folate intakes were computed using data from the diet diary. The bioavailability of food folate is lower than that of FA added to fortified foods and dietary supplements. The DFE conversion of TFF was therefore used to account for differential bioavailability. In Australia, the National Health and Medical Research Council (NHMRC, 2006) calculates DFE as:
DFE = ( Food Folate μg ) + ( Folic acid μg × 1.67) . DFE, TFF, and FA were used in the analysis of folate intake in this study. Statistical Analysis.—All analyses were performed using the Statistical Package for Social Sciences (SPSS version 21.0, IBM, Sydney, NSW, Australia). Average dietary folate (TFF, FA, and DFE), serum folate, and plasma homocysteine concentration means were determined by descriptive analysis. The relationship between migraine clinical variables (migraine frequency, MIDAS, and migraine pain severity), biochemical variables (serum folate, serum B6, B12, and plasma folate), and dietary folate consumption (DFE, FF, and TFF) was analyzed using Pearson’s correlation test. Linear regression analysis was performed using the “Enter” method, to determine the predictors of migraine frequency, severity, and disability. All tests
304 were two-tailed. Significance was defined as P < .05 level.
RESULTS Diet diaries were successfully collected from 141 participants who were then genotyped for the MTHFR C677T polymorphism. In the current study, 181 participants had the C allele, and 101 participants had the T allele for the MTHFR C677T variant (MTHFR C677T genotype frequency- CC: 63, CT:55, TT: 23). Biomarker correlations were only performed on diet diaries that were completed within 2 weeks of blood tests. The dietary folate intake of the participants calculated using FoodWorks was 551.78 ± 19.7 DFE and well above the required daily intake (RDI) of 400 μg DFE. The ratio (%) of individuals who consumed more than RDI of DFE 400 μg were 70.2% (n = 99). The DFE intake ranges from 161 μg to 1286 μg. The mean homocysteine level for the population tested in this study was 11.65 μmol/L. A positive Pearson’s correlation was found between DFE consumption and serum folate (r = 0.209, P = .019). An inverse positive correlation was also observed between DFE consumption and migraine frequency (r = −0.200, P = .024). MIDAS was positively correlated to migraine pain severity (r = 0.175, P = .039). An inverse significant relation was also observed between serum folate levels and plasma homocysteine levels (r = −0.276, P = .002). Regression analysis was used to test if folate consumption and the MTHFR genotype significantly predicted serum folate levels. The results of the regression indicated that DFE consumption predicted 4.4% of the variance (R2 = 0.044, P = .019, 95% confidence interval [CI] [−0.002, −0.20]), FA consumption predicted 3.7% of the variance (R2 = 0.037, P = .059, 95% CI [−0.01, −0.020]), and TFF consumption predicted 2.6% of the variance in serum folate levels (R2 = 0.026, P = .073, 95% CI [−0.001, 0.023]). Folate consumption models were also run including MTHFR genotype as a possible confounding factor in a multivariate analysis; however, the MTHFR genotype was not a significant contributor for serum folate levels. Univariate analysis was performed on variables: B6, B12, serum folate, and the MTHFR C677T geno-
February 2015 type to determine significant predictors of plasma homocysteine levels. Serum B12 (R2 = 0.053, CI [−0.011, −0.002] P = .007) and serum folate (R2 = 0.165, CI [−0.222, −0.048] P = .003] levels were significant factors in determining plasma homocysteine levels. However, when multivariate analysis was performed including all variables in regression using the “Enter” method, only serum folate was found to be a significant factor of plasma homocysteine levels (R2 = 0.173, B = −0.122, P = .063, 95% CI [−0.219, −0.024]). Folate consumption (DFE, FA, and TFF) was not significantly related to plasma homocysteine levels (P > .05). When folate consumption and migraine outcomes were tested in univariate regression analysis, it was observed that DFE consumption (R2 = 0.040, P = .024, CI [−0.002, −0.002]) and in particular FA consumption (R2 = 0.080, P = .004, CI [−0.006, −0.001]) was significantly related to migraine frequency (Table 1). In multiple regression analysis, DFE (R2 = 0.201, B = −0.002, P = .045, 95% CI [−0.004, −0.001]) and FA (R2 = 0.255, B = −0.005, P = .036, 95% CI [−0.005, −0.001]) consumption adjusted for B6, B12, plasma homocysteine levels, and the MTHFR genotype were significantly related to migraine frequency (Figs. 1 and 2). Serum folate levels adjusted for serum B6 and B12, plasma homocysteine levels, and the MTHFR genotype were not significantly related to migraine frequency (R2 = 0.025, P = .956, CI [−0.04, 0.031]).
Table 1.—Folate Consumption Predictors of Migraine Frequency in Univariate Regression Analysis
95% CI
Model
DFE FA TFF
R2
B
P
Lower
Higher
0.040 0.080 0.024
−0.001 −0.004 −22.915
.024* .004* .080
−0.002 −0.006 −48.818
−0.002 −0.001 2.989
*Significance was taken at P ≤ .05. Univariate regression analysis of folate consumption and migraine frequency showed significant links between DFE and FA consumption and migraine frequency. 95% CI = 95% confidence interval; DFE = dietary folate equivalent; FA = folic acid; TFF = total food folate.
Headache
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10.00
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y= 2.31 + -8.78E – 4*X
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2.00
2.00
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250.00
500.00
750.00
y= 2.25 + -3.38E – 3*X
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1000.00 1250.00
100.00
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Fig 1.—A scatterplot showing the relationship between dietary folate equivalents (DFE) consumption and migraine frequency. A regression analysis with the “Enter” method between independent (B6, B12, plasma homocysteine levels, DFE consumption, and methylenetetrahydrofolate reductase [MTHFR] C677T genotype) was performed on migraine frequency data. The analysis revealed that DFE consumption significantly affects migraine frequency.
Fig 2.—A scatterplot showing the relationship between folic acid (FA) consumption and migraine frequency. A regression analysis with the “Enter” method between independent variables (B6, B12, plasma homocysteine levels, FA consumption, and methylenetetrahydrofolate reductase [MTHFR] C677T genotype) was performed on migraine frequency data. The analysis revealed that FA consumption significantly affects migraine frequency.
When data were stratified by the MTHFR genotype, it was observed that T allele carriers had consumed the most amounts of DFE (561.3 μg) and TFF (486.83 μg), but the least amount of FA (139.8 μg) compared with the CC genotype carriers (DFE: 541.3 μg, TFF: 455.13 μg, FA: 161.62 μg). It was also observed that the T allele carriers had higher plasma homocysteine levels (12.0 μmol/L) and lower serum B12 (309.03 pmol/L) and serum folate levels (29.1 nmol/L) compared with the CC genotype carriers (homocysteine: 11.4 μmol/L, B12: 314.36 pmol/L, serum folate: 30.3.nmol/L). The percentage of T allele carriers experiencing an average of 3 to 6 migraines in 3 months was 32.4% compared with 24.5% in the CC genotype carriers. Linear regression did not observe a significant relationship between MTHFR C677T genotype and biochemical variables (P > .05) or migraine outcomes (P > .05). Further regression analysis observed that in individuals with the CC genotype, migraine frequency was significantly inversely related to FA consumption (R2 = 0.106, B = −0.0004, P = .029, CI [−0.007, −0.004)]) (Table 2).
Table 2.—Linear Regression Analysis Exploring the Influence of Folate Consumption on Migraine Frequency, When Stratified by the MTHFR C677T Genotype
95% CI
Model
CC
CT
TT
DFE FA TFF DFE FA TFF DFE FA TFF
R2
P
B
Lower
Higher
0.058 0.106 0.036 0.047 0.278 0.034 0.002 0.124 0.003
.069 .029* .156 .137 .078 .202 .872 .687 .812
−0.001 −0.004 −0.001 −0.001 −0.004 −0.001 0.0002 0.003 0.003
−0.002 −0.007 −0.003 −0.003 −0.009 −0.004 −0.002 −0.011 −0.003
0.0001 −0.0004 0.0005 0.0004 0.0005 0.001 0.003 0.016 0.003
*Significance was taken at P ≤ .05. Linear regression analysis of folate consumption and migraine frequency, stratified by the methylenetetrahydrofolate reductase gene variant C677T (MTHFR C677T) showed that in individuals with the CC genotype, migraine frequency is inversely related to FA consumption. 95% CI = 95% confidence interval; DFE = dietary folate equivalent; FA = folic acid; TFF = total food folate.
306 Univariate analysis and multivariate analysis adjusted for B6, B12, plasma homocysteine levels, and the MTHFR C677T genotype did not identify a significant relationship between folate consumption (DFE, FA, TFF) or serum folate levels and migraineassociated disabilities such MIDAS and migraine pain severity scores (P > .05).
DISCUSSION A possible interplay between genetic and environmental factors has been the basis of migraine pathophysiology hypothesized to date.15 Recent studies have reported a higher incidence of the co-occurrence of ischemic cardiocerebrovascular disease (CVD) and migraine.16-20 This incidence seems to be increased in MA sufferers.21 Elevated plasma Hcy levels have been reported to increase the risk of atherosclerotic vasculopathy and act as an independent risk factor for CVD.22 An increase in total Hcy concentration has also been recently reported in migraineurs, specifically in those with MA.23 It is plausible that homocysteine levels may be involved in both MA and CVD pathophysiology. Reasons for hyperhomocysteinemia may include mutations in homocysteine metabolizing genes such as MTHFR, MTRR, and cystathionin beta synthase (CBS), or possible nutritional deficiencies in cofactors involved in homocysteine metabolism.12 The current study investigated the association between dietary folate consumption, MTHFR genotype, and migraine-associated disability in a cohort of female MA sufferers. This may be the only study of its kind to analyze food folate consumption by examining folate consumption in 3 different components: TFF, fortified FA content of foods, and DFE in food consumed in migraineurs. The results of this study observed a significant link between increasing serum folate levels and increasing DFE consumption, and this significance most probably contributed to the total consumption of FA more than TFF. However, the results also show that DFE consumption and FA consumption only account for 4.4% and 3.7%, respectively, of serum folate levels. Serum folate levels were significantly correlated to decreasing plasma homocysteine levels as expected in the
February 2015 current study; however, there was no significant link observed between serum folate levels and migraine frequency, MIDAS, or pain severity. Folate mainly taken up by developing erythrocytes reflects timeintegrated intake and is therefore considered to be a measure of long-term folate status, whereas serum folate predominantly reflects transitory changes in folate concentration.24 Red blood cell folate may have provided a more accurate measurement of folate levels in the participants of this trial compared with serum folate measurement. Other influencing factors of serum folate levels are the differing acute post-absorptive plasma kinetics of folate and the challenges in accurately determining dietary folate bioavailability.25 In the migraine population tested in the current study, a significant correlation between folate consumption and plasma homocysteine levels was not observed. This finding is contrary to many previous studies that have observed a significant inverse relationship between folate consumption and homocysteine levels in plasma.26-28 The “FoodWorks” software utilized in this trial did not have several food items that were popular among the participants of this trial. The closest food match was selected, for example, “Raw spinach” was selected in place of rocket. This may have affected folate consumption measurement as food substitutes used may not have been the most accurate comparisons. Second, the lack of confounding factors analyzed in the current trial may have also influenced the lack of association observed between plasma homocysteine levels and dietary folate intake. The study did not collect demographic information such as participant smoking habits, blood pressure, or blood cholesterol levels. Dyslipidemia, hypertension, and smoking have been shown to affect serum folate and plasma homocysteine levels.29 A major finding of this study is the significant inverse relation observed between DFE and, more specifically, FA consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, increasing levels of FA consumption significantly related to decreasing migraine frequency. T allele carriers who had, on average, consumed less FA than CC genotype
Headache carriers showed no correlation between FA consumption and migraine frequency. Although the current study did not find a significant correlation between the MTHFR C677T genotype and migraine frequency or homocysteine levels, it was observed that the T allele carriers, albeit by a small margin, had higher migraine frequency and plasma homocysteine levels compared with individuals with the CC genotype for the MTHFR C677T variant. Naturally occurring folate found in food is present in the reduced, polyglutamated form with methyl or formyl as the one carbon substitution.30,31 FA found in fortified food and supplements is a synthetic, fully oxidized monoglutamate form of folate and is reduced to tetrahydrofolate prior to its participation in a metabolic reaction.32 Relative to naturally found folate, FA has a higher bioavailability, which is defined as the proportion of ingested folate that is absorbed and can be used for metabolic processes.33 Although there is a broad consensus that FA is more bioavailable than naturally found food folate, there are some intervention trials with food that have reported similar improvements in folate status compared with equimolar or small doses of FA from fortified foods or supplements.34-36 Previous clinical trials by Lea et al and Menon et al have provided evidence that homocysteine lowering by FA supplementation can reduce migraine disability, and that this effect may be modified by polymorphisms in genes coding for key enzymes that affect folate metabolism, such as the MTHFR genotype.13,14 T allele carriers of the MTHFR C677T genotype with a 35-70% reduction in their enzymatic rate are genetically slower in homocysteine metabolism.37 The findings of the current study, as well as those from previous migraine trials, suggest that individuals who carry the T allele for the MTHFR C677T variant may require an increase in folate consumption, specifically the more bioavailable FA,38,39 to experience reduced migraine frequency and possibly other associated symptoms as well.13,14 Certain limitations of this study include the small participant sample size, which may not offer a good representation of the female Caucasian population suffering from MA and their dietary folate levels. It
307 should also be noted that the dietary information from the migraineurs of the current study was also obtained prior to the implementation of the mandatory folate fortification in Australia. Mandatory folate fortification was implemented in Australia in September 2009. Brown et al reported a significant increase in serum folate levels between prefortification folate serum concentrations, and serum folate concentrations taken approximately 6 months postfortification.40 A recent study by Dugbaza et al that investigated the levels of total dietary FA intake following the mandatory FA fortification reported an increase of 2.5 times in FA consumption in the Australian adult population.41 Among females with low intake of folate (represented by the tenth percentile of intake), mandatory fortification has increased their estimated intake from less than 5% to 75% of the recommended amount.41 The premise of mandatory folate fortification policy was to reduce the incidence of neural tube defects; however, secondary health benefits may result, such as the potential role of decreasing homocysteine concentrations in reducing cardiovascular disease as well as migraine frequency and disability. A long-term follow-up population study is necessary to investigate if mandatory folate fortification results in a decrease in migraine incidence in Australia and if it successfully translates to a decrease in migraine-associated disability (work place absenteeism due to migraine) and positively impacts on the financial burden of migraine in the country’s economy.
CONCLUSION An increase in dietary folate, especially the more bioavailable FA consumption levels, may effectively reduce migraine frequency in female migraineurs in the investigated study. If this is a causative relationship, then the T allele carriers of the MTHFR C677T variant may need higher levels of FA compared with CC genotype carriers to experience a significant reduction in their migraine frequency. Acknowledgments: We would like to acknowledge all participants who consented and volunteered to be involved in this study.
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STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths (b) Acquisition of Data Saras Menon; Sarah Ingle; Michelle Sutherland; Shirley Wee (c) Analysis and Interpretation of Data Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths Category 2 (a) Drafting the Manuscript Saras Menon; Rodney A. Lea; Lyn R. Griffiths (b) Revising It for Intellectual Content Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths Category 3 (a) Final Approval of the Completed Manuscript Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths
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February 2015 5. Lea RA, Ovcaric M, Sundholm J, MacMillan J, Griffiths LR. The methylenetetrahydrofolate reductase gene variant C677T influences susceptibility to migraine with aura. BMC Med. 2004;12:2-3. 6. Di Rosa G, Attina S, Spano M, et al. Efficacy of folic acid in children with migraine, hyperhomocysteinemia and MTHFR polymorphisms. Headache. 2007;47:1342-1344. 7. Kara I, Sazci A, Ergul E, Kaya G, Kilic G. Association of the C677T and A1298C polymorphisms in the 5,10 methylenetetrahydrofolate reductase gene in patients with migraine risk. Brain Res Mol Brain Res. 2003;111:84-90. 8. Aguilar B, Rojas JC, Collados MT. Metabolism of homocysteine and its relationship with cardiovascular disease. J Thromb Thrombolysis. 2004;18:7587. 9. Klerk M, Verhoef P, Clarke R, Blom HJ, Kok FJ, Schouten EG. MTHFR 677C-->T polymorphism and risk of coronary heart disease: A meta-analysis. JAMA. 2002;288:2023-2031. 10. Moschiano F, D’Amico D, Usai S, et al. Homocysteine plasma levels in patients with migraine with aura. Neurol Sci. 2008;29(Suppl. 1):S173-S175. 11. Johnson ES. A basis for migraine therapy – the autonomic theory reappraised. Postgrad Med J. 1978; 54:231-243. 12. Silaste ML, Rantala M, Sampi M, Alfthan G, Aro A, Kesaniemi YA. Polymorphisms of key enzymes in homocysteine metabolism affect diet responsiveness of plasma homocysteine in healthy women. J Nutr. 2001;131:2643-2647. 13. Lea R, Colson N, Quinlan S, Macmillan J, Griffiths L. The effects of vitamin supplementation and MTHFR (C677T) genotype on homocysteinelowering and migraine disability. Pharmacogenet Genomics. 2009;19:422-428. 14. Menon S, Lea RA, Roy B, et al. Genotypes of the MTHFR C677T and MTRR A66G genes act independently to reduce migraine disability in response to vitamin supplementation. Pharmacogenet Genomics. 2012;22:741-749. 15. Mulder EJ, Van Baal C, Gaist D, et al. Genetic and environmental influences on migraine: A twin study across six countries. Twin Res. 2003;6:422-431. 16. Gudmundsson LS, Scher AI, Aspelund T, et al. Migraine with aura and risk of cardiovascular and all cause mortality in men and women: Prospective cohort study. BMJ. 2010;341:c3966.
Headache 17. Kurth T, Diener HC, Buring JE. Migraine and cardiovascular disease in women and the role of aspirin: Subgroup analyses in the Women’s Health Study. Cephalalgia. 2011;31:1106-1115. 18. Rockett FC, Perla Ada S, Perry ID, Chaves ML. Cardiovascular disease risk in women with migraine. J Headache Pain. 2013;14:75. 19. Schürks M, Rist PM, Bigal ME, Buring JE, Lipton RB, Kurth T. Migraine and cardiovascular disease: Systematic review and meta-analysis. BMJ. 2009;339:b3914. 20. Sinclair AJ, Matharu M. Migraine, cerebrovascular disease and the metabolic syndrome. Ann Indian Acad Neurol. 2012;15(Suppl. 1):S72-S77. 21. MacClellan LR, Giles W, Cole J, et al. Probable migraine with visual aura and risk of ischemic stroke: The stroke prevention in young women study. Stroke. 2007;38:2438-2445. 22. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA. 1995;274:1049-1057. 23. Isobe C, Terayama Y. A remarkable increase in total homocysteine concentrations in the CSF of migraine patients with aura. Headache. 2010;50:1561-1569. 24. Devalia V, Hamilton MS, Molloy AM. Guidelines on the investigation and diagnosis of cobalamin and folate deficiencies. A publication of the British Committee for Standards in Haematology. BCSH General Haematology Test Force. Clin Lab Haematol. 1994;16:101-115. 25. Ohrvik VE, Witthoft CM. Human folate bioavailability. Nutrients. 2011;3:475-490. 26. Parkins KJ, Poets CF, O’Brien LM, Stebbens VA, Southall DP. Lowering blood homocysteine with folic acid based supplements: Meta-analysis of randomised trials. Homocysteine Lowering Trialists’ Collaboration. BMJ. 1998;316:894-898. 27. Wald DS, Bishop L, Wald NJ, et al. Randomized trial of folic acid supplementation and serum homocysteine levels. Arch Intern Med. 2001;161:695700. 28. Ward M, McNulty H, McPartlin J, Strain JJ, Weir DG, Scott JM. Plasma homocysteine, a risk factor for cardiovascular disease, is lowered by physiological doses of folic acid. QJM. 1997;90:519-524. 29. El Oudi M, Aouni Z, Ouertani H, Mazigh C, Machghoul S. Effect of lipopenic and hypotensive
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treatment on homocysteine levels in type 2 diabetics. Vasc Health Risk Manag. 2010;25;6:327-332. Perry J, Chanarin I. Intestinal absorption of reduced folate compounds in man. Br J Haematol. 1970; 18:329-339. Scott JM, Weir DG. Folate composition, synthesis and function in natural materials. Clin Haematol. 1976;5:547-568. Futterman S. Enzymatic reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid. J Biol Chem. 1957;228:1031-1038. Caudill MA. Folate bioavailability: Implications for establishing dietary recommendations and optimizing status. Am J Clin Nutr. 2010;91:1455S-60S. Fenech M, Noakes M, Clifton P, Topping D. Aleurone flour increases red-cell folate and lowers plasma homocyst(e)ine substantially in man. Br J Nutr. 2005;93:353-360. Vahteristo L, Kariluoto S, Barlund S, et al. Functionality of endogenous folates from rye and orange juice using human in vivo model. Eur J Nutr. 2002; 41:271-278. Winkels RM, Brouwer IA, Siebelink E, Katan MB, Verhoef P. Bioavailability of food folates is 80% of that of folic acid. Am J Clin Nutr. 2007;85:465-473. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111-113. Ashfield-Watt PA, Pullin CH, Whiting JM, et al. Methylenetetrahydrofolate reductase 677C-->T genotype modulates homocysteine responses to a folate-rich diet or a low-dose folic acid supplement: A randomized controlled trial. Am J Clin Nutr. 2002;76:180-186. Hickling S, Knuiman M, Jamrozik K, Hung J. A rapid dietary assessment tool to determine intake of folate was developed and validated. J Clin Epidemiol. 2005;58:802-808. Brown RD, Langshaw MR, Uhr EJ, Gibson JN, Joshua DE. The impact of mandatory fortification of flour with folic acid on the blood folate levels of an Australian population. Med J Aust. 2011;194: 65-67. Dugbaza J, Cunningham J. Estimates of total dietary folic acid intake in the Australian population following mandatory folic acid fortification of bread. J Nutr Metab. 2012;2012:492353. doi: 10.1155/2012/492353. Epub 2012 Aug 23.
Headache © 2015 American Headache Society
Research Submission Effects of Dietary Folate Intake on Migraine Disability and Frequency Saras Menon, PhD; Rodney A. Lea, PhD; Sarah Ingle, MSc; Michelle Sutherland, BSc; Shirley Wee, PhD; Larisa M. Haupt, PhD; Michelle Palmer, PhD; Lyn R. Griffiths, PhD
Background.—Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The methylenetetrahydrofolate reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid (FA), vitamin B6, and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However, the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyze the association of dietary folate intake in the form of dietary folate equivalent, FA, and total food folate (TFF) on migraine frequency, severity, and disability. Methods.—A cohort of 141 adult females of Caucasian descent with MA was genotyped for the MTHFR C677T variant using restriction enzyme digestion. Dietary folate information was collected from all participants and analyzed using the “FoodWorks” 2009 package. Folate consumption was compared with migraine frequency, severity, and disability using linear regression. Results.—A significant inverse relation was observed between dietary folate equivalent (R2 = 0.201, B = −0.002, P = .045, 95% confidence interval [CI] [−0.004, −0.001]) and FA (R2 = 0.255, B = −0.005, P = .036, 95% CI [−0.009, −0.002]) consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, migraine frequency was significantly linked to FA consumption (R2 = 0.106, B = −0.004, P = .029, 95% CI [−0.007, −0.004]). Conclusions.—The results from this study indicate that folate intake in the form of FA may influence migraine frequency in female MA sufferers. Key words: migraine with aura, methylenetetrahydrofolate reductase C677T, folic acid, dietary folate equivalent, homocysteine Abbreviations: CBS cystathionin beta synthase, CVD cardiocerebrovascular disease, DFE dietary folate equivalent, FA folic acid, MA migraine with aura, MIDAS migraine disability assessment score, MO migraine without aura, MTHFR methylenetetrahydrofolate reductase, MTRR methionine synthase reductase, TFF total food folate (Headache 2015;55:301-309)
From the Genomics Research Centre, Institute for Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia (S. Menon, R.A. Lea, M. Sutherland, L.M. Haupt, and L.R. Griffiths); Griffith Health Institute, Griffith University, Gold Coast, QLD, Australia (S. Ingle, S. Wee, and M. Palmer). Address all correspondence to L.R. Griffiths, Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland University of Technology, Musk Ave, Kelvin Grove, QLD 4059, Australia, email: [email protected] Accepted for publication October 9, 2014. Conflict of Interest: None. Financial Support: This study was supported by funding from the Nutricia Research Foundation project grant and the Department of Innovation, Industry, Science and Research Funding agreement: International Science Linkages grant. R.A. Lea was supported partially by a Corbett Research Fellowship.
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302 Migraine is an extremely debilitating and highly prevalent neurovascular disorder presenting multiple symptoms.1 Migraine affects a significant proportion of the Australian population, thus placing a large economic burden on the economy.2 The International Headache Society has subdivided migraine into 2 broad categories: migraine with aura (MA) and migraine without aura (MO).3 MA has been linked to the methylenetetrahydrofolate reductase (MTHFR) gene mutation C677T.4,5 Individuals with the MTHFR C677T variant have shown diminished capacity to remethylate homocysteine to methionine, consequently inducing the increase in circulating plasma homocysteine levels.4-7 Elevated homocysteine levels have been shown to be an independent risk factor for cardiovascular disease as well as being associated with endothelial dysfunction.8,9 Although alteration in vascular function and cerebral blood flow has been identified in MA sufferers, the exact role vascular dysfunction plays in migraine pathogenesis is yet to be unravelled.10,11 The B group vitamins are crucial coenzymes in the maintenance of homocysteine homeostasis via the methylation cycle and deficiencies in these vitamins have been implicated in hyperhomocysteinemia.12 Folic acid (FA), B6, and B12 supplementation have been previously shown to reduce migraine disability, frequency, and severity in MA sufferers.6,13,14 However, the effects of dietary B vitamin consumption on migraine severity, frequency, and disability are yet to be determined. The aims of the current study were to analyze the consumption of dietary folate among a cohort of adult females with MA and to determine if there is an association among the levels of folate consumption, MTHFR genotype, migraine frequency, severity, and disability prevalence.
METHODS Study Design and Participant Group.—This study involved the secondary analysis of data collected from a randomized, double-blind, placebo-controlled clinical trial on the effects of vitamin B supplementation and MTHFR and methionine synthase reductase (MTRR) genotype on migraine disability, severity, and frequency.14 The clinical trial investigating the effects of vitamin B supplementation and
February 2015 MTHFR and MTRR genotype of migraine disability, severity, and frequency was registered with the Australian New Zealand Clinical Trial Registry and had previously obtained ethical approval from the Griffith University Ethics Committee for experimentation on human subjects. The analysis of the current study was included in the approval. The intervention period for this trial was 6 months with serum folate and plasma homocysteine levels recorded at baseline and at the end of the trial. MTHFR genotype was determined, and migraine disability and frequency were also recorded. Participants of the clinical trial were asked to complete diet diaries, which were used to analyze the composition of dietary folate intake (total food folate [TFF], FA, and dietary folate equivalents [DFE]). Pretrial data (baseline measurement) from the participants were used for the dietary analysis. The baseline folate intake of the participants was compared with the baseline measurements of plasma homocysteine, serum folate, MTHFR C677T variant, migraine severity, frequency, and disability. Patient Group.—In total, 245 Caucasian females with MA of European ancestry between the ages of 18 and 65 years were recruited from Australia and were randomly assigned either to the placebo group or to the vitamin-treated group. A detailed inclusion criterion for participant recruitment for the migraine trial is described in the study by Menon et al.14 Participants were included if they had suffered from migraine for more than 5 years and had a current diagnosis of MA (>90% of their migraine were associated with aura), and a 1-year history of severe, long-lasting attacks of MA episodes with severe headaches lasting 4-72 hours and had a family history of migraine. Participants were excluded if they were pregnant or suffered from a clinically recognized cardiovascular or neuropsychiatric condition (eg, depression and anxiety) as reported by the participant at the time of the screening process to identify their eligibility for the trial. All participants were instructed to cease all vitamin supplementation 2 weeks prior to commencement of the trial.14 Three participants dropped out before the commencement of the trial and the remaining 242 participants received baseline assessment and commenced the trial. There were 119
Headache participants in the vitamin-treated group and 123 participants in the placebo group. Of these, 44 participants were lost to follow-up because of lack of compliance, and 162 participants completed the trial (76 vitamin and 86 placebo). Diet diaries were successfully completed for 141 participants. The remaining participants either did not complete the diet diary or the diary was not successfully retrieved from the participants after the trial ended. Baseline Assessment and Genotyping.—Total fasting plasma homocysteine and serum folate concentrations were taken at baseline and at the conclusion of the 6-month trial intervention. The plasma homocysteine (μmol/L), serum folate (nmol/L), serum B6 (nmol/L), and serum B12 (pmol/L) were measured in an accredited laboratory. Genotype assessment was performed by extracting DNA from the participants’ blood and analyzed using standard techniques described in Lea et al.5 Participants were assessed for migraine disability using the Migraine Disability Assessment Score (MIDAS) Instrument, which provides a measure of productive days lost to migraine headache in the previous 3 months. Questions 6 and 7 of the MIDAS instrument were on migraine frequency and head pain severity, respectively. Participants were asked to complete a MIDAS prior to starting the trial, 3 months after starting the trial, and at the end of the 6-month intervention of the trial. Dietary Assessment Tool.—The dietary assessment tool used in this trial was designed to focus on foods that were rich sources of folate, >200 μg/100 g according to the Nutrient Tables for Use in Australia 2006 Composition Table and was created to document the subject’s daily food consumption, with an emphasis on folate consumption. A day consisted of a breakfast, lunch, dinner, and snack section. Participants were instructed to indicate what they ate at each corresponding meal time and to write down “other food” items that they had consumed that were not on the list of the foods given in the diet diary provided. Subjects were advised that one serve of food was equal to 100 g and were instructed to indicate if fruits and vegetable consumed were cooked (fried, boiled, baked) or raw. Participants were advised to complete one diary before commencing
303 the trial and one diary for every fortnight of the trial duration. Diet Diary Analysis.—Analysis of estimated dietary folate intake was performed using the nutrient analysis software FoodWorks Professional (Version 6, 2009, Xyris Software, Brisbane, QLD, Australia). The TFF, FA, and DFE content of each diet diary completed by each participant was analyzed. In instances where participants did not specify cooking techniques, an unspecified option (Not Specified – NFS) was always selected. If serve sizes were not specified, a “Standard” serve size was selected as specified by FoodWorks. In instances where FoodWorks did not have a specific food item in its database, the closest possible option was selected. All assumptions were made to be as consistent as possible. Diet diary data were excluded if folate intake was not reported within 2 weeks of relevant biomarker measurements. Assessment of Folate Intake.—Total folate intakes were computed using data from the diet diary. The bioavailability of food folate is lower than that of FA added to fortified foods and dietary supplements. The DFE conversion of TFF was therefore used to account for differential bioavailability. In Australia, the National Health and Medical Research Council (NHMRC, 2006) calculates DFE as:
DFE = ( Food Folate μg ) + ( Folic acid μg × 1.67) . DFE, TFF, and FA were used in the analysis of folate intake in this study. Statistical Analysis.—All analyses were performed using the Statistical Package for Social Sciences (SPSS version 21.0, IBM, Sydney, NSW, Australia). Average dietary folate (TFF, FA, and DFE), serum folate, and plasma homocysteine concentration means were determined by descriptive analysis. The relationship between migraine clinical variables (migraine frequency, MIDAS, and migraine pain severity), biochemical variables (serum folate, serum B6, B12, and plasma folate), and dietary folate consumption (DFE, FF, and TFF) was analyzed using Pearson’s correlation test. Linear regression analysis was performed using the “Enter” method, to determine the predictors of migraine frequency, severity, and disability. All tests
304 were two-tailed. Significance was defined as P < .05 level.
RESULTS Diet diaries were successfully collected from 141 participants who were then genotyped for the MTHFR C677T polymorphism. In the current study, 181 participants had the C allele, and 101 participants had the T allele for the MTHFR C677T variant (MTHFR C677T genotype frequency- CC: 63, CT:55, TT: 23). Biomarker correlations were only performed on diet diaries that were completed within 2 weeks of blood tests. The dietary folate intake of the participants calculated using FoodWorks was 551.78 ± 19.7 DFE and well above the required daily intake (RDI) of 400 μg DFE. The ratio (%) of individuals who consumed more than RDI of DFE 400 μg were 70.2% (n = 99). The DFE intake ranges from 161 μg to 1286 μg. The mean homocysteine level for the population tested in this study was 11.65 μmol/L. A positive Pearson’s correlation was found between DFE consumption and serum folate (r = 0.209, P = .019). An inverse positive correlation was also observed between DFE consumption and migraine frequency (r = −0.200, P = .024). MIDAS was positively correlated to migraine pain severity (r = 0.175, P = .039). An inverse significant relation was also observed between serum folate levels and plasma homocysteine levels (r = −0.276, P = .002). Regression analysis was used to test if folate consumption and the MTHFR genotype significantly predicted serum folate levels. The results of the regression indicated that DFE consumption predicted 4.4% of the variance (R2 = 0.044, P = .019, 95% confidence interval [CI] [−0.002, −0.20]), FA consumption predicted 3.7% of the variance (R2 = 0.037, P = .059, 95% CI [−0.01, −0.020]), and TFF consumption predicted 2.6% of the variance in serum folate levels (R2 = 0.026, P = .073, 95% CI [−0.001, 0.023]). Folate consumption models were also run including MTHFR genotype as a possible confounding factor in a multivariate analysis; however, the MTHFR genotype was not a significant contributor for serum folate levels. Univariate analysis was performed on variables: B6, B12, serum folate, and the MTHFR C677T geno-
February 2015 type to determine significant predictors of plasma homocysteine levels. Serum B12 (R2 = 0.053, CI [−0.011, −0.002] P = .007) and serum folate (R2 = 0.165, CI [−0.222, −0.048] P = .003] levels were significant factors in determining plasma homocysteine levels. However, when multivariate analysis was performed including all variables in regression using the “Enter” method, only serum folate was found to be a significant factor of plasma homocysteine levels (R2 = 0.173, B = −0.122, P = .063, 95% CI [−0.219, −0.024]). Folate consumption (DFE, FA, and TFF) was not significantly related to plasma homocysteine levels (P > .05). When folate consumption and migraine outcomes were tested in univariate regression analysis, it was observed that DFE consumption (R2 = 0.040, P = .024, CI [−0.002, −0.002]) and in particular FA consumption (R2 = 0.080, P = .004, CI [−0.006, −0.001]) was significantly related to migraine frequency (Table 1). In multiple regression analysis, DFE (R2 = 0.201, B = −0.002, P = .045, 95% CI [−0.004, −0.001]) and FA (R2 = 0.255, B = −0.005, P = .036, 95% CI [−0.005, −0.001]) consumption adjusted for B6, B12, plasma homocysteine levels, and the MTHFR genotype were significantly related to migraine frequency (Figs. 1 and 2). Serum folate levels adjusted for serum B6 and B12, plasma homocysteine levels, and the MTHFR genotype were not significantly related to migraine frequency (R2 = 0.025, P = .956, CI [−0.04, 0.031]).
Table 1.—Folate Consumption Predictors of Migraine Frequency in Univariate Regression Analysis
95% CI
Model
DFE FA TFF
R2
B
P
Lower
Higher
0.040 0.080 0.024
−0.001 −0.004 −22.915
.024* .004* .080
−0.002 −0.006 −48.818
−0.002 −0.001 2.989
*Significance was taken at P ≤ .05. Univariate regression analysis of folate consumption and migraine frequency showed significant links between DFE and FA consumption and migraine frequency. 95% CI = 95% confidence interval; DFE = dietary folate equivalent; FA = folic acid; TFF = total food folate.
Headache
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y= 2.31 + -8.78E – 4*X
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750.00
y= 2.25 + -3.38E – 3*X
.00
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Fig 1.—A scatterplot showing the relationship between dietary folate equivalents (DFE) consumption and migraine frequency. A regression analysis with the “Enter” method between independent (B6, B12, plasma homocysteine levels, DFE consumption, and methylenetetrahydrofolate reductase [MTHFR] C677T genotype) was performed on migraine frequency data. The analysis revealed that DFE consumption significantly affects migraine frequency.
Fig 2.—A scatterplot showing the relationship between folic acid (FA) consumption and migraine frequency. A regression analysis with the “Enter” method between independent variables (B6, B12, plasma homocysteine levels, FA consumption, and methylenetetrahydrofolate reductase [MTHFR] C677T genotype) was performed on migraine frequency data. The analysis revealed that FA consumption significantly affects migraine frequency.
When data were stratified by the MTHFR genotype, it was observed that T allele carriers had consumed the most amounts of DFE (561.3 μg) and TFF (486.83 μg), but the least amount of FA (139.8 μg) compared with the CC genotype carriers (DFE: 541.3 μg, TFF: 455.13 μg, FA: 161.62 μg). It was also observed that the T allele carriers had higher plasma homocysteine levels (12.0 μmol/L) and lower serum B12 (309.03 pmol/L) and serum folate levels (29.1 nmol/L) compared with the CC genotype carriers (homocysteine: 11.4 μmol/L, B12: 314.36 pmol/L, serum folate: 30.3.nmol/L). The percentage of T allele carriers experiencing an average of 3 to 6 migraines in 3 months was 32.4% compared with 24.5% in the CC genotype carriers. Linear regression did not observe a significant relationship between MTHFR C677T genotype and biochemical variables (P > .05) or migraine outcomes (P > .05). Further regression analysis observed that in individuals with the CC genotype, migraine frequency was significantly inversely related to FA consumption (R2 = 0.106, B = −0.0004, P = .029, CI [−0.007, −0.004)]) (Table 2).
Table 2.—Linear Regression Analysis Exploring the Influence of Folate Consumption on Migraine Frequency, When Stratified by the MTHFR C677T Genotype
95% CI
Model
CC
CT
TT
DFE FA TFF DFE FA TFF DFE FA TFF
R2
P
B
Lower
Higher
0.058 0.106 0.036 0.047 0.278 0.034 0.002 0.124 0.003
.069 .029* .156 .137 .078 .202 .872 .687 .812
−0.001 −0.004 −0.001 −0.001 −0.004 −0.001 0.0002 0.003 0.003
−0.002 −0.007 −0.003 −0.003 −0.009 −0.004 −0.002 −0.011 −0.003
0.0001 −0.0004 0.0005 0.0004 0.0005 0.001 0.003 0.016 0.003
*Significance was taken at P ≤ .05. Linear regression analysis of folate consumption and migraine frequency, stratified by the methylenetetrahydrofolate reductase gene variant C677T (MTHFR C677T) showed that in individuals with the CC genotype, migraine frequency is inversely related to FA consumption. 95% CI = 95% confidence interval; DFE = dietary folate equivalent; FA = folic acid; TFF = total food folate.
306 Univariate analysis and multivariate analysis adjusted for B6, B12, plasma homocysteine levels, and the MTHFR C677T genotype did not identify a significant relationship between folate consumption (DFE, FA, TFF) or serum folate levels and migraineassociated disabilities such MIDAS and migraine pain severity scores (P > .05).
DISCUSSION A possible interplay between genetic and environmental factors has been the basis of migraine pathophysiology hypothesized to date.15 Recent studies have reported a higher incidence of the co-occurrence of ischemic cardiocerebrovascular disease (CVD) and migraine.16-20 This incidence seems to be increased in MA sufferers.21 Elevated plasma Hcy levels have been reported to increase the risk of atherosclerotic vasculopathy and act as an independent risk factor for CVD.22 An increase in total Hcy concentration has also been recently reported in migraineurs, specifically in those with MA.23 It is plausible that homocysteine levels may be involved in both MA and CVD pathophysiology. Reasons for hyperhomocysteinemia may include mutations in homocysteine metabolizing genes such as MTHFR, MTRR, and cystathionin beta synthase (CBS), or possible nutritional deficiencies in cofactors involved in homocysteine metabolism.12 The current study investigated the association between dietary folate consumption, MTHFR genotype, and migraine-associated disability in a cohort of female MA sufferers. This may be the only study of its kind to analyze food folate consumption by examining folate consumption in 3 different components: TFF, fortified FA content of foods, and DFE in food consumed in migraineurs. The results of this study observed a significant link between increasing serum folate levels and increasing DFE consumption, and this significance most probably contributed to the total consumption of FA more than TFF. However, the results also show that DFE consumption and FA consumption only account for 4.4% and 3.7%, respectively, of serum folate levels. Serum folate levels were significantly correlated to decreasing plasma homocysteine levels as expected in the
February 2015 current study; however, there was no significant link observed between serum folate levels and migraine frequency, MIDAS, or pain severity. Folate mainly taken up by developing erythrocytes reflects timeintegrated intake and is therefore considered to be a measure of long-term folate status, whereas serum folate predominantly reflects transitory changes in folate concentration.24 Red blood cell folate may have provided a more accurate measurement of folate levels in the participants of this trial compared with serum folate measurement. Other influencing factors of serum folate levels are the differing acute post-absorptive plasma kinetics of folate and the challenges in accurately determining dietary folate bioavailability.25 In the migraine population tested in the current study, a significant correlation between folate consumption and plasma homocysteine levels was not observed. This finding is contrary to many previous studies that have observed a significant inverse relationship between folate consumption and homocysteine levels in plasma.26-28 The “FoodWorks” software utilized in this trial did not have several food items that were popular among the participants of this trial. The closest food match was selected, for example, “Raw spinach” was selected in place of rocket. This may have affected folate consumption measurement as food substitutes used may not have been the most accurate comparisons. Second, the lack of confounding factors analyzed in the current trial may have also influenced the lack of association observed between plasma homocysteine levels and dietary folate intake. The study did not collect demographic information such as participant smoking habits, blood pressure, or blood cholesterol levels. Dyslipidemia, hypertension, and smoking have been shown to affect serum folate and plasma homocysteine levels.29 A major finding of this study is the significant inverse relation observed between DFE and, more specifically, FA consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, increasing levels of FA consumption significantly related to decreasing migraine frequency. T allele carriers who had, on average, consumed less FA than CC genotype
Headache carriers showed no correlation between FA consumption and migraine frequency. Although the current study did not find a significant correlation between the MTHFR C677T genotype and migraine frequency or homocysteine levels, it was observed that the T allele carriers, albeit by a small margin, had higher migraine frequency and plasma homocysteine levels compared with individuals with the CC genotype for the MTHFR C677T variant. Naturally occurring folate found in food is present in the reduced, polyglutamated form with methyl or formyl as the one carbon substitution.30,31 FA found in fortified food and supplements is a synthetic, fully oxidized monoglutamate form of folate and is reduced to tetrahydrofolate prior to its participation in a metabolic reaction.32 Relative to naturally found folate, FA has a higher bioavailability, which is defined as the proportion of ingested folate that is absorbed and can be used for metabolic processes.33 Although there is a broad consensus that FA is more bioavailable than naturally found food folate, there are some intervention trials with food that have reported similar improvements in folate status compared with equimolar or small doses of FA from fortified foods or supplements.34-36 Previous clinical trials by Lea et al and Menon et al have provided evidence that homocysteine lowering by FA supplementation can reduce migraine disability, and that this effect may be modified by polymorphisms in genes coding for key enzymes that affect folate metabolism, such as the MTHFR genotype.13,14 T allele carriers of the MTHFR C677T genotype with a 35-70% reduction in their enzymatic rate are genetically slower in homocysteine metabolism.37 The findings of the current study, as well as those from previous migraine trials, suggest that individuals who carry the T allele for the MTHFR C677T variant may require an increase in folate consumption, specifically the more bioavailable FA,38,39 to experience reduced migraine frequency and possibly other associated symptoms as well.13,14 Certain limitations of this study include the small participant sample size, which may not offer a good representation of the female Caucasian population suffering from MA and their dietary folate levels. It
307 should also be noted that the dietary information from the migraineurs of the current study was also obtained prior to the implementation of the mandatory folate fortification in Australia. Mandatory folate fortification was implemented in Australia in September 2009. Brown et al reported a significant increase in serum folate levels between prefortification folate serum concentrations, and serum folate concentrations taken approximately 6 months postfortification.40 A recent study by Dugbaza et al that investigated the levels of total dietary FA intake following the mandatory FA fortification reported an increase of 2.5 times in FA consumption in the Australian adult population.41 Among females with low intake of folate (represented by the tenth percentile of intake), mandatory fortification has increased their estimated intake from less than 5% to 75% of the recommended amount.41 The premise of mandatory folate fortification policy was to reduce the incidence of neural tube defects; however, secondary health benefits may result, such as the potential role of decreasing homocysteine concentrations in reducing cardiovascular disease as well as migraine frequency and disability. A long-term follow-up population study is necessary to investigate if mandatory folate fortification results in a decrease in migraine incidence in Australia and if it successfully translates to a decrease in migraine-associated disability (work place absenteeism due to migraine) and positively impacts on the financial burden of migraine in the country’s economy.
CONCLUSION An increase in dietary folate, especially the more bioavailable FA consumption levels, may effectively reduce migraine frequency in female migraineurs in the investigated study. If this is a causative relationship, then the T allele carriers of the MTHFR C677T variant may need higher levels of FA compared with CC genotype carriers to experience a significant reduction in their migraine frequency. Acknowledgments: We would like to acknowledge all participants who consented and volunteered to be involved in this study.
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STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths (b) Acquisition of Data Saras Menon; Sarah Ingle; Michelle Sutherland; Shirley Wee (c) Analysis and Interpretation of Data Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths Category 2 (a) Drafting the Manuscript Saras Menon; Rodney A. Lea; Lyn R. Griffiths (b) Revising It for Intellectual Content Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths Category 3 (a) Final Approval of the Completed Manuscript Saras Menon; Rodney A. Lea; Sarah Ingle; Michelle Sutherland; Shirley Wee; Larisa M. Haupt; Michelle Palmer; Lyn R. Griffiths
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