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Original Paper
Neuroimmunomodulation 2008;15:145–152
DO I: 10 .1159/0 00151527
Effect of Folic Acid Combined with Fluoxetine
in Patients with Major Depression on Plasma
Homocysteine and Vitamin B
12
, and Serotonin
Levels in Lymphocytes
G u s t a v o R e s l e r
a
Renée Lavie
a
Julio Campos
a
Salvador Mata
a
Mary Urbina
b
Alberto García
c
Rafael Apitz
c
Lucimey Lima
b
a Servicio de Psiquiatría, Hospital Vargas de Caracas, y
b Laboratorio de Neuroquímica, y
c Laboratorio de
Trombosis Experimental, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas,
Caracas , Venezuela
score was significantly lower in patients receiving fluoxetine
and folic acid compared with those receiving fluoxetine and
placebo after 6 weeks of treatment (7.43 8 1.65 v s. 11.43 8
1.31, respectively; p = 0.04). Plasma homocysteine statisti-
cally significant decreased after folic acid (p = 0.02), but no
significant changes were observed in vitamin B
12
. Results:
Serotonin was significantly reduced after fluoxetine either
with folate (p = 0.03) or placebo (p = 0.01) probably by the
effect of transporter blockade. 5-Hydroxyindoleacetic acid
was lower in lymphocytes of patients receiving folate (p =
0.04), indicating a reduced turnover rate, thus accumulating
serotonin in the cells. A significant negative correlation was
noted between homocysteine and folate. No significant cor-
relations were present among biochemical parameters and
depression severity. Conclusion: Modifications due to treat-
ment with fluoxetine and folic acid may alter lymphocyte
function in depression probably indirectly by reducing ho-
mocysteine levels and directly on lymphocytes by modify-
ing the serotonergic system. Copyright © 20 08 S. Karger AG, Basel
Key Words
Folic acid Fluoxetine Homocysteine Ly mphocytes
Major depression Serotonin
A b s t r a c t
O b j e c t i v e ( s ) : Folic acid, a micronutrient supporting the nat-
ural defense system, may elevate antidepressant responses,
although the lymphocyte serotonergic system has not been
explored in folate-supplemented depressed patients. Meth-
ods: Twenty-seven patients were randomly assigned to
groups receiving fluoxetine (20 mg) and folic acid (10 mg/
day) or fluoxetine and placebo for 6 weeks. Clinical outcome
was assessed according to the Hamilton Depression Rating
Scale (HDRS) at the beginning, during and at the end of treat-
ment. Blood samples were taken, plasma was separated, and
lymphocytes were obtained by density gradient centrifuga-
tion with Ficoll/Hypaque and differential adhesion to plastic
dishes. Fifteen healthy subjects served as controls. Plasma
folate, homocysteine and vitamin B
12
, and serotonin concen-
tration in lymphocytes were determined by HPLC. The HDRS
Re ceived: January 25, 200 8
Accepted afte r revision: May 9, 2008
Published o nline: August 21, 2008
Dr. Lucimey Lima
Laborator io de Neuroquímic a, Centro de Biofísica y Bioquím ica
Instituto Venezolano de Investigaciones Científ icas
Apdo. 21827, Caracas 1020 -A (Venezuela)
Tel. +58 212 504 1213, Fax +58 212 504 1295, E-Mail lli ma@ivic.ve
© 20 08 S. Karger AG, Bas el
1021–7401/08/0153–0145$24.50/0
Accessible online at:
www.karger.com/nim
Resler /Lavie /Campos /Mata /Urbina /
García
/Api tz /Lima
Neuroimmunomodulation 2008;15:145–152
146
Introduction
The administration of vitamins and other natural
stimulants for the treatment of depressive symptoms has
been common practice for several years. Physicians ob-
served empirically the clinical improvement without
knowledge of the underlying biological mechanisms
[1] .
More recently, vitamins have been found to act as cofac-
tors of enzymatic reactions, drawing attention to their
involvement in various diseases, e.g. in psychiatric disor-
ders
[2–5] .
Major depressive disorders are one of the main causes
of d isability in the world, with significant negative socio-
economic consequences
[6, 7] . Based on the theory ex-
plaining mood disorders, especially depression, with im-
balanced biogenic amine synthesis, e.g. serotonin (5-HT)
and catecholamines, and ascribing folic acid the role of
an indirect cofactor, it is reasonable to think that their
supplementation could result in increased neurotrans-
mitter synthesis and thus improve the clinical response
to antidepressant treatment
[3, 4, 8–14] .
In humans, fruits and vegetables constitute the pri-
mary nutritious source of folate, with a recommended
daily intake of 400 g, but this requirement is elevated
during periods of high metabolic demand, for example in
pregnancy
[15, 16] . Tetrahydrofolate (THF) is transport-
ed to the cell interior by a specific transporter, and the
safety of daily supplements of 5–15 mg folic acid has been
reported
[17 ] .
The main function of folic acid is to trigger biochemi-
cal reactions to transfer 1-carbon group of methyl and
formyl. Serine is the main source of these groups reacting
with THF to produce glycine and N-5,10-methylene-
THF. These by-products constitute a source of carbon
group donors tr ansfe rring it to a n appropr iate receptor to
form metabolic intermediates and finally precursors for
de novo synthesis of macromolecules, e.g. purines, de-
oxythymidylate monophosphate and methionine. The
latter is formed by the transfer of the methyl group from
N-5-methyl-THF to form methyl-homocysteine. Folate
deficiency restricts the methylation of homocysteine to
methionine, a reaction which needs vitamin B
12 [18 , 19] .
In vit ro studies have revealed that folate deficiency in-
creases the risk of depression
[11, 14, 20] . Folate metabo-
lism is linked to biopterin-dependent neurotransmitter
synthesis, to biogenic amine methylation and to phos-
pholipid synthesis in the central nervous system
[5, 18] .
Homocysteine or its metabolites have excitotoxic effects
through glutamate N-methyl-
D -aspartate receptors or by
the inhibition of S-adenosyl-methionine-dependent bio-
genic amine methylation and the formation of S-adeno-
syl-homocysteine. Some authors proposed that S-adeno-
syl-methionine could be superior to placebo in the treat-
ment of depression
[20] .
Pauling [21] presented the hypothesis that many men-
tal illnesses are due to insufficient concentrations of cere-
bral cofactors as a result of an insufficient consumption
of micronutrients and that some cerebral dysfunctions
may be due to mutations that affect the affinity constant
of the enzyme substrates. For instance, the polymorphism
677C ] T of methylene-THF reductase has been exam-
ine d in p atient s w ith s chiz ophr enia , major dep ress ive di s-
orders and bipolar disorders. The TT gene variant of the
methylene-THF reductase was found in 12% of 419 healthy
controls, 21% of 297 patients with schizophrenia, 28% of
32 patients with major depression and 13% of 40 patients
with bipolar disorders
[22] . Melancholic depressions oc-
cur more often in subjects with decreased levels, having a
bad response to fluoxetine administration
[2, 3, 12, 14,
18]
. Recently, the relationship between folate and depres-
sion has been considered a neglected problem
[23] .
For the above reasons and the lack of information on
cellular peripheral 5-HT in folate-supplemented pa-
tients, the main objective of the present work was to ex-
plore possible modifications in 5-HT turnover in lym-
phocytes induced by folic acid supplementation which
may be related to its immune function. In addition, sec-
ondary aims were: (1) to study the effect of folic acid ad-
ministration on the antidepressant response to fluox-
etine, and (2) to determine plasma levels of folate, homo-
cysteine and vitamin B
12
before and after 6 weeks of
treatment, in order to correlate these parameters with
the severity of depression.
Patients and Methods
From 76 patients consulting for depression at the Servicio de
Psiquiatría, Hospital Vargas de Caracas, from May to December
2006, 27 outpatients (23 women and 4 men) (21–58 years old,
35.04 8 2 .63) recently dia gnosed with a major depressive epis ode,
in the absence of other psychiatric or medical conditions, and
without psychotic symptoms or risk of suicide were studied. Pa-
tients treated with any antidepressant or vitamin during the 30
days prior to study entry were excluded. Each patient was in-
formed about the aims and implications of the study and was re-
quested to sign an informed consent according to the Ethic Com-
mittees of the Hospital and the Institute. Diagnosis was estab-
lished usi ng the clinica l version (SCID-I) of the depression sec tion
of the Structured Clinical Interview for Axis I Disorders (Spanish
version) of the Diagnostic and Statistical Manual of Mental Dis-
orders (DSM-IV)
[24] . This questionnaire is supported by the di-
Effect of Folic Acid Supplementation on
Major Depression
Neuroimmunomodulation 2008;15:145–152
147
agnost ic criteria for major depression of the America n Psychiatric
Association (DSM-IV)
[25] . The clinical severity of depressive
symptoms was assessed at the beginning and every other week (0,
3 and 6 weeks) using the 17-item Hamilton Depression Rating
Scale (H DRS)
[26] , validated i n Spain, in which a score 6 18 po int s
suggests mild, moderate or severe episodes
[27] . A 50% reduction
in the HDRS score with respect to the basal score was considered
as response
[28] . The control group was composed of 15 appar-
ently healthy subjects (9 women and 6 men; 26–49 years old,
mean: 34.13 8 2.05) without any personal or family history of
psychiatric diseases. According to the modified Graffar classifi-
cation, all subjects belonged to class III (low middle class) or IV
(worki ng cla ss)
[29] . Consumption of caffeine was moderate
(maximally two small cups per day), alcohol occasional and to-
bacco sporadic in a few of them.
Patients were randomly assigned to two groups in a blind
manner using a lottery procedure. One of the two groups com-
prised 14 individuals, who received 20 mg/day of fluoxetine plus
10 mg of folic acid during 6 weeks, while the second group, 13 pa-
tients, was treated with 20 mg of fluoxetine plus placebo during
the same t ime period. Fluoxeti ne was given in the form of g rooved
tablets of 20 mg of fluoxetine hydrochloride. Folic acid and pla-
cebo were administered as gelatin capsules containing 10 mg of
the vitamin
[2] or a similar quantity of inert powder. They were
provided by the physician every 2 weeks in plastic bags prepared
by non-medical personal and containing the exact amount to be
taken during that period of time.
Folic Acid, Homocysteine and Vitamin B
12
Determination in
Plasma
The determinations were performed at the beginning (day 0)
and at the end of the study (week 6). The samples were labeled by
continuous numeration for blindness of biochemical perfor-
mance and calculations. Peripheral vein blood was sampled be-
tween 7 and 8 a.m. and collected in a BD Vacutainer tube with
heparin as anticoagulant (1,000 U/ml). After centrifugation at
1,500 g , plasma was obtained and further centrifuged at 4
° C,
38,000 g , for 10 min to obtain platelet-poor plasma. The samples
were frozen at –80
° C until assayed. Folic acid, homocysteine and
vitamin B
12
were determined by high-performance reverse-phase
liquid chromatography (HPLC), as previously described
[30] .
Serotonin and 5-Hydroxyindoleacetic Acid in Lymphocytes
Blood (20 ml) was centrifuged at 500 g with a vasculant rotor
for 10 min at room temperature. The layer of white plus some red
blood cells was taken and transferred to tubes with 0.1
M phos-
phate-buffered saline solution (PBS, pH 7.4) and centrifuged at
160 g for 10 min at room temperature. The white layer was col-
lected, suspended in 10 ml of PBS, placed on 5 ml of Ficoll/
Hypaque (1,077 g/l) and, af ter centrifugat ion at 1,500 g for 30 min,
peripheral blood mononuclear cells were collected and placed on
plastic tissue culture dishes to obtain nonadherent lymphocytes
(85 8 5% CD3+). Cells were counted in a hemocytometer and
di lut ed i n R PMI 1640 to rea ch a spe cifi c ce ll c onc ent rat ion . In teg-
rity of the membrane was determined by trypan blue dye exclu-
sion (viability 6 96%)
[31] . The cells were suspended in HPLC
buffer solution, homogeni zed in Tissumiz er (Tekmar, Cincinnat i,
Ohio, USA) and centrifuged at 38,000 g for 10 min; the superna-
tant was analyzed for 5-HT and 5-hydroxyindoleacetic acid (5-
HIAA). The system was composed of an integrator and a pump
(Waters HPLC model 600; Waters, Minnesota, USA), an auto-
matic injector (Waters model 7171), a Supelco LC-18 column (15
cm ! 4.6 mm inside diameter, average particle size: 5 m) and
an electrochemical detector (Waters model 464; +0.7 V, 0.2 nA).
The mobile phase was 0.02
M sodium acet ate, 0.0125 M citric acid,
1 m
M EDTA, 1.52 m M octanyl sulfonate, pH 3.9, plus 10% aceto-
nitri le
[31] . The a mount present in the sample was ca lculated from
the area under the curve of samples and external standards with
the program Millenium (Waters). Concentrations are expressed
in nanograms per 10
6
cells.
Statistical Analysis
At the end of the study, the identification of patients with cor-
responding samples was done for performing corresponding
analy sis. Data were expressed a s means 8 SEM, and p ^ 0.05 was
considered statistically significant with a confidence interval of
95%. Data before treatment and differences between groups were
analyzed using Student’s t test. The nonparametric Mann-Whit-
ney U test was utilized to analyze treatment effects. Linear cor-
relation ana lysis was performed b etween parameters a nd between
parameters and severity of depression. The data were classified
and analyzed using the Statistical Package for Social Sciences
(SPSS, Chicago, Ill., USA).
R e s u l t s
P a t i e n t s
Depressive patients with concomitant medical illness-
es, e.g. hypertension, diabetes or allergies, were not in-
clu de d i n t he pr es en t s tu dy. Twe nt y of t he 27 pa ti en ts co m-
0
5
10
15
20
25
HDRS score
024
Placebo
Folic acid
6
*
Weeks of treatment
F i g . 1 . Mean HDRS scores at the beginning, and 2, 4 and 6 weeks
after t reatment with f luoxetine (20 mg/day) and placebo, or f luox-
etine (20 mg/day) and folic acid (10 mg/day) * p ! 0.05 vs. place-
bo.
Resler /Lavie /Campos /Mata /Urbina /
García
/Api tz /Lima
Neuroimmunomodulation 2008;15:145–152
148
pleted the 6-week study; the remainder abandoned the
study but reported partia l amelioration of symptoms after
3–4 weeks of treatment, with a 30% reduction in the
HDRS score. Main symptoms were sadness, anhedonia,
anorexia, insomnia and anxiety. No side effects were re-
ported by this group of patients. At the beginning of the
study, the median HDRS scores were 22.50 8 0.98 for the
folate group and 21.85 8 0.94 for the placebo group (p =
0.32). At the end of the study (week 6), both groups pre-
sented clinical improvement, with a statistically signifi-
cant decline in the HDRS score in both groups ( fig. 1 ). The
average final HDRS scores were 7.43 8 1.65 for the folate
group and 11.43 8 1.31 for the placebo group (p = 0.04).
Plasma Levels of Folic Acid, Homocysteine and
Vitamin B
12
There were no significant differences in the plasma
folate concentrations between the groups at the begin-
ning of the study. Folate concentration was markedly in-
creased in the folate-supplemented group, with values of
9.22 8 1.97 to 47.81 8 6.66 n
M (p = 0.0005) versus 9.10
8 1.66 and 11.61 8 3.53 n
M in the placebo group (non-
significant), but the 6-week folate concentration was sig-
nificantly lower in the placebo group compared with the
folate-supplemented group (p = 0.0006; fig. 2 ). The dif-
ference in plasma homocysteine concentrations among
the groups at study entry was not statistically significant
0
10
20
30
40
50
60
Folic acid (n )
M
Placebo Folic acid Control
Before
After *
0
2
4
8
10
12
14
Homocysteine (µ )
M
Placebo Folic acid Control
Before
After
*
6
0
400
200
600
800
1,200
1,400
Vitamin B (p )
12 M
Placebo Folic acid Control
Before
After
1,000
F i g . 2 – 4 . Folic acid ( 2 ), homocysteine ( 3 ) and vitamin B
12
concen-
trations (
4 ) in t he plasma of the control group and i n the depressed
patients treated with fluoxetine (20 mg/day) and placebo, or
fluoxetine (20 mg/day) and folic acid (10 mg/day) for 6 weeks.
Plasma was obtained before treatment initiation and at the end of
week 6. * p ! 0.05 vs. before.
2 3
4
Effect of Folic Acid Supplementation on
Major Depression
Neuroimmunomodulation 2008;15:145–152
149
(9.49 8 0.7 p M) . However, levels were significantly de-
creased in the folate group at study completion (7.35 8
0.61 p
M; p = 0.02; fig. 3 ). Vitamin B
12
demonstrated intra-
and intergroup variations, but no statistically significant
differences were found ( fig. 4 ).
5-HT and 5-HIAA Concentrations in Lymphocytes
5-HT did not significantly differ among the placebo,
folic acid or control groups. The administration of fluox-
etine with (p = 0.03) or without (p = 0.01) folate supple-
mentation reduced the 5-HT concentration in lympho-
cytes ( fig. 5 a). 5-HIAA was not significantly different
between the patients and the controls; however, the me-
tabolite was markedly decreased in the folate-supple-
mented group (p = 0.04; fig. 5 b). The 5-HT/5-HIAA ratio
was lower in depressed patients; however, there was a
wide variation in the control group, with no effect of
treatment ( fig. 5 c).
Discussion
The majority of patients included in this study were
female, in agreement with epidemiological findings on
a worldwide basis indicating that the number of women
diagnosed with depressive disorders surpasses that of
0
500
1,500
2,000
3,000
4,000
5,000
Placebo Folic acid Control
Before
After
*
1,000
2,500
4,500
3,500
*
5-HT (pg/10 cells)
6
a0
100
200
300
400
500
600
Placebo Folic acid Control
Before
After
*
5-HIAA (pg/10 cells)
6
b
0
20
40
60
80
100
120
5-HT/5-HIAA
Placebo Folic acid Control
Before
After
c
F i g . 5 . Concentrations of 5-HT ( a ) and 5-HIAA ( b ), and 5-HT/5-
HIAA ratio (
c ) in lymphocytes of the control group and in the
depressed patients treated with fluoxetine (20 mg/day) and pla-
cebo, or fluoxetine (20 mg/day) and folic acid (10 mg/day) for 6
weeks. * p ! 0.05 vs. corresponding value before treatment.
Resler /Lavie /Campos /Mata /Urbina /
García
/Api tz /Lima
Neuroimmunomodulation 2008;15:145–152
150
men and that women are more comfortable seeking help
[32, 33] . The severity of depressive symptoms was com-
parable between both groups and significant differences
in HDRS scores were lacking. In patients receiving fo-
late supplementation, symptoms more often improved,
but the difference was only significant at the end of
treatment (week 6), in agreement with previous reports
[2, 34] , although the timing of response differed [18]
since the severity of depression did not differ between
both groups at the middle of the evaluation. Thus, in
this study, combination therapy with fluoxetine and fo-
lic did not accelerate the antidepressant response, but
augmented it. Possibly a longer treatment duration may
increase the beneficial effects of folate. In addition, in
contrast to other studies
[11, 12, 18] , folic acid concen-
tration in plasma was not decreased in our depressive
patients.
Folic acid supplementation may beneficially affect
chronic inflammatory diseases probably via a reduction
in homocysteine levels; in addition, it has been associated
with the prevention of non-fatal strokes, preservation of
cognitive function, immune function and psychiatric
disorders, e.g. depression
[23, 35] . This vitamin is re-
quired for pterin biosynthesis, and in depressed patients
there is a positive correlation between pterins and folate
[5] ; therefore, depressed patients might need folic acid
supplementation.
In our study, homocysteine levels were inversely pro-
portional to the levels of folate, which may be explained
by the need of methyl groups (e.g. from folate) for me-
thionine synthesis and homocysteine methylation. How-
ever, an association between homocysteine levels and
clinical changes was not found, which is in contrast to a
previous study indicating that high levels of homocyste-
ine may be neurotoxic and induce symptoms associated
with decreased biogenic amine synthesis
[20] , and, con-
sequently, decreased homocysteine levels may improve
symptoms and prevent the risk of cardiovascular disease,
which is frequently present in depressed patients
[36 –
39]
.
Interestingly, in a multiple regression analysis of an-
ger attacks adjusted for symptoms of depression, creati-
nine, vitamin B
12
, folate, age, smoking and alcohol, se-
rum levels of homocysteine were positively correlated
with the duration of the depressive episode
[40] . Defi-
ciencies in folate, vitamin B
12
, iron, zinc and selenium
we re more fr eque ntly rep or te d in d epr es sed p atie nts
[41] .
In addition, previous studies reported associations be-
tween low serum folate, but not vitamin B
12
and homo-
cysteine levels, and delayed onset of response to fluox-
etine in major depression [18] , the risk of relapse and re-
sistance to antidepressive treatment
[38, 39] . Serum and
red blood cell folate as well as plasma vitamin B
12
levels
were decreased and plasma homocysteine levels were in-
creased in depression
[42] . Moreover, white matter hy-
persensitivity in the brain was related to high homocys-
teine levels and negatively associated with folate
[43] . It
is interesting to note that vitamin B
6
, a cofactor of amino
acid transamination and decarboxylation, was also in-
volved in depression, since low plasma levels of vitamin
B
6
were associated with depression score, which encour-
age examining the role of the B complex in depression
and probably in other psychiatric disorders, too. Al-
though the therapeutic dose of folate may vary consider-
ably
[17, 23 ] , high doses may mask vitamin B
12
deficiency,
an effect not observed in the present work using pharma-
cological supplementation of folic acid. Although fur-
ther studies on the beneficial effects of folate are re-
quired, it has been suggested that 2 mg per day should be
given in major depression
[11] . This relatively elevated
dose of folate for patients with major depression may be
explained on the one hand by their lower dietary folate
intake according to their social class, and on the other
hand the raised need for folate due to depression. The
cap ac it y of h um an s f or p re se rv ing fola te is hi gh , a nd nor-
mal plasma values may be maintained even with cell de-
ficiency.
Fluoxetine administration resulted in decreased 5-HT
levels in lymphocytes after 6 weeks, despite the synaptic
increase in 5-HT availability by transporter blockade; the
observed results could be the consequence of its increased
catabolism to 5-HIAA and the corresponding excretion.
Increased levels of 5-HT in the culture medium of lym-
phocytes from depressed patients resulted in increased
proliferation
[44, 45] . However, only in lymphocytes of
patients receiving folic acid, a reduction in 5-HIAA was
noted, probably indicating the protection of 5-HT from
metabolism. Recent biochemical observations indicate a
modification of lymphocyte serotonergic system by folic
acid at the level of turnover rate, but obviously further
research is needed to document functional effects, since
these parameters have not been studied in immune cells
of depressed patients.
Thus, the reduction in 5-HT turnover may affect lym-
phocyte function via autocrine effects on 5-HT receptors.
Previously, increased lymphocyte proliferation probably
due to 5-HT
1A
receptor activation [44] , in addition to a
decreased 5-HT turnover rate and a decreased number of
5-HT transporters, were reported
[46] , possibly affecting
the immune response mediated by these cells. If folate
Effect of Folic Acid Supplementation on
Major Depression
Neuroimmunomodulation 2008;15:145–152
151
administration reduces homocysteine levels and also
modifies the effect of 5-HT on the immune system, co-
administration of folate in antidepressant treatment may
be of increasing interest.
A c k n o w l e d g m e n t s
This work was supported by the Fondo de Ciencia Tecnología
e Innovación (FONACIT) G-1387, Venezuela. We appreciate the
secretarial assistance of Mrs. Carolina Flores.
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