B Vitamin and/or ω-3 Fatty Acid Supplementation and Cancer

Randomized Trial of Folic Acid Supplementationand Serum Homocysteine LevelsDavid S. Wald, MRCP; Lucy Bishop, MRCP; Nicholas J. Wald, DSc(Med); Malcolm Law, FRCP;Enid Hennessy, MSc; Donald Weir, FRCP; Joe McPartlin, PhD; John Scott, ScD

Background: Lowering serum homocysteine levels withfolic acid is expected to reduce mortality from ischemicheart disease. Homocysteine reduction is known to bemaximal at a folic acid dosage of 1 mg/d, but the effectof lower doses (relevant to food fortification) is unclear.

Methods:Werandomized151patientswith ischemicheartdisease to 1 of 5 dosages of folic acid (0.2, 0.4, 0.6, 0.8, and1.0 mg/d) or placebo. Fasting blood samples for serum ho-mocysteine and serum folate analysis were taken initially,after 3 months of supplementation, and 3 months after fo-lic acid use was discontinued.

Results:Median serum homocysteine level decreased withincreasing folic acid dosage, to a maximum at 0.8 mg offolic acid per day, when the homocysteine reduction (pla-cebo adjusted) was 2.7 µmol/L (23%), similar to the knowneffect of folic acid dosages of 1 mg/d and above. The higher

a person’s initial serum homocysteine level, the greater wasthe response to folic acid, but there were statisticallysignificant reductions regardless of the initial level. Serumfolate level increased approximately linearly (5.5 nmol/Lfor every 0.1 mg of folic acid). Within-person fluctuationsover time in serum homocysteine levels, measured in theplacebo group, were large compared with the effect offolic acid, indicating that monitoring of the reduction inan individual is impractical.

Conclusions: A dosage of folic acid of 0.8 mg/d ap-pears necessary to achieve the maximum reduction in se-rum homocysteine level across the range of homocyste-ine levels in the population. Current US food fortificationlevels will achieve only a small proportion of the achiev-able homocysteine reduction.

Arch Intern Med. 2001;161:695-700

T AKING FOLIC acid lowers se-rum homocysteine levels. Adosage of 1 mg/d has beenshown in a meta-analysis1

to produce the maximumhomocysteine reduction, with no furtherreduction with higher dosages, up to 5mg/d. This maximum reduction is about25% (or 3 µmol/L from an average of 12µmol/L).1 Given the size of the associa-tion between serum homocysteine leveland ischemic heart disease shown in a co-hort study,2 this decrease in serum homo-cysteine level would be expected to lowerischemic heart disease mortality rates byabout 15%. Increasing average folic acidconsumption in the population is there-fore likely to be important in reducing theincidence of ischemic heart disease.

The effect of lower doses of folic acidon serum homocysteine concentration re-mains uncertain. In the meta-analysis therewere no data regarding whether a dose lowerthan 1.0 mg would produce a maximumreduction.1 In view of the current interestin fortifying food with folic acid, it is nec-

essary to determine the lowest dose of folicacid that will produce the maximum re-duction in serum homocysteine level. Tohelp answer this question, we carried outa randomized, double-blind, placebo-con-trolled trial of folic acid supplementation,to test the serum homocysteine–loweringeffect of dosages of 0.2, 0.4, 0.6, 0.8, and 1.0mg of folic acid per day in patients withknown ischemic heart disease. The studyalso aimed to assess whether there was anyresidual effect on serum homocysteine re-duction 3 months after folic acid supple-mentation had been stopped.

RESULTS

Table 1 shows the initial median (andmean) values and the values at the 10thand 90th percentiles for serum homocys-teine and serum folate concentrations.Figure 1 shows the distribution of ini-tial serum homocysteine values in the 151patients; this was positively skewed. About10% had serum homocysteine levelsgreater than 20 µmol/L, compared with

ORIGINAL INVESTIGATION

From the Department ofCardiology, St Richard’sHospital, Chichester, WestSussex, England (Drs D. Waldand Bishop); Wolfson Instituteof Preventive Medicine, London,England (Drs N. Wald and Lawand Ms Hennessy); Departmentof Clinical Medicine, St James’sHospital, Dublin, Ireland(Drs Weir and McPartlin); andDepartment of Biochemistry,Trinity College, Dublin(Dr Scott).

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about 2% to 3% of individuals without ischemic heart dis-ease of the same age.2 These values remained high in theplacebo group at the 3- and 6-month visits, indicatingthat they were genuinely high and not simply the result

of individual fluctuations or measurement error. Be-cause of the skewed distribution and the presence of out-lying values, the median was used instead of the meanas a measure of central tendency.

Figure 2A shows the median increase in serum fo-late level between the initial and the 3-month visit ac-cording to supplementation group (148 patients). Re-sults were placebo-adjusted (subtracting the medianchange in the placebo group from the median change ineach supplementation group). Serum folate level in-creased with increasing folic acid dose; for each 0.1 mgof folic acid, serum folate level increased by about 5.5nmol/L. Figure 2B shows the placebo-adjusted medianreduction in serum homocysteine levels between the ini-tial and 3-month visit according to supplementationgroup. Serum homocysteine levels showed a continu-ous decline with increasing folic acid dose up to a dos-age of 0.8 mg/d, where the median serum homocysteinereduction was 2.7 µmol/L (23% of the median startinghomocysteine level in that supplementation group). TheSEs of the changes in serum homocysteine level from alinear regression analysis of the change in serum homo-cysteine level on folic acid dose were about 0.7 µmol/L,and while the trial did not have the statistical power toshow statistically significant differences between adja-cent folic acid dosage groups, the trend in increasing ho-mocysteine reduction in relation to increasing folic aciddosages up to 0.8 mg/d was significant. The high serumhomocysteine response in the 0.8-mg and 1.0-mg dos-age groups was not due to a chance preponderence of sub-jects with very high (.20 µmol/L) initial serum homo-cysteine levels in these 2 dosage groups (the 0.8-mg grouphad none of these). The homocysteine reduction with the1-mg folic acid dose was slightly less than that with 0.8

20

10

15

5

0 105 15 25 35 4020 30 45Serum Homocysteine, µmol/L

No. o

f Per

sons

Figure 1. Distribution of serum homocysteine concentrations at initial visit.

Table 1. Median, Mean, and 10th and 90th Percentilesof Serum Homocysteine and Serum Folate Levelsat Initial Visit (n = 151)

Median Mean10th

Percentile90th

Percentile

Serum homocysteine, µmol/L 13.4 14.6 9.4 20.8Serum folate, nmol/L* 15.4 18.4 9.1 31.2

*To convert nanomoles per liter to micrograms per liter, divide by 2.266.

PATIENTS AND METHODS

Patients at St Richard’s Hospital, Chichester, En-gland, who were known to have ischemic heart dis-ease (previous myocardial infarction or angina) wereinvited to participate in the study. The trial was ap-proved by the local research ethics committee. Pa-tients already taking vitamin supplements or takinganticonvulsant therapy were excluded from partici-pating in the study. Patients with a myocardial infarc-tion in the previous 3 months were also excluded, asserum homocysteine level increases in the acute phaseafter a myocardial infarction and then decreases.3

A total of 151 patients were randomized to 5 folicacidsupplementationgroupsandaplacebogroup. Iden-tical tablets containingno folic acid (placebo)anddosesof 0.2, 0.4, 0.6, 0.8, and 1.0 mg of folic acid (Cantas-sium Vitamins, London, England) were used. The trialwas double-blind. Bottles of tablets were numbered inrandom order and dispensed in numerical order by thepharmacy. The tablets were taken for 3 months.

Fasting blood samples for serum folate and se-rum homocysteine measurement were taken from eachpatient on 3 occasions: before commencing the tab-lets, after taking them for 3 months, and after stoppingthem for 3 months. The blood samples were placed onice immediately after collection and centrifuged at 4°Cwithin 2 hours, and the serum was stored at −20°C. Se-rumhomocysteineassayswereperformedatTrinityCol-lege,Dublin, Ireland,by isocratichigh-performance liq-uid chromatography,4 with the use of a fluorescentconjugate (SBD-1).5 A 1:10 dilution of 0.5 mL of wholeblood in 1-g/L sodium ascorbate was also stored at−20°C, until analyzed for serum folate by microbio-logic assay.6 Laboratory staff were unaware of the ran-domization group to which patients were assigned.

Of the 151 patients (mean age, 65 years), 125 weremale; 84 had had a myocardial infarction and the other67 had angina. There were 25 patients each in the pla-cebo, 0.4-mg, 0.6-mg, and 0.8-mg groups; 27 pa-tients in the 0.2-mg group; and 24 patients in the1.0-mg group. Three patients were unavailable for fol-low-up at 3 months (1 in each of the placebo, 0.2-mg,and 0.4-mg groups), and an additional 5 patients wereunavailable for follow-up at 6 months (2 in both theplacebo and 0.4-mg groups and 1 in the 0.2-mg group).In the other groups, no patients were unavailable forfollow-up. The effect of folic acid after 3 months wasdetermined from the 148 patients who attended thefirst and second visits, and the effect of stopping folicacid supplementation was determined from the 143patients who attended all 3 visits.

At the clinic visit after 3 months of supplemen-tation, all patients said they had taken their tabletsregularly. Counts of unused tablets in the contain-ers showed a maximum of 5 (of 91 initially), and morethan half of the patients had taken all their tablets.



mg; this is probably due to chance, but it does suggestthat the effect of 1 mg is unlikely to be greater than thatof 0.8 mg. The serum homocysteine–lowering effect offolic acid was similar in older and younger patients. Themean reduction in each supplementation group was a littlegreater than the median, and at higher doses it was about3 µmol/L (21% of the mean starting homocysteine level),similar to the effect of folic acid dosages between 1 and5 mg/d, shown in the meta-analysis of the Homocyste-ine Lowering Trialists’ Collaboration.1

Table2 shows the placebo-adjusted reduction in se-rum homocysteine concentrations according to tertilegroups (thirds) of initial values. The size of the serum ho-mocysteine reduction increased with initial serum homo-cysteine level, as shown previously.1 The effect of folic acidin lowering serum homocysteine level was statistically sig-nificant in all 3 tertile groups (P,.001, P,.001, and P=.04from a linear regression analysis in the highest, middle,and lowest tertile groups, respectively). A folic acid dos-age of 0.8 mg/d achieved the maximum median reduc-tion in serum homocysteine level in all 3 tertile groups,but in the highest group a dosage of 0.4 mg/d appearedsufficient to achieve the maximum effect. The results sug-gest that the higher the initial homocysteine level, the lowerthe folic acid dose needed to attain the maximum reduc-tion. This trend was confirmed in the 10% of patients withvery high initial homocysteine levels ($20 µmol/L). Thisgroup showed a dramatic response to folic acid supple-mentation, even in the lowest dosage group, with a me-dian serum homocysteine reduction of 7.1 µmol/L (meanreduction, 9 µmol/L).

Table 3 shows the extent to which the effect of folicacid on serum folate and serum homocysteine levels wassustained 3 months after folic acid supplementation wasstopped (143 patients in total). Serum folate concentra-tion decreased after cessation but (apart from the 0.2-mgdosage group) did not return to initial levels; it was 9.3nmol/L higher in the 0.8-mg/d supplementation group and12.4 nmol/L higher in the 1.0-mg/d supplementation group(the latter representing a 75% return to the initial level).Serum homocysteine concentrations returned to near ini-tial levels for all supplementation groups, suggesting thata sustained serum homocysteine reduction relies on con-tinued folic acid supplementation.

The 3 sets of measurements in 22 placebo-treated sub-jects allowed calculation (from an analysis of variance) ofthe within- and between-person SDs for serum homocys-teine and serum folate levels (Table 4). For serum ho-mocysteine, the between-person SD (6.8 µmol/L) is largein relation to the within-person SD (2.3 µmol/L), indicat-ing that the variation across a single set of measure-ments is mainly due to true differences between in-

Table 2. Reduction in Median Serum Homocysteine LevelAfter 3 Months of Folic Acid Supplementation*

Randomization Group,mg of Folic Acid

Initial Serum Homocysteine, µmol/L

,11.7 11.7-15.0 .15.0

0 (Placebo) . . . . . . . . .0.2 −0.1 (1)† −2.5 (19) −2.2 (13)0.4 −1.1 (11) −1.5 (12) −4.8 (25)0.6 0 (0) −3.0 (22) −3.8 (21)0.8 −1.9 (19) −3.7 (28) −5.0 (32)1.0 −1.8 (18) −3.6 (29) −3.5 (18)

*Ellipses indicate not applicable. Adjusted (by subtraction) for change inplacebo group, according to dose of folic and initial serum homocysteinelevel.

†Values are absolute reductions in micromoles per liter, with percentagereductions in parentheses. Percentage reductions are placebo-adjustedmedian of differences divided by initial median.

Table 3. Median Difference From Initial Median SerumFolate and Median Serum Homocysteine Concentrations3 Months After Stopping Folic Acid Supplementation(Placebo-Adjusted), According to Dose of Folic Acid

Folic Acid Dose, mgSerum Folate,

nmol/L*Serum

Homocysteine, µmol/L

Initial concentration 15.4 13.4Change in concentration 3 mo

after stopping folic acid0.2 −0.2 0.40.4 4.8 −0.50.6 5.0 −0.50.8 9.9 0.31.0 12.4 −0.6


60

30

40

50

20

10

0

0 0.40.2 0.6 0.8 1.0Randomization Group, mg of Folic Acid

Seru

m F

olat

e, n

mol

/L

0.0

–1.5

–1.0

–0.5

–2.5

–2.0

0.0

0

–3.0

0.4

–1.3 (–10%)

0.2

–1.2 (–9%)

55.3

46.0

31.5

26.1

10.2

0

0.6

–1.8 (–12%)

0.8

–2.7 (–23%)

1.0

–2.5 (–20%)

Randomization Group, mg of Folic Acid

Seru

m H

omoc

yste

ine,

µm

ol/L

A B

Figure 2. A, Median change in serum folate concentration between initial and 3-month visits, adjusted for changes in placebo group. To convert nanomoles perliter to micrograms per liter, divide by 2.266. B, Median change (percentage change) in serum homocysteine concentrations between initial and 3-month visits,adjusted for changes in placebo group.



dividuals rather than to random variation in the sameindividual. Applying the data in Table 4 to prospective(cohort) studies of homocysteine and the incidence ofheart disease and stroke, the “regression dilution cor-rection factor” (the ratio of total to between-person vari-ance) for serum homocysteine is 1.12 ([2.332+6.762]/6.762), a low value, indicating that the observed regressioncoefficient (slope) of cardiovascular disease on homo-cysteine needs to be increased by only 12% to allow forthe diluting effect of random fluctuations in homocys-teine level.7,8

The data in Table 4 can also be used to assess thevalue of using homocysteine measurements before andafter the start of folic acid treatment to monitor the ho-mocysteine reduction. This might be worthwhile if theeffect of treatment were large in relation to the within-person fluctuation in homocysteine level. In fact it is rela-tively small, as shown by the “monitoring factor” for ho-mocysteine of 0.45, so monitoring in this way is not useful.This monitoring factor is the median reduction from 0.8mg of folic acid per day, divided by 2.56 times the within-person SDs (which estimates the 10th to 90th percentilerange of values in an individual over time). For serumfolate level, on the other hand, the monitoring factor islarge (3.5).

COMMENT

Our results show a clear effect of increasing folic acid doseon serum homocysteine reduction. The maximum ef-fect was attained at 0.8 mg/d, where the serum homo-cysteine reduction was 2.7 µmol/L, a 23% reduction, simi-lar to the effect shown for folic acid dosages between 1and 5 mg/d.1

The results confirm that the higher the initial se-rum homocysteine level, the more sensitive is the re-sponse to folic acid. The study had the statistical powerto show that there was a significant serum homocysteine–lowering effect of folic acid in people with relatively lowinitial serum homocysteine levels, indicating some ben-efit in folic acid supplementation regardless of serum ho-mocysteine level. Although a dosage of 0.4 mg of folicacid per day is sufficient to attain close to the maximumserum homocysteine reductions in those with higher ini-tial serum homocysteine concentrations, it will achieveonly about half of the smaller reductions in those withlower serum homocysteine levels—overall, about 75%of the total effect. The higher dosage appears necessaryfor the full benefit. The consistent pattern of our re-sults, both unstratified by initial serum homocysteine level(Figure 2B) and stratified according to initial serum ho-mocysteine level (Table 2), suggests that we can be rea-sonably confident of the pattern of the dose-response re-lationship observed between serum homocysteine leveland supplemental folic acid despite the fairly wide con-fidence intervals that apply to any one dosage group orsubgroup within a dosage group.

Table 5 summarizes the results of published stud-ies that have investigated the effect on serum homocys-teine level of folic acid supplements (in pills or fortifiedbreakfast cereal) in dosages less than 1.0 mg/d.9-15 Twostudies in selected populations with very high serum ho-mocysteine concentrations (.40 µmol/L) were ex-cluded.16,17 We also restricted our analysis to studies inwhich the mean age was more than 40 years, becauseanalysis of the published trials shows that the increasein serum folate for a specified increase in folic acid in

Table 4. Within- and Between-Person SDs for SerumHomocysteine and Serum Folate Levels

SerumHomocysteine

SerumFolate

Within-person SD (W) 2.3 µmol/L 5.3 nmol/L*Between-person SD (B) 6.8 µmol/L 8.7 nmol/L*Regression dilution

correction factor[(B2+W2)/B2]

1.12 1.37

Monitoring factor(median change achievedby 0.8 mg of folic aciddivided by 2.56W)

0.45 3.4


Table 5. Serum Homocysteine Reduction in Trials Using Folic Acid at Dosages Below 1 mg/d,in Which Mean Age of Subjects Was More Than 40 Years

Source* Folic Acid, mg Mean Age, y

Serum Homocysteine, µmol/L

Initial Change† Ratio, Final to Initial % Reduction

Ward et al9 0.1 45 9.0 −0.9 0.91 9Malinow et al10‡ 0.13 64 10.0 −0.7 0.93 7Schorah et al11‡ 0.2 45 10.2 −1.3 0.87 13Ward et al9 0.2 45 9.0 −1.4 0.84 16Ward et al9 0.4 45 9.0 −1.9 0.79 21Lobo et al12 0.4 61 14.0 −4.0 0.71 29Riddell et al13 0.45 53 11.7 2.1 0.82 18Malinow et al10‡ 0.5 66 11.4 −2.0 0.82 18Den Heijer et al14 0.5 53 12.2 −2.1 0.83 17Coppen and Bailey15 0.5 42 9.5 −2.1 0.78 22Malinow et al10‡ 0.67 63 14.6 4.2 0.71 29

*All studies were randomized except those of Ward et al 9 and Lobo et al.12

†All values are placebo-adjusted except for those from Ward et al.9

‡Cereal fortification.



take is greater in older than in younger people. It is likely,therefore, that for a given intake of folic acid, homocys-teine levels will decrease more in older than in youngerpeople. The data support our own results in suggestingthat folic acid dosages of 0.1 and 0.2 mg/d do not attainthe full effect and that dosages of 0.4 and 0.5 mg/d re-duce serum homocysteine levels by about 20%—achieving most but not all of the known maximum ef-fect of about 25%. There is little previously publishedinformation on dosages between 0.6 and 1.0 mg/d.

The data in Table 4 show that the within-person fluc-tuation in serum homocysteine level over time is largein comparison with the maximum average decrease inserum homocysteine level produced by folic acid. It istherefore impractical to monitor the change in serum ho-mocysteine level produced by folic acid in an individualpatient (as opposed to a group); the relatively modest truereduction would be obscured.

On the other hand, the within-person SD of homo-cysteine is small in relation to the between-person SD (2.3vs 6.8 µmol/L), indicating that, in cohort studies of se-rum homocysteine and cardiovascular disease, the “dilut-ing” effect of fluctuations in homocysteine will be small.The reduction in ischemic heart disease mortality that canbe expected through serum homocysteine reduction shouldbe close to the observed reduction in ischemic heart dis-ease mortality in cohort studies (systematic underestima-tion of the true effect will be only about 10%). This com-pares with the larger effect of within-person fluctuationin serum cholesterol level, where the effect of serum cho-lesterol reduction on ischemic heart disease is about 50%greater than that estimated in cohort studies.7,8

In an analysis of variance, such as that used to pro-duce the results in Table 4, it may be better to adopt 2 modi-fications: to use logarithms (because the variation is likelyto be proportional to the mean) and to minimize the effectof outliers by estimating the SD from the 10th to the 90thpercentiles in the placebo group to calculate the between-person SD. Analyzing the data in this way yields estimatesof 0.31 and 0.13 for the between- and within-person SDsof homocysteine (units are loge), and 0.38 and 0.26, re-spectively, for serum folate. These numbers can be madeinterpretable in the original units of measurement by tak-ing the antilogarithm and using this together with a meanvalue to calculate the range defined by the mean±1 SD. Forexample, the range of mean±1 within-person SD for ho-mocysteine with a mean of 10 µmol/L would be 7.3 µmol/L(10 divided by antiloge 0.31) to 13.6 µmol/L (10 times an-tiloge 0.31). This compares with a range of 7.7 to 12.3µmol/L using the estimate in Table 4.

Our estimate for the homocysteine regression dilu-tion correction factor (1.12 from Table 4 or 1.18 from theabove estimates) is close to the estimate of 1.14 from Clarkeand colleagues,18 but their estimates of both within- andbetween-person SD were lower. This may reflect the lowerhomocysteine values in that study (because the subjectsdid not have ischemic heart disease). An analysis of vari-ance using logarithms largely reconciles the differences be-tween the 2 studies. In another study, the within-personSD of homocysteine was small (0.6 µmol/L), but the du-ration of the study was short (4 weeks); the between-person SD was 2.8 µmol/L.19

Public health initiatives that fortify food with folicacid are likely to be the most effective means of increas-ing folic acid consumption in the population. This is aninexpensive and simple strategy. Fortification hasalready been introduced in the United States to preventneural tube defects. The level of fortification mandatedby the US government (0.14 mg of folic acid per 100 gof cereal grain, with the intention of supplementinga person’s diet by about 0.1 mg of folic acid per day)20

will achieve only a small proportion of the maximumserum homocysteine–lowering effect. In the UnitedKingdom, the relevant government advisory committeehas recommended the universal fortification of flour ata level of 0.24 mg per 100 g of flour.21 Even this willhave only a partial effect in lowering serum homocyste-ine level.

The advantages in selecting a study population withischemic heart disease were that they represent a signifi-cant proportion of people who stand to benefit most froma reduction in serum homocysteine level through food for-tification. As expected, they had higher serum homocys-teine concentrations than unaffected persons of the sameage (median, 13.4 µmol/L compared with about 12 µmol/L)and included a higher proportion of people with particu-larly high serum homocysteine levels (likely to be causedby genetic variants, such as homozygotes for the 677 me-thyltetrahydofolate reductase variant). This important high-risk group stands to benefit considerably from serum ho-mocysteine reduction; their median serum homocysteinereduction of 7 µmol/L would be expected to reduce riskby about 25%.2 Randomized trials of the efficacy of folicacid supplements in the prevention of ischemic heart dis-ease are ongoing, but the existing evidence of an effect ispersuasive.1 It would be reasonable for clinicians to con-sider advising patients with ischemic heart disease to take0.8 mg of folic acid each day.

Accepted for publication October 23, 2000.This study was supported by the British Medical As-

sociation, London, England (Brackenbury Research Award),and EU Biomed Project PL963549, Brussels, Belgium.

WethankJeremyQuiney,FRCP,RayLyon,MRPharmS,and staff in the biochemistry, hematology, and pharmacydepartments at St Richard’s Hospital, Chichester, England,and Lynn George and Tiesheng Wu at the Wolfson Instituteof Preventive Medicine, London, England. We also thank Rob-ert Woodward, Melvyn Thomas, and Baldeo Ramsarran ofCantassium Vitamins for making and supplying the folic acidtablets.

Corresponding author and reprints: David S. Wald,MRCP, Department of Cardiology, St Richard’s Hospital,Spitalfield Lane, Chichester PO19 4SE, West Sussex,England (e-mail: [email protected]).

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homocysteine in acute myocardial infarction: homocysteine-lowering effect offolic acid. J Intern Med. 1995;237:381-388.

4. Ubbink JB, Hayward Vermaak WJ, Bissport S. Rapid high-performance liquidchromatographic assay for total homocysteine levels in human serum. J Chro-matogr. 1991;565:441-446.

5. Araki A, Sako Y. Determination of free and total serum homocysteine in humanplasma by high performance liquid chromatography with fluorescence detec-tion. J Chromatogr. 1987;422:43-52.

6. O’Broin S, Kelieher B. Microbiological assay on microtitre plates of folate in se-rum and red cells. J Clin Pathol. 1992;45:344-347.

7. Chen Z, Peto R, Collins R, MacMahon S, Lu J, Li W. Serum cholesterol concen-tration and coronary heart disease in population with low cholesterol concen-trations. BMJ. 1991;303:276-282.

8. Law MR, Wald NJ, Wu T, Hackshaw A, Bailey A. Systematic underestimation ofassociation between serum cholesterol concentration and ischaemic heart dis-ease in observational studies: data from the BUPA study. BMJ. 1994;308:363-366.

9. Ward M, McNulty H, McParthin J, et al. Plasma homocysteine, a risk factor forcardiovascular disease, is lowered by physiological doses of folic acid. Q J Med.1997;90:519-524.

10. Malinow MR, Duell PB, Hess DL, et al. Reduction of plasma homocysteine lev-els by breakfast cereal fortified with folic acid in patients with coronary heart dis-ease. N Engl J Med. 1998;338:1009-1015.

11. Schorah CJ, Devitt H, Lucock M, et al. The responsiveness of plasma homocys-teine to small increase in dietary folic acid: a primary care study. Eur J Clin Nutr.1998;52:407-411.

12. Lobo A, Naso A, Arheart K, et al. Reduction of homocysteine levels in coronary

artery disease by low-dose folic acid combined with vitamins B6 and B12. Am JCardiol. 1999;83:821-825.

13. Riddell LJ, Chisholm A, Williams S, Mann JI. Dietary strategies for lowering ho-mocysteine concentrations. Am J Clin Nutr. 2000;71:1448-1454.

14. Den Heijer M, Brouwer IA, Bos GMJ, et al. Vitamin supplementation reduces bloodhomocysteine levels: a controlled trial in patients with venous thrombosis andhealthy volunteers. Arterioscler Thromb Vasc Biol. 1998;18:356-361.

15. Coppen A, Bailey J. Enhancement of the antidepressant action of fluoxetine byfolic acid: a randomised, placebo controlled trial. J Affect Disord. 2000;60:121-130.

16. Guttormsen AB, Velan PM, Nesthus I, et al. Determinants and vitamin respon-siveness of intermediate hyperhomocysteinemia ($40 µmol/L). J Clin Invest. 1996;98:2174-2183.

17. Ubbink JB, van der Merwe A, Vermaak WJH, Becker PJ, Delport R, Potgieter HC.Vitamin requirements for the treatment of hyperhomocysteinemia in humans.J Nutr. 1994;124:1927-1933.

18. Clarke R, Woodehouse P, Ulvik A, et al. Variability and determinants of total ho-mocysteine concentrations in plasma in an elderly population. Clin Chem. 1998;44:102-107.

19. Garg UC, Zheng Z-J, Folsom AR, et al. Short-term and long-term variability ofplasma homocysteine measurement. Clin Chem. 1997;43:141-145.

20. Jacque PF, Selhub J, Bostem AG, Wilson PW, Rosenberg IH. The effect of folicacid fortification on plasma folate and total homocysteine concentrations. N EnglJ Med. 1999;340:1449-1454.

21. Department of Health. Folic Acid and the Prevention of Disease: Report of theCommittee on Medical Aspects of Food and Nutrition Policy. London, England:Dept of Health; 2000.



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B Vitamin and/or ω-3 Fatty Acid Supplementation and Cancer