Effect of riboflavin supplementation on plasma homocysteine in

elderly people with low riboflavin status

McKinley, M., McNulty, H., McPartlin, J., Strain, J. J., Weir, D. G., & Scott, J. M. (2002). Effect of riboflavin
supplementation on plasma homocysteine in elderly people with low riboflavin status. European Journal of
Clinical Nutrition, 56(9), 850-856.

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European Journal of Clinical Nutrition

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 European Journal of Clinical Nutrition (2002) 56, 850–856
ß 2002 Nature Publishing Group All rights reserved 0954–3007/02 $25.00
www.nature.com/ejcn

PAPER
Effect of riboflavin supplementation on plasma
homocysteine in elderly people with low riboflavin
status
MC McKinley1, H McNulty1*, J McPartlin2, JJ Strain1 and JM Scott3

1
Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine, Northern Ireland; 2Department of Clinical
Medicine, Trinity College Dublin, Republic of Ireland; and 3Department of Biochemistry, Trinity College Dublin, Republic of Ireland

Objective: To investigate the effect of riboflavin supplementation on plasma homocysteine (tHcy) concentrations in healthy
elderly people with sub-optimal riboflavin status.
Design: A double-blind, randomized, placebo-controlled riboflavin supplementation trial.
Setting: Community based study in Northern Ireland.
Subjects: From a screening sample of 101 healthy elderly people, 52 had sub-optimal riboflavin status (erythrocyte glutathione
reductase activation coefficient, EGRAC
1.20) and were invited to participate in the study.
Intervention: The intervention had two parts. Part 1 was a 12 week randomized double blind, placebo-controlled intervention
with riboflavin (1.6 mg=day). Following completion of part 1, the placebo group went on to part 2 of the study which involved
supplementation with folic acid (400 mg=day) for 6 weeks followed by folic acid and riboflavin (1.6 mg=day) for a further 12
weeks, with a 16 week washout period post-supplementation. The purpose of part 2 was: (a) to address the possibility that
homocysteine-lowering in response to riboflavin may be obscured by a much greater effect of folate, and that, once folate status
was optimized, a dependence of homocysteine on riboflavin might emerge; and (b) to demonstrate that these subjects had
homocysteine concentrations which could be lowered by nutritional intervention.
Results: Although riboflavin supplementation significantly improved riboflavin status in both parts 1 and 2 of the study
(P < 0.001 for each), tHcy concentrations were unaffected (P ¼ 0.719). In contrast, folic acid supplementation (study part 2)
resulted in a homocysteine lowering of 19.6% (P ¼ 0.001).
Conclusion: Despite the metabolic dependency of tHcy on riboflavin, it did not prove to be an effective homocysteine-lowering
agent, even in the face of sub-optimal riboflavin status.

European Journal of Clinical Nutrition (2002) 56, 850 – 856. doi:10.1038=sj.ejcn.1601402

Keywords: riboflavin; homocysteine; dietary supplementation; elderly people; folic acid

*Correspondence: Professor H McNulty, Northern Ireland Centre for Food
and Health (NICHE), University of Ulster, Coleraine BT52 1SA, Northern
Ireland.
E-mail: H.McNulty@ulst.ac.uk
Guarantor: Professor H McNulty.
Contributors: MMcK participated in hypothesis formulation and study
design, was responsible for study execution, sample collection,
laboratory analysis, data analysis, data interpretation and did the main
writing of the paper. HMcN was involved in formulating the hypothesis
and participated in study design, data analysis, data interpretation and
writing the paper. JMcP participated in laboratory analysis of samples and
writing the paper, JJS and JMS participated in hypothesis formulation,
study design and writing the paper.
Received 5 July 2001; revised 4 December 2001;
accepted 10 December 2001
Introduction
Mild elevations in total plasma homocysteine (tHcy) are
associated with an increased risk of cerebro-, peripheral-
and cardio-vascular disease (Eikelboom et al, 1999). Homo-
cysteine is formed by the demethylation of methionine.
Once formed, homocysteine is either remethylated to
methionine, or undergoes a trans-sulphuration reaction to
form cysteine. The remethylation of homocysteine is cata-
lysed by the enzyme methionine synthase which requires
vitamin B12, in the form of methylcobalamin (Finkelstein,
1990), and folate in the form of 5-methyltetrahydrofolate
(5-MTHF; Finkelstein, 1990), as co-factor and co-substrate
respectively. In the trans-sulphuration pathway, the

pyridoxal 50 -phosphate (vitamin B6)-dependent enzyme,
cystathionine b-synthase (CBS), catalyses the conversion of
homocysteine to cysteine (Mudd et al, 1995). In addition to
the roles played by vitamins B6, B12 and folate in either the
trans-sulphuration or remethylation pathways, a fourth B-
vitamin, riboflavin, is required for the function of both
pathways. The co-substrate for methionine synthase, 5-
MTHF, is generated by the action of the enzyme methylene-
tetrahydrofolate reductase (MTHFR) which in turn requires
riboflavin, in the form of flavin adenine dinucleotide (FAD)
as a prosthetic group (Bates & Fuller, 1986). In the trans-
sulphuration pathway, riboflavin, in the form of flavin
mononucleotide (FMN), is required for the generation of
the active co-enzyme form of vitamin B6, pyridoxal 50 -
phosphate (PLP; McCormick, 1989), which serves as a co-
factor for CBS.
Low riboflavin status is frequently found in older popula-
tions, with 49 – 78% (Madigan et al, 1998; Bailey et al, 1997)
of elderly subjects classified as having sub-optimal riboflavin
status (erythrocyte glutathione reductase activation coeffi-
cient, EGRAC
1.20). It is also widely recognized that
plasma homocysteine (tHcy) concentrations increase with
age (Selhub et al, 1993; Andersson et al, 1992). In support of
the possibility that riboflavin may be an important determi-
nant of tHcy, Lakshmi et al (1990) noted a 2 – 4-fold increase
in the concentration of tHcy in the skin of rats in response to
an experimentally induced riboflavin deficiency. Sparse and
conflicting data exist, however, on the effect of riboflavin
intake and status on plasma tHcy in humans. A large
(n ¼ 2435) cross-sectional study from The Netherlands (De
Bree et al, 2001) recently observed that dietary folate intake
was the only B-vitamin independently inversely associated
with plasma tHcy. Jacques et al (2001) observed only a
modest (but significant) association between dietary ribofla-
vin and tHcy in the Framingham Offspring cohort
(n ¼ 1960). In contrast, Shimakawa et al (1997) reported a
strong inverse relationship between dietary intake of ribo-
flavin and plasma tHcy concentrations after multivariate
adjustment (r ¼ 70.763, P < 0.01). In addition, Hustad et al
(2000) found plasma riboflavin to be an independent deter-
minant of plasma tHcy in a cross-sectional study of 423
healthy blood donors.
Riboflavin has previously been included in only two
interventions in which tHcy was the primary end-point
(Olszewski et al, 1989; Lakshmi & Ramalakshmi, 1998). In
the first study (Olszewski et al, 1989), 12 myocardial infarc-
tion patients were treated with a combination of troxerutin,
choline, vitamin B6, vitamin B12, folate and riboflavin for 21
days. A study design such as this makes it impossible to
determine the effect of riboflavin alone on fasting tHcy
levels. In the second study (Lakshmi & Ramalakshmi,
1998), 20 women with clinical and marked biochemical
riboflavin deficiency (EGRAC ¼ 1.80) were given a pharma-
cological dose of riboflavin (10 mg) for 15 days. This resulted
in a significant improvement in riboflavin status but there
was no significant change in plasma tHcy concentration.
Effect of riboflavin supplementation
MC McKinley et al
851
This study, however, was not blinded and had no placebo
group or washout period.
Although the effect of supplementation with other rele-
vant B-vitamins on plasma tHcy concentration is well docu-
mented, there is clearly a lack of information regarding the
effect of riboflavin. Therefore, the aim of this study was to
investigate the effect of riboflavin supplementation on fast-
ing tHcy concentrations in a group of healthy elderly people
who were pre-screened in order to select individuals with
sub-optimal riboflavin status.
Methods
Subjects
Ethical approval was granted by the University of Ulster
Research Ethical Committee and subjects gave written,
informed consent. Subjects aged 60 y and over were recruited
between January 1998 and April 1998 through senior citizen
groups and local ‘Folds’ (which provide sheltered accommo-
dation but no medical support for healthy elderly people
who live independently within a housing complex and take
care of themselves). All potential subjects were interviewed,
using a short medical questionnaire, regarding general
health, drug and supplement use. The exclusion criteria
were: B-vitamin supplementation; gastrointestinal disease;
haematological disorders; drugs known to affect vitamin B6
or riboflavin metabolism (antacids containing magnesium or
ioniazid); vascular, hepatic or renal disease; impaired cogni-
tive function (score < 7 on Hodkinson 10-Point Mental State
Questionnaire; Quereshi & Hodkinson, 1974); serum creati-
nine concentration
130 mmol=l; or a serum vitamin B12
concentration less than 111 pmol=l (150 ng=l).
Study design
The adequacy of sample size was established from power
calculations using typical variances from other similar inves-
tigations (Madigan et al, 1998). All potential subjects were
initially screened for riboflavin status. Only individuals with
sub-optimal riboflavin status (erythrocyte glutathione reduc-
tase activation coefficient, EGRAC
1.20) were invited to
participate in the intervention.
Figure 1 shows the study design. A laboratory technician,
who was not involved with the study in any other way,
randomly assigned subjects to two groups (1 or 2). Both
group 1 and group 2 participated in the first part of this
intervention (weeks 0 – 12); only group 1 went on to partici-
pate in the second part of the intervention (weeks 12 – 46).
Part 1 of the intervention was a randomized, double-blind,
placebo-controlled trial during which group 1 took a placebo
supplement (weeks 0 – 12), while group 2 received riboflavin
(1.6 mg) supplementation daily for 12 weeks. Following
completion of part 1 of the study (the placebo controlled
trial), the involvement of group 2 was complete. Group 1
(placebo group from part 1 of the study) went on to part 2 of
the study and received low dose folic acid (400 mg=day) for
European Journal of Clinical Nutrition
 Effect of riboflavin supplementation
MC McKinley et al
852
Figure 1 Study design.
the next 6 weeks (weeks 12 – 18), followed by folic acid and
riboflavin supplementation for the last 12 weeks (weeks 18 –
30). All group 1 subjects gave a washout blood sample that
was taken at least 16 weeks after the intervention had ended.
Thus, for part 2 of the study, each individual acted as his=her
own control.
For this study, we used low doses of both riboflavin and
folic acid in order to make the results more relevant for any
future public health policies aimed at the prevention of
hyperhomocysteinemia through modification of dietary
intakes, probably via the fortification of foods. A supplemen-
tation level of 1.6 mg=day riboflavin for 12 weeks was chosen
based on data from a previous riboflavin intervention (Madi-
gan et al, 1998) carried out at this centre (but not concerned
with tHcy), which demonstrated that this amount signifi-
cantly improved riboflavin status in elderly people. We
therefore chose the same regimen in order to observe what
effect, if any, a change in riboflavin status would have on
plasma tHcy. In the case of folic acid, a previous study (Ward
et al, 1997) from our laboratory demonstrated that
200 mg=day for 6 weeks produced significant tHcy lowering
in young adults. In the current study in elderly people we
doubled this dose (400 mg=day) in order to ensure that tHcy
concentrations in this group were, in the event that ribo-
flavin supplementation was unsuccessful, indeed lowerable.
In order to maximize compliance throughout both parts of
the study, subjects were visited in their own homes every 3
weeks and supplied with the exact number of supplements;
any unused tablets from the previous 3 weeks were returned
at this stage and counted. Subjects were instructed to follow
their usual diet and refrain from commencing any form of
European Journal of Clinical Nutrition
vitamin supplementation throughout the intervention and
until after the washout blood sample.
Blood sampling
Group 1 subjects provided six 20 ml fasting blood samples in
total: a screening sample, then samples at baseline (week 0),
week 12; week 18; week 30; and after a 16 week washout (ie
week 46). Group 2 subjects gave three 20 ml fasting blood
samples in total: a screening sample, then samples at base-
line (week 0) and week 12. All blood samples were collected
after an overnight fast, in the subjects’ own home with
subjects in the sitting position at the time of collection.
After collection and processing, samples were stored at
770 C for batch analysis.
Laboratory measurements
Total plasma homocysteine was measured by immunoassay
(Nexo et al, 2000); red cell folate (Molloy & Scott, 1997),
serum folate (Molloy & Scott, 1997) and serum vitamin B12
(Kelleher & O’Brien, 1991) were measured by microbiological
assay. Measurement of EASTAC (Mount et al, 1987) and
EGRAC (Powers et al, 1983) were by enzyme assay on the
Cobas Fara centrifugal analyzer (Roche Diagnostics, Welwyn
Garden City, UK). For all assays, samples were analysed
blind, in duplicate (except for EGRAC where triplicate sam-
ples were measured), and within 6 months after sampling.
Average values of duplicate or triplicate measurements are
reported. Samples were re-analysed if the CV between
duplicate or triplicate measurements was greater than 10%.
 Effect of riboflavin supplementation
MC McKinley et al
853
Quality control was provided by repeated analysis of stored Table 1 General characteristics and baseline blood measurementsa
(770 C) batches of pooled erythrocytes (for EASTAC and
Reference Group 1 Group 2c
EGRAC), plasma (tHcy), serum (vitamin B12, folate) or red range
b
(n ¼ 23) (n ¼ 23)
cell folate lysates (red cell folate) covering a wide range of
values. MTHFR genotype was identified by polymerase chain Age (y) — 66.91  4.07 69.91  5.29
Number males, (%) — 5 (21.7) 7 (30.4)
reaction (PCR) amplification followed by HinF1 restriction BMI (kg=m )
2 d
— 26.7  4.1 26.5  3.1
digestion (Goyette et al, 1994). Serum creatinine (mmol=l) 40 – 130 94.35  14.15 97.70  20.53
Plasma tHcy (mmol=l)e  15.00 11.51  2.55 12.00  5.46
EGRACf 1.00 – 1.20 1.26  0.06 1.26  0.05
Red cell folate (nmol=l) 340 – 2266 1078  346 1032  553
Statistics Serum folate (nmol=l) 6.80 – 45.32 17.43  8.09 14.60  5.78
All statistical analysis was performed using the Statistics EASTAC
g
1.00 – 2.00 1.71  0.10 1.72  0.10
Package for the Social Sciences (SPSS) computer software Serum vitamin B12 (pmol=l) 111 – 738 303.2  137.5 248.9  57.9
package (Chertsey, UK). Groups 1 and 2 were compared at a
Values are mean  s.d.
baseline using independent samples t-test. The response of b
Clinical biochemistry reference ranges are given for all parameters except for
subjects in groups 1 and 2 to the placebo-controlled stage of plasma tHcy where the cut-off for hyperhomocysteinemia defined by Kang
the intervention was examined using two-way ANOVA. et al (1992) has been used.
c
Groups 1 and 2 not significantly different for any of the variables at baseline
Response of group 1 to riboflavin supplementation when when examined using independent samples t-test.
given after repletion with folic acid was examined using d
Body mass index.
e
repeated measures ANOVA with LSD; P-values < 0.05 were tHcy, total homocysteine.
f
considered significant. EGRAC, erythrocyte glutathione reductase activation coefficient, a functional
indicator of riboflavin status.
g
EASTAC, erythrocyte aspartate aminotransferase activation coefficient, a
functional indicator of vitamin B6 status.
Results
A total of 101 healthy elderly people were initially recruited
and screened for riboflavin status. Some 52 (51.5%) subjects
were found to have sub-optimal riboflavin status changed during the 12 week period. In response to 1.6 mg
(EGRAC
1.20) and were invited to participate in the inter- riboflavin daily for 12 weeks riboflavin status improved
vention, of which 48 agreed to participate and 45 volunteers significantly in group 2, but no significant change in fasting
(mean age 68.4 y) completed the study. By week 12, one tHcy was observed. When the homocysteine response to
individual (group 1) had died and one individual (group 2) riboflavin supplementation was examined in the upper half
had dropped out because of illness; by week 30 a further of baseline tHcy concentrations (data not shown), no sig-
subject (group 1) withdrew because of the long duration of nificant (P ¼ 0.318) homocysteine-lowering effect was found
the study. Throughout the study, the compliance of all (placebo group (group 1), n ¼ 12: pre-tHcy (mmol=l) ¼
subjects was excellent (ie only one subject missed more 13.44  1.59, post-tHcy (mmol=l) ¼ 13.33  3.51; riboflavin
than two tablets during the intervention period, that subject supplementation group (group 2), n ¼ 11: pre-tHcy (mmol=l) ¼
was included in the analysis). The general characteristics of 15.40  6.31, post-tHcy (mmol=l) ¼ 16.22  3.51).
groups 1 and 2 are shown in Table 1. There were no In part 2 of the intervention, group 1 was followed (weeks
significant differences in any of the variables examined 12 – 46) to investigate the effect of riboflavin supplementa-
between groups 1 and 2 at baseline. tion on plasma tHcy when given after optimization of folate
Baseline plasma tHcy concentrations ranged from 5.36 – status. After taking a placebo supplement daily during the
33.07 mmol=l with 42 of 46 participants classified as normo- first part of the intervention (weeks 0 – 2), group 1 took folic
homocysteinaemic at baseline (ie plasma tHcy  15 mmol=l; acid (400 mg=day) for 6 weeks (weeks 12 – 18) which resulted
Kang et al, 1992). Serum folate was significantly correlated in a significant homocysteine-lowering of 19.6%, from
with plasma tHcy at baseline (r ¼ 70.376, P ¼ 0.010), but no 11.42  3.46 to 9.18  1.96 mmol=l, as shown in Table 3. In
significant correlation with tHcy was found for red cell response to riboflavin (1.6 mg=day) in combination with
folate, serum vitamin B12, EASTAC or EGRAC (data not folic acid (400 mg=day) for the next 12 weeks of the study
shown). Of the 45 subjects who completed all stages of the (weeks 18 – 30), the results showed that, although riboflavin
intervention study, n ¼ 5 (11.1%), n ¼ 21 (46.7%) and n ¼ 19 supplementation successfully improved riboflavin status, it
(42.2%) were found to be homozygous, heterozygous and failed to lower plasma tHcy (Table 3). Although the folate
wild-type, respectively, for the C?T677 (thermolabile) var- status of group 1 continued to increase significantly during
iant of MTHFR. the riboflavin intervention (owing to continued supplemen-
Table 2 shows the response of plasma tHcy and B-vitamin tation with folic acid during weeks 18 – 30), this did not
status to 12 weeks placebo (group 1) or riboflavin affect plasma tHcy concentrations which had reached a
(1.6 mg=day) supplementation (group 2; ie part 1 of the plateau by week 18, before riboflavin supplementation com-
study). As expected, neither the riboflavin status nor menced. Following a 16 week washout, tHcy, EGRAC and red
plasma tHcy concentrations of the placebo group (group 1) cell folate concentrations had all returned to baseline.

European Journal of Clinical Nutrition

 Effect of riboflavin supplementation
MC McKinley et al
854
Table 2 Study part 1: response of plasma tHcy and B-vitamin statusa to 12 weeks riboflavin supplementation
(1.6 mg=day) in 46 free-living healthy elderly people with sub-optimal riboflavin status (EGRAC
1.20)b

Group 1 — placebo (n ¼ 23) Group 2 — riboflavin (n ¼ 23)

c
Before (week 0) After (week 12) Before (week 0) After (week 12) P-value
d
Plasma tHcy (mmol=l) 11.51  2.55 11.42  3.46 12.00  5.46 12.57  6.06 0.719
b e
EGRAC 1.26  0.06 1.24  0.06 1.26  0.05 1.10  0.07 0.000
Red cell folate (nmol=l) 1078  346 1216  477 1032  553 1057  654 0.595
Serum folate (nmol=l) 17.43  8.09 18.72  10.97 14.60  5.78 15.66  9.00 0.552
EASTACf 1.71  0.10 1.67  0.07 1.72  0.10 1.66  0.07 0.619
Serum vitamin B12 (pmol=l) 303.2  137.5 308.3  122.7 248.9  57.9 253.7  70.1 0.973
a
Values are mean  s.d.
b
EGRAC, erythrocyte glutathione reductase activation coefficient, a functional indicator of riboflavin status. EGRAC
1.20 indicates
sub-optimal riboflavin status.
c
Response to intervention within and between groups examined using two-way ANOVA.
d
tHcy, total homocysteine.
e
Significantly different from placebo group and pre-riboflavin supplementation values.
f
EASTAC, erythrocyte aspartate aminotransferase activation coefficient, a functional indicator of vitamin B6 status. EASTAC
2.0
indicates sub-optimal vitamin B6 status.

Table 3 Study part 2: response of plasma homocysteine and B-vitamin statusa to 12 weeks (weeks 18 – 30) riboflavin supplementation
(1.6 mg=day) given after optimization of folate statusb (weeks 12 – 18) in 22 free-living healthy elderly people (group 1) with sub-optimal
riboflavin status (EGRAC
1.20)c
d
Week 0 Week 46
e
(baseline) Week 12 Week 18 Week 30 (washout) P-value
f { {
Plasma tHcy (mmol=l) 11.51  2.55* 11.42  3.46* 9.18  1.96 9.11  2.01 11.64  3.42* 0.001
c {
EGRAC 1.26  0.06* 1.24  0.06* 1.24  0.06* 1.12  0.06 1.26  0.05* 0.000
Red cell folate (nmol=l) 1078  346* 1216  477* 1729  436{ 2227  412{ 1142  348* 0.000
a
Values are mean  s.d.
b
During weeks 0 – 12 subjects took a placebo supplement. During weeks 12 – 18 subjects took folic acid (400 mg=day). During weeks 18 – 30 subjects took folic acid
(400 mg=day) plus riboflavin (1.6 mg=day). All supplementation was discontinued during weeks 30 – 46.
c
EGRAC, erythrocyte glutathione reductase activation coefficient, a functional indicator of riboflavin status. EGRAC
1.20 indicates sub-optimal riboflavin status.
d
Washout blood sample taken 16 weeks post-intervention.
e
Response to intervention was examined using repeated measures ANOVA. Values across rows with different symbols are significantly different (P < 0.05; least-
squares difference).
f
tHcy, total homocysteine.

For inclusion in this intervention we used an
EGRAC
1.20 as a cut-off to define sub-optimal riboflavin
status in our screening sample. Even after applying stricter
cut-off values for riboflavin status of either EGRAC
1.25 or
EGRAC
1.30, the results showed no change in plasma tHcy
in response to riboflavin supplementation (EGRAC
1.25,
n ¼ 24; tHcy before ¼ 11.00  5.20 mmol=l, tHcy after -
¼ 11.12  5.94 mmol=l; EGRAC
1.30, n ¼ 13: tHcy
before ¼ 9.87  2.31 mmol=l, tHcy after ¼ 9.70  2.32 mmol=l;
for the purpose of this additional analysis individuals who
received riboflavin either before (group 1) or after (group 2)
folic acid were grouped together).
Discussion
This study investigated the potential role of riboflavin as a
homocysteine-lowering agent, which, to date, has been
largely overlooked in the literature. The results of the first
part of this study (ie the 12 week randomized, placebo-
controlled trial) show that, despite the metabolic depen-
dency of homocysteine on both FAD and FMN, riboflavin
supplementation in highly compliant subjects did not result
in any homocysteine-lowering effect. This was also found to
be true when the response of tHcy to riboflavin supplemen-
tation was examined in individuals who had the highest
tHcy concentrations at baseline. Also, there was no signifi-
cant correlation between riboflavin intake and tHcy concen-
trations in the baseline sample of 101 elderly people.
In the second part of this riboflavin intervention study,
the placebo group (group 1) was followed for a further 6
weeks with folic acid (400 mg=day), then a combination of
folic acid (400 mg=day) and riboflavin (1.6 mg=day) for a final
12 weeks. This part of the study examined the possibility that
homocysteine-lowering in response to riboflavin may be
obscured by a much greater effect of folate status. The
hypothesis addressed in part 2 was therefore that once
folate status had been optimized, a dependence of tHcy on
riboflavin might emerge, similar to the dependence of tHcy

European Journal of Clinical Nutrition


on vitamin B12 that emerges following folic acid supplemen-
tation, as recently reported by our group (Quinlivan et al,
2002). The results show that, in response to folic acid
supplementation, tHcy concentrations decreased signifi-
cantly (by 19.6%), demonstrating that baseline tHcy con-
centrations were indeed lowerable by nutritional
intervention in these subjects. Riboflavin supplementation,
however, even when given after optimization of folate status,
again proved to have no significant homocysteine-lowering
effect. By 16-weeks post-supplementation, riboflavin status,
folate status and tHcy concentrations had all returned to
baseline values. It could be argued that as tHcy had already
shown such a marked response to folic acid supplementa-
tion, a further lowering, in response to riboflavin supple-
mentation, would have been unlikely. We have recently
demonstrated (McKinley et al, 2001), however, that low-
dose vitamin B6 supplementation significantly lowered
plasma tHcy concentrations in healthy elderly subjects
when given after optimization of folate status.
In this study, low-dose folic acid was a very effective
homocysteine-lowering agent, even though all subjects
were generally healthy, had normal red cell and serum
folate status, and 42 out of 46 (91.3%) subjects were
normo-homocysteinaemic. Previous studies have demon-
strated that doses of folic acid as low as 200 mg=day appear
to effectively lower homocysteine concentrations in both
hyperhomocysteinaemic (Guttormsen et al, 1996) and nor-
mohomocysteinaemic subjects (Ward et al, 1997). If homo-
cysteine is viewed as a functional indicator of folate status,
then it seems probable that current definitions of ‘normal’
folate status may need to be revised as folic acid-responsive
homocysteine concentrations are found in individuals who
have what is currently defined as ‘normal’ folate status.
It is possible that riboflavin supplementation had no
effect on tHcy because riboflavin status at baseline was not
low enough to adversely affect the activity of MTHFR, the
FAD-dependent enzyme involved in homocysteine remethy-
lation. As demonstrated by Bates and Fuller (1986), MTHFR
activity in rats was not adversely affected at EGRAC values of
1.27 and 1.31, but was affected when much higher EGRAC
values of 1.81 and above (indicative of more severe defi-
ciency) were attained. Therefore, we looked at the homo-
cysteine response to riboflavin supplementation in a sub-
sample of individuals after applying a stricter cut-off for low
riboflavin status (EGRAC
1.25 and EGRAC
1.30), but still
observed no homocysteine-lowering effect. The effect of
riboflavin supplementation on tHcy in individuals with
severe riboflavin deficiency could not be examined in the
current study since no subject had an EGRAC value greater
than 1.40.
Several years ago a C?T677 polymorphism in the MTHFR
gene was discovered (Frosst et al, 1995). This polymorphism,
which is commonly referred to as thermolabile MTHFR, is
autosomal recessive in nature, exhibits a lower specific activ-
ity in lymphocytes (  30% of wild-type) and decreased
activity after in vitro heating to 46 C in individuals posses-
Effect of riboflavin supplementation
MC McKinley et al
855
sing the TT (homozygous) genotype (Frosst et al, 1995). The
in vitro work of Guenther et al (1999) found that the mutant
enzyme was approximately 10 times more likely than the
wild-type enzyme to dissociate from its FAD prosthetic group
and become inactivated. This in vitro work, therefore, sug-
gests that MTHFR activity in individuals with the TT geno-
type may be particularly sensitive to riboflavin status.
Indeed, data from 2 recent cross-sectional studies (Hustad
et al, 2000; McNulty et al, 2001) indicate that riboflavin is a
determinant of tHcy, but the effect is driven by subjects who
are homozygous for the thermolabile variant of MTHFR. This
raises the possibility that riboflavin supplementation may
only effectively lower tHcy when given to individuals with
the TT genotype. Only five such subjects were found in the
current sample. These TT subjects were randomized to dif-
ferent treatment groups at the start of the study, making it
impossible, therefore, to examine the effect of riboflavin
supplementation on tHcy in different genotype groups for
thermolabile MTHFR. Placebo-controlled studies are, there-
fore, necessary to investigate the effect of riboflavin supple-
mentation on plasma tHcy in individuals who are
homozygous for the thermolabile (C?T677) variant of
MTHFR.
In conclusion, despite the metabolic basis for this study,
riboflavin did not prove to be an effective homocysteine-
lowering agent in this group of elderly people who had sub-
optimal riboflavin status at baseline. Further investigation is
required, however, to determine its effect in different popu-
lation sub-groups, in individuals with severe riboflavin
deficiency, and, importantly, in individuals who are homo-
zygous for thermolabile MTHFR (5 – 18% of healthy popula-
tions; Schneider et al, 1998).
Acknowledgements
We wish to acknowledge Clonmel Healthcare, Tipperary,
Ireland for providing us with the folic acid supplements,
and the volunteers who kindly participated in the study. This
study was supported by EU Project BMH 4983549 and Abbott
Germany.
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