BRIEF REPORTS
A Pilot Study of Vitamins to
Lower Plasma
Homocysteine Levels in
Alzheimer Disease
Paul S. Aisen, M.D.
Susan Egelko, Ph.D.
Howard Andrews, Ph.D.
Ramon Diaz-Arrastia, M.D.
Myron Weiner, M.D.
Charles DeCarli, M.D.
William Jagust, M.D.
Joshua W. Miller, Ph.D.
Ralph Green, M.D.
Karen Bell, M.D.
Mary Sano, Ph.D.
Objective: Authors determined the impact of high-
dose vitamin supplements on plasma homocysteine
levels in patients with Alzheimer disease (AD). Meth-
ods: Authors used an open-label trial of folic acid, vi-
tamin B12, and vitamin B6, in combination for 8
weeks, with measurement of plasma homocysteine
levels in the fasting state and after methionine-
loading. A total of 69 subjects with AD were enrolled,
including 33 who were taking standard multivitamin
supplements; 66 were available at 8-week follow-up.
Results: The high-dose vitamin regimen was associ-
ated with a significant reduction in fasting and post–
methionine-loading homocysteine. Reductions were
greater in the subgroup not using multivitamins, but
were also significant in the multivitamin users. Con-
clusion: High-dose vitamin supplementation reduces
homocysteine levels in patients with AD. The effect of
supplementation on rate of cognitive decline will be
assessed later in a randomized, double-blind study.
(Am J Geriatr Psychiatry 2003; 11:246–249)
Received November 18, 2002; revised December 23, 2002, January 3, 2003; accepted January 3, 2003. From the Department of Neurology,
Georgetown University Medical Center, Washington, DC. Address correspondence to Paul S. Aisen, M.D., Department of Neurology, Georgetown
University Medical Center, Building D, Suite 207, 4000 Reservoir Road NW, Washington, DC 20007.
Copyright 䉷 2003 American Association for Geriatric Psychiatry
246
E levated plasma homocysteine, which is associated
with increased risk of vascular disease, may also be
associated with risk1,2 and radiologically demonstrated
progression1 of Alzheimer disease (AD). In vitro and in
vivo studies suggest that homocysteine contributes to
excitotoxic damage3 and augments amyloid toxicity,4
and thus may play an important role in AD-related neu-
rodegeneration. Reduction of homocysteine levels with
vitamin supplements thus represents a plausible thera-
peutic strategy.
Previous studies in persons not diagnosed with AD
suggest that high-dose vitamin supplementation can re-
duce plasma levels of homocysteine by 25%.5 In the
United States, grain products have been fortified with
folic acid since 1998, an intervention aimed at reducing
neonatal neural tube defects known to be linked to folic
acid deficiency. This fortification has reduced mean ho-
mocysteine levels by 7%6 and thus may have reduced
the potential impact of high-dose vitamin supplemen-
tation.
In preparation for a large multicenter trial to deter-
mine whether homocysteine reduction slows progres-
sion in persons with AD, we conducted the present
study to determine the impact of high-dose vitamin sup-
plementation on fasting and post–methionine-loading
homocysteine in American subjects with AD. In partic-
ular, we sought to determine whether high-dose vitamin
supplements reduce plasma homocysteine despite folic
acid fortification of grain products in the United States
and whether high-dose supplements reduce homocys-
teine levels in users of daily multivitamins.
METHODS
Study Design
The 8-week study used an open-label design to de-
termine the impact of high-dose vitamin supplementa-
tion on homocysteine levels. The treatment regimen
consisted of folic acid 5 mg (given as five 1-mg tablets
taken simultaneously), plus vitamin B12 1 mg, plus vi-
tamin B6 50 mg, administered together each morning;
Am J Geriatr Psychiatry 11:2, March-April 2003

this regimen was selected to provide optimal reduction
in homocysteine.5
Medically stable subjects with probable AD7 were
eligible for enrollment regardless of level of cognitive
impairment if a caregiver was available to supervise ad-
ministration of the vitamin regimen. Subjects were ex-
cluded if there was evidence of significant renal insuf-
ficiency (creatinine ⬎1.5 mg/dL), a history of B12 or
folate deficiency, use of vitamin supplements containing
greater than 400 lg of folic acid, regular use of vitamin
B12 injections, or use of medications known to influence
homocysteine metabolism (e.g., methotrexate, azathio-
prine, phenytoin). Subjects who were taking multivita-
mins at the time of enrollment continued these multi-
vitamins in addition to the study regimen during the
trial.
The cognitive/behavioral assessments included the
Folstein Mini-Mental State Exam (MMSE),8 the Alzhei-
mer’s Disease Assessment Scale9 cognitive subscale
(ADAScog), and the Geriatric Depression Scale (GDS).10
Blood specimens were obtained for measurement of ho-
mocysteine in the fasting state and 2 hours after admin-
istration of L-methionine (0.1 g/kg body weight).
Laboratory Measures
Plasma homocysteine was determined by high-
pressure liquid chromatography with post-column fluo-
rescence detection.11 Normal quality control (mean 7.5
lmol/L): intra-assay coefficient of variation (CV): 1.3%,
inter-assay CV: 4.2%; high quality control (mean 27.8
lmol/L): intra-assay CV: 1.1%, inter-assay CV: 3.0%.
Vitamin B12 was measured by automated chemilu-
minescence assay (ACS 180, Bayer Diagnostics, Tarry-
town, NY); red blood cell folate, by radioassay (Quan-
taphase II, BioRad Diagnostics, Hercules, CA); and
plasma pyridoxal-5’-phosphate (an indicator of tissue
stores of vitamin B6) by radioenzymatic assay (ALPCO,
Windham, NH). MTHFR genotype was determined by
the method of Frosst.12
Statistical Analysis
The primary analysis was a comparison by paired t-
tests of the mean change in fasting and post–methionine-
loading homocysteine levels from baseline to the 8-week
study visit. Further analyses examined the relationship
among demographic, clinical, and genetic factors and
homocysteine levels, by calculating zero-order Pearson
Am J Geriatr Psychiatry 11:2, March-April 2003
Aisen et al.
correlation coefficients among these variables as well as
conducting a simultaneous multiple-regression analysis
predicting change in fasting levels of homocysteine.
RESULTS
Participant Flow and Follow-Up
A total of 69 subjects (44 women) were enrolled at
the four sites; 54 were taking cholinesterase inhibitors
at the time of enrollment, and 33 were taking multivi-
tamins. The typical multivitamin used by study partici-
pants contained 400 lg folic acid, 25 lg vitamin B12,
and 3 mg–25 mg vitamin B6. The mean age was 70.8
years (standard deviation [SD]: 9.4), and the mean du-
ration of AD was 3.3 (SD: 2.5) years. At enrollment, the
mean MMSE score was 19.3 (SD: 7.0); mean ADAScog
score was 34.9 (SD: 9.5); and mean GDS score was 6.8
(SD: 5.5). There were no significant differences in base-
line characteristics between multivitamin users and
non-users.
Pill counts at the Week 8 visit indicated that com-
pliance with each component of the high-dose supple-
ment regimen exceeded 80%.
Table 1 shows the effect of treatment on fasting and
post–methionine-loading homocysteine levels. Overall,
there was a decline in both fasting and post–methionine-
loading homocysteine. As expected, change was greater
among non-users of multivitamins than users in fasting
homocysteine level (t[45.6]⳱2.80; p⳱0.008, using
a separate variance estimate), as well as in post–
methionine-loading homocysteine level (t[46.9]⳱2.31;
p⳱0.03, also using a separate variance estimate). The
change in fasting homocysteine level was shown to be
related to change in red blood cell folate (Pearson
r[64]⳱0.29; p⳱0.02), but not with change in B12 or
pyridoxal-5’-phosphate.
A paired t-test comparing baseline and 8-week
MMSE scores showed no treatment effect on MMSE:
mean baseline MMSE was19.2 (SD: 7.0), versus 8-week
MMSE of 19.3 (SD: 7.7); (t[62]⳱0.25; p⳱0.80).
In zero-order correlations conducted among study
variables, there was a correlation between baseline fast-
ing homocysteine level and age (r[66]⳱0.23; p⳱0.06);
there was no correlation between fasting homocysteine
levels and MMSE, ADAScog, or GDS. In a simultaneous
multiple-regression equation in which fasting homocys-
247
Vitamins and Homocysteine in AD

teine level, vitamin use, age, and gender were entered
as predictors, the most significant predictor of decline
in fasting homocysteine at 8 weeks was baseline fasting
homocysteine (partial correlation: r[60]⳱0.73;
p⳱0.001). In this equation, after controlling for base-
line fasting homocysteine, multivitamin use was not re-
lated to change in fasting homocysteine (r⳱ –0.15;
p⳱0.25), but age (r⳱0.28; p⳱0.03) and gender (r⳱
–0.174; p⳱0.05) each showed predictive value.
Subjects were divided into two groups by median
baseline fasting homocysteine level (cutpoint of 8.575
lmol/L). There was a significant decline in mean fasting
homocysteine levels with treatment in both the high
group (baseline: 11.7 [SD: 3.2] lmol/L; supplemented:
9.0 [SD: 2.4] lmol/L; t[32]⳱5.18; p⳱0.001; change:
23%) and the low group (baseline: 7.4 [SD: 1.1] lmol/
L; supplemented: 6.7 [SD: 1.3] lmol/L; t[32]⳱3.28;
p⳱0.003; change: 10%) in paired t-tests.
MTHFR analysis revealed that 29 (42%) of subjects
carried the CC genotype; 34 (49%) were heterozygous
CT, and 6 (9%) were homozygous TT. There was a sig-
nificant association between MTHFR genotype and
baseline fasting homocysteine levels in the overall one-
way ANOVA across the three genotype groups
(F[2,66]⳱5.67; p⳱0.005). In the post-hoc Tukey HSD
test to identify pairwise group differences, as expected,
the TT genotype group had a significantly higher mean
fasting homocysteine level (13.6 [SD: 4.8]), compared
with both the CC (9.0 [SD: 2.9]) and CT (9.4 [SD: 2.8])
groups. MTHFR genotype was not a significant predic-
tor of change in fasting homocysteine level with treat-
ment in an ANCOVA, with baseline homocysteine level
as a covariate: F[2,62]⳱1.71; p⳱0.189.
Adverse Events
There were no serious adverse events, and in no
instance was the vitamin regimen discontinued as a re-
sult of adverse events. In no instance was an adverse
symptom judged likely to be related to the study inter-
vention.
DISCUSSION
Treatment of patients with AD, living in the United
States, with a regimen of high-dose folic acid, B6, and
B12 for 8 weeks reduced plasma homocysteine. Fasting
and post–methionine-loading levels declined with this
treatment. The extent of decline was related to baseline
homocysteine levels. Subjects who were using multivi-
tamins at the time of enrollment showed decline in
plasma homocysteine with the treatment regimen, al-
though the decline was greater in non-users of multi-
vitamins. Similarly, subjects with baseline plasma ho-
mocysteine levels below and above the median showed

TABLE 1. Homocysteine and vitamin levelsa before and after supplementation in multivitamin users and non-users (Nⴔ66)
Baseline Supplemented Mean Change p
Fasting homocysteine 9.6 (3.2) 7.8 (2.3) 1.7 (2.5) 0.001
Post–methionine-loading homocysteine (n⳱62) 27.0 (10.2) 21.4 (5.9) 5.6 (9.3) 0.001
Red blood cell folate 434 (166) 819 (259) 385 (255) 0.001
Plasma B12 560 (343) 1,158 (514) 598 (408) 0.001
Plasma pyridoxal-5⬘-phosphate 107 (120) 314 (171) 207 (157) 0.001
Multivitamin non-users (n⳱35)
Fasting homocysteine 10.4 (3.8)b 7.9 (2.6) 2.5 (3.1) 0.001
Post–methionine-loading homocysteine (n⳱33) 29.9 (12.2)c 21.9 (5.7) 8.0 (11.2) 0.001
Red blood cell folate 361 (127) 834 (289) 473 (287) 0.001
Plasma B12 514 (345) 1,174 (567) 660 (434) 0.001
Plasma pyridoxal-5⬘-phosphate 55 (57) 284 (169) 229 (174) 0.001
Multivitamin users (n⳱31)
Fasting homocysteine 8.7 (2.1)b 7.8 (1.9) 0.9 (1.2) 0.005
Post–methionine-loading homocysteine (n⳱29) 23.7 (6.0)c 20.8 (6.2) 2.9 (5.3) 0.005
Red blood cell folate 516 (168) 802 (225) 286 (168) 0.001
Plasma B12 613 (339) 1,140 (456) 527 (363) 0.001
Plasma pyridoxal-5⬘-phosphate 166 (143) 349 (168) 183 (135) 0.001
Note: Values are mean (standard deviation).
a
Units: homocysteine: lmol/L; red blood cell folate, ng/ml; B12, pg/ml; pyridoxal-5⬘-phosphate, nmol/L; p values for paired t-tests.
b
Multivitamin non-users had higher fasting homocysteine levels at baseline than multivitamin users: t[62.1]⳱1.98; p⳱0.05.
c
Multivitamin non-users had higher methionine-loaded homocysteine levels at baseline than multivitamin users: t[52.0]⳱2.33; p⳱0.02.

248 Am J Geriatr Psychiatry 11:2, March-April 2003


decline in levels with the treatment regimen. Thus, de-
spite fortification of grain products with folic acid and
use of multivitamins containing the recommended daily
allowances of folic acid, B6, and B12, there was a reduc-
tion in plasma homocysteine in this population with the
use of high-dose supplements. Baseline homocysteine
levels were higher in C677T homozygotes (TT); how-
ever, significant decline in fasting and post–methionine-
loading homocysteine levels occurred in subjects with
each of the three genotypes.
The present study confirms that homocysteine re-
duction is feasible in the American AD population. Max-
imal reduction occurred with supplementation in sub-
jects with relatively high baseline levels. However,
reduction occurred in unselected AD patients, includ-
ing those taking multivitamin supplements. Whether
homocysteine reduction will slow AD progression must
be tested in a long-term, randomized, controlled, pro-
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