Down-regulation of reduced folate carrier may result in
folate malabsorption across intestinal brush border
membrane during experimental alcoholism
Abid Hamid
1
, Nissar Ahmad Wani
1
, Satyavati Rana
2
, Kim Vaiphei
3
, Akhtar Mahmood
4
and
Jyotdeep Kaur
1
1 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Biochemistry, Panjab University, Chandigarh, India
Keywords
alcoholism; brush border membrane; crypt–
villus axis; methylation; reduced folate
carrier
Correspondence
J. Kaur, Department of Biochemistry,
Postgraduate Institute of Medical Education
and Research, Chandigarh 160 012, India
Fax: +91 172 2744401 ⁄ 2745078
Tel: +91 172 2747585 5181
E-mail:

derived from ethanol-fed rats. Parallel changes were observed in reduced
folate carrier protein levels in brush border membrane along the entire
crypt–villus axis. In addition, immunohistochemical staining for reduced
folate carrier protein showed that, in alcoholic conditions, deranged
reduced folate carrier localization was observed along the entire crypt–vil-
lus axis, with a more prominent effect in differentiating crypt base stem
cells. These changes in functional activity of the membrane transport sys-
tem were not caused by a general loss of intestinal architecture, and hence
can be attributed to the specific effect of ethanol ingestion on the folate
transport system. The low folate uptake activity observed in ethanol-fed
rats was found to be associated with decreased serum and red blood cell
folate levels, which might explain the observed jejunal genomic hypomethy-
lation. These findings offer possible mechanistic insights into folate mal-
absorption during alcoholism.
Abbreviations
BBM, brush border membrane; BBMV, brush border membrane vesicle; LAP, leucine aminopeptidase; RBC, red blood cell;
RFC, reduced folate carrier; SAM, S-adenosyl methionine.
FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS 6317
The mechanism of folate transport is under extensive
investigation because mammals require the ingestion
and absorption of preformed folates in order to
meet their needs for one-carbon moieties to sustain
key biosynthetic reactions [1]. In addition, the cellu-
lar concentration of folate cofactors, in different
oxidative states, governs the intricate network of
methylation reactions of DNA, RNA, proteins and
phospholipids [2]. The most well-characterized folate
transporter, the reduced folate carrier (RFC), is an
integral membrane protein of  65 kDa that medi-
ates the cellular uptake of reduced folates and anti-

(BBM) were calculated, and the mRNA and protein
expression of a major folate transporter, RFC, was
studied. The investigation of the regulation of folate
transport via RFC expression in absorptive epithelia
may aid in the development of future therapeutic
strategies targeting the regulatory protein. In addi-
tion to alcoholism, folate malabsorption has also
been reported to occur in several intestinal diseases,
congenital disorders of the folate transport system,
drug interactions and intestinal resection, and may
involve similar mechanisms.
Results
There was no significant decrease in body weight of
ethanol-fed rats relative to the control group during
the course of the experiment. At the time of killing,
the mean body weights of rats in control and ethanol-
fed groups were 201 ± 8 and 196 ± 9 g, respectively.
Estimation of blood alcohol levels
In order to establish the suitability of the rat model
for studies on experimental alcoholism using our
experimental set-up, the blood alcohol level was a pre-
requisite parameter. It was found that the alcohol level
was 88% higher ( P < 0.001) in the chronic ethanol-
fed group than in the control group. The mean
blood alcohol levels were 15.04 ± 1.96 and
1.77 ± 0.34 mgÆdL
)1
in the ethanol-fed and control
groups, respectively.
Purity of membrane vesicles

incubating BBMVs for various time intervals, was
found to be at a maximum at 30 s in both control and
ethanol-fed groups, as described previously [15]. For
further experiments, a 30 s time interval was chosen for
the determination of the initial uptake. Moreover,
[
3
H]folic acid uptake revealed no significant difference
between fresh and frozen vesicles. In the control group,
the uptake was observed to be 36.20 ± 3.20 and
35.29 ± 2.20 pmolÆ(30 s)
)1
Æmg
)1
protein in fresh and
frozen BBMVs, respectively, in comparison with
19.69 ± 1.90 and 19.06 ± 2.81 pmolÆ(30 s)
)1
Æmg
)1
protein in the ethanol-fed group. Therefore, for further
studies, frozen reconstituted vesicles were used.
Intestinal folate malabsorption in alcoholism A. Hamid et al.
6318 FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS
[
3
H]Folic acid uptake
For all the assays, except folic acid transport during
the course of the study, BBMVs were isolated at the
end of 3 months of treatment.

and V
max
for folic acid transport were determined
from the Lineweaver–Burk plot (Fig. 3, inset). The K
m
values for control and ethanol-fed groups were found
to be 0.90 ± 0.08 and 1.53 ± 0.09 lm (P < 0.01),
respectively. The V
max
values for control and ethanol-
fed groups were found to be 100 ± 5.60 and
83 ± 3.65 pmolÆ(30 s)
)1
Æmg
)1
protein (P < 0.05),
respectively.
Folate transport across the crypt–villus axis
of the intestine
The cell fractions (F1–F9) were isolated from the
small intestine of both groups of rats at the end of
AB
Fig. 1. Electron micrographs (· 60 000) of
representative BBMVs with uniform shape
showing sealed outer surfaces and ‘right
side out’ orientation: (A) control group;
(B) ethanol-fed group.
0
10
20

M
)
***
**
***
**
V(pmol/30 sec/mg protein)
Fig. 3. [
3
H]Folic acid uptake in intestinal BBMVs as a function of
substrate concentration (inset Lineweaver–Burk plot). Uptake was
measured by varying the [
3
H]folic acid concentration from 0.125 to
1.50 l
M in an incubation medium of pH 5.5 after incubating BBMVs
for 30 s. Each data point is the mean ± standard deviation of eight
separate uptake determinations carried out in triplicate. **P < 0.01,
***P < 0.001 versus control.
A. Hamid et al. Intestinal folate malabsorption in alcoholism
FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS 6319
3 months of treatment, and were characterized by an
approximate eight-fold decrease in specific activity of
the villus cell marker enzyme alkaline phosphatase
from F1 (villus tip) to F9 (crypt base) (data not
shown). In addition, isolated epithelial cells were
characterized by measuring the DNA content and
[
3
H]thymidine incorporation into DNA of various

internal control); products of 489 and 588 bp for RFC
and b-actin, respectively, were obtained on electropho-
resis using a 1.2% agarose gel. From densitometric
analysis, it was deduced that the expression of mRNA
coding for RFC was three-fold lower during chronic
ethanol feeding (Fig. 4A,B). Thus, ethanol imparts its
effect through transcriptional regulation of RFC at the
primary absorptive site of folic acid, i.e. the small
intestine.
Expression of the RFC protein in BBM of the
intestine
The effect of chronic alcoholism on the level of expres-
sion of the RFC protein at the BBM surface was
studied by western blot analysis. Analysis of purified
BBMVs was performed to identify RFC using
polyclonal antibodies raised against a specific region of
rat RFC; reactivity was found at approximately
65 kDa. Moreover, there was no cross-reaction of
RFC antibodies against any protein in the vesicular
preparations used. Antisera against the leucine amino-
peptidase (LAP) showed reactivity at 80 kDa, which
served as an internal control. LAP is a membrane-
bound aminopeptidase whose activity has been found
to be unaffected by chronic ethanol feeding [18,19].
The expression of the RFC protein was observed to be
2.3-fold higher in BBMVs from the control group rela-
tive to those from the chronic ethanol-fed group
(Fig. 5A,B). When studied along the crypt–villus axis
in BBMVs isolated from different cell types from the
two groups of rats, maximum RFC expression in the

experiments. ***P < 0.001 versus control. Lanes 1, 2, control;
lanes 3–5, ethanol; lane 6, negative control.
Intestinal folate malabsorption in alcoholism A. Hamid et al.
6320 FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS
RFC distribution and localization across the
intestinal vertical axis
The distribution pattern of RFC protein was deter-
mined by immunohistochemical localization. In control
rats, localization of RFC was mainly seen along the
epithelial cells of the villus lining; stronger expression
was localized along the tip epithelial cells and towards
the enterocyte brush border, and positive cells were
visible up to the base of the villi, i.e. at the villus–crypt
junction. However, there was a gradual decrease in
intensity from villus to crypt cells (Fig. 7A). In etha-
nol-fed rats, there was a marked decrease in the inten-
sity of positively stained cells; only a few cells along
the tip of the villi and mid-villus showed positivity
(Fig. 7B). No staining was detected in the sections
incubated with only secondary antibody. Furthermore,
RFC protein was not detected in the lamina propria,
muscularis mucosa, submucosa, muscularis externa or
smooth muscle cells of the small intestine (data not
shown).
Histochemical assessment of jejunal sections
After visualizing the slides under a light microscope,
no changes in intestinal architecture were observed
in the intestinal tissues from control (Fig. 8A) and
ethanol-fed (Fig. 8B) rats. However, ethanol-fed
rats showed mucodepletion and an increase in intra-

fold more SAM relative to that from the control
80kDa
12
~65kDa
Control
0
1
2
3
4
5
Ethanol
***
Mean relative RFC protein
levels (RFC/LAP)
A
B
Fig. 5. (A) Western blot analysis of intestinal BBMVs using anti-
RFC (65 kDa) and anti-LAP (80 kDa) IgG. (B) Densitometric analysis
representing the relative change in RFC protein levels. Data shown
are the mean of eight separate sets of experiments. Lane 1, con-
trol; lane 2, ethanol. ***P < 0.001 versus control.
80kDa
~65kDa
12345
Villus tip
Mid villus
Crypt base
Control
1.00

fed rats.
Discussion
Chronic alcoholism is often associated with folate defi-
ciency, which is mainly a result of malabsorption of
folate across the intestinal membrane [12,20]. In a rat
model of experimental alcoholism, we examined the
mechanism of the regulation of folate transport medi-
ated by RFC, the major folate transporter protein in
the intestine. It was observed that a significant concen-
tration of blood alcohol was maintained when deter-
mined 24 h after the last dose of ethanol of 1 gÆkg
)1
body weight per day at the end of a 3 month course.
Such a dose was chosen according to earlier studies
[21], which suggested that the ethanol concentration of
jejunal tissue should not exceed 6% in animal experi-
ments in order to be relevant to the human intestine.
In the present study, 1 gÆkg
)1
body weight of ethanol
AB
Fig. 7. Immunohistochemical analysis of rat
jejunal sections exposed to anti-RFC IgGs,
showing relative localization and distribution
pattern of RFC protein (as depicted by
brown counterstaining of haematoxylin)
along the intestinal absorptive axis. Figures
(· 450) shown are representative of each
group: (A) control; (B) ethanol.
AB

clinical signs of intoxication [22].
A significant decrease in folic acid uptake by
BBMVs in the chronic ethanol-fed group, which
appeared even after 1.5 months of treatment, suggests
that ethanol feeding has a profound malabsorptive
effect on folate uptake, which may be of biological sig-
nificance. The decrease was associated with an increase
in K
m
and a decrease in V
max
, suggesting that both the
affinity of the transporter and the number of trans-
porter sites on BBMVs are reduced after chronic etha-
nol ingestion. The increase in K
m
may also suggest
that an alternative route of folate transport is opera-
tional after chronic ethanol feeding. These observa-
tions confirmed an earlier study which was carried out
at toxic blood alcohol levels in the micropig model of
chronic alcoholism [20]. In order to evaluate the mech-
anism of reduced folate uptake, the expression profile
of RFC was of prime importance, as RFC is believed
to be a major folate carrier responsible for intestinal
folate absorption [15,23], although recently a proton-
coupled folate transporter has been found to play an
essential role in folate absorption in the intestine [24].
The decreased V
max

data). Earlier studies carried out in models of dietary
folate deficiency support our findings that transcrip-
tional regulatory mechanisms operate in the folate
transport system via RFC [17,26].
The role of RFC regulation across the crypt–villus
axis during alcoholism was evaluated. It was observed
that the apical membrane folate transport activity was
greatest in differentiated upper villus cells, followed by
differentiating mid-villus cells, and lowest in proliferat-
ing cells, and the proportional distribution of the RFC
protein was found along the entire crypt–villus axis.
These results were in accordance with earlier studies
[27], where a similar RFC distribution was shown to
exist across the crypt–villus axis. Importantly, chronic
ethanol feeding decreased RFC protein expression
along the entire crypt–villus axis. In addition, the
higher level of RFC protein in villus tip cells suggests
that a larger number of folate transporters are
expressed at the villus tip and that the redistribution
of RFC occurs with the maturation of intestinal stem
cells. Such findings correlate with the observed higher
rate of folate uptake in villus tip cells relative to crypt
base cells. A similar distribution has been reported
previously for biotin uptake [28].
Consistent with immunoblot analysis, immunohisto-
chemical staining revealed RFC localization along the
entire crypt–villus axis; moreover, staining was signifi-
cantly more intense in epithelial cells lining the villus
tip and decreased towards the crypt–villus junction in
the control group. A stronger expression was observed

ciated with hyperhomocysteinaemia and lower levels of
SAM. The low folate levels result in low SAM levels
which, in turn, may influence DNA methylation, as
reflected by the observed hypomethylated jejunal DNA
in alcohol-fed rats. Our study is in agreement with that
of Choi et al. [29], who observed hypomethylation of
colonic mucosal DNA in rats after chronic ethanol
ingestion, although no systemic folate reduction was
observed, by contrast with our study. Such a discrep-
ancy may be attributed to the different methods
employed for ethanol administration and the restric-
tion of the study to 4 weeks only, in comparison with
3 months in our investigation. Regardless of how
chronic ethanol ingestion produces genomic DNA
hypomethylation of jejunal tissue in rats, it may have
implications regarding the mechanism(s) by which
chronic alcohol exposure increases the risk of different
cancers in humans.
Taken together, the results show that chronic etha-
nol ingestion leads to decreased intestinal BBM folic
acid uptake and reduced jejunal mRNA levels encoded
by RFC, resulting in low RFC protein levels and
recruitment along the entire BBM of the crypt–villus
axis. The decreased transport efficiency of intestinal
BBM is reflected in reduced serum and RBC folate lev-
els, which may result in the observed hypomethylation
of jejunal DNA.
Experimental procedures
Chemicals
Radiolabelled [3¢,5¢,7,9-

membrane filters (0.45 lm) were obtained from Millipore
Corporation (Bedford, MA, USA).
Animals
Young adult male albino rats (Wistar strain), weighing
100–150 g, were obtained from the Postgraduate Institute
of Medical Education and Research’s Central Animal
House (Chandigarh, India). The rats were housed in clean
wire mesh cages with controlled temperature (23 ± 1 °C)
and humidity (45–55%) and with a 12 h ⁄ 12 h dark ⁄ light
cycle throughout the study. The rats were randomized into
two groups of eight animals each, such that the mean body
weights and range of body weights for each group of ani-
mals were similar. The rats in group I were given 1 gÆkg
)1
body weight of ethanol (20% solution) per day for
3 months, and those in group II received an isocaloric
amount of sucrose (36% solution) orally by Ryle’s tube
daily for 3 months. Such a dose does not produce a toxic
blood alcohol concentration [21] and is therefore relevant
to human studies. The rats were fed a commercially avail-
able pellet diet (Ashirwad Industries, Chandigarh, India)
containing 2 mgÆkg
)1
folic acid and water ad libitum. The
body weights of the rats were recorded twice weekly.
Animals from both groups were killed under anaesthesia
using sodium pentothal, and blood was drawn from the tail
vein for alcohol and folate estimations. Starting from the
ligament of Treitz, two-thirds of the small intestine was
removed, flushed with ice-cold saline and processed for the

6324 FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS
d,l-dithiothreitol in NaCl ⁄ P
i
, and kept at 37 °C in a shaker
at 100 r.p.m. for 30 min; the solution was then collected for
the isolation of total enterocytes. Furthermore, small intes-
tinal epithelial cells enriched in enterocytes of different ori-
gins along the crypt–villus axis were also isolated. In this
case, different cell fractions were collected after filling the
intestine for different time intervals. Fractions 1–3 were col-
lected at 4, 2 and 2 min intervals, fractions 4–6 at 3, 4 and
5 min intervals, and fractions 7–9 at 7, 10 and 15 min inter-
vals. Each consecutive three fractions were pooled and
represented the villus tip, mid-villus and crypt base cells,
respectively. The collected cells were centrifuged at 800 g
for 15 min. The pellet contents were mixed with a Pasteur
pipette and centrifuged at 800 g for 10 min after the addition
of 5 mL of cold NaCl ⁄ P
i
. Two more NaCl ⁄ P
i
washings were
performed. These cells were then used for BBM isolation.
Preparation of BBMVs from isolated intestinal
epithelial cells
BBMVs were prepared from isolated total intestinal cells
from control and ethanol-fed rats at different time intervals
during the course of treatment at 4 °C by the method of
Kessler et al. [32] with some modifications. The final pellet
containing cells was homogenized by adding 2 mm

membrane vesicles by transmission electron
microscopy
The final BBMV preparations obtained were suspended in
NaCl ⁄ P
i
and centrifuged at 27 000 g for 30 min. Vesicular
suspensions were fixed at 4 °C in 3% buffered glutaralde-
hyde for 5–6 h and centrifuged at 10 000 g for 10 min.
Suspensions were gently rinsed twice with 0.2 m NaCl ⁄ P
i
at
4 °C and postfixed for 1 h at 4 °C with 1% buffered
osmium tetroxide. After dehydration in 70%, 90% and
absolute ethanol for 2 h, 20 min and 1 h, respectively, the
suspensions were treated with propylene oxide at room tem-
perature. The preparations were embedded in epoxy resin
TAAB-812 (TAAB Laboratories, Aldermaston, UK) and
polymerized for 24 h at 60 °C. Semi-thin sections were
placed on microslides, stained with 0.5% alkaline toluidine
blue and examined under a light microscope to verify the
areas of intensity. Ultrathin sections (60 nm) were cut,
placed on metal grids, stained on ultracut E (Reichert-Jung,
Nuslock, Germany) and double stained with uranyl acetate
and lead citrate. The microslides were then examined under
a Zeiss EM-906 transmission electron microscope (Carl
Zeiss, Dresden, Germany).
Transport of [
3
H]folic acid
Uptake studies were performed at 37 °C using incubation

RT-PCR analysis
Total RNA from all animals was isolated from the upper
1 cm of jejunal tissues following the method of Chomeczyn-
ski and Sacchi [35]. cDNA synthesis was carried out from
the purified and intact total RNA, according to the manu-
facturer’s instructions (MBI Life Sciences). Expression of
RFC and b-actin was evaluated using sequence-specific
primers corresponding to the sequence in the open reading
frame. A 20 lL PCR mixture was prepared in 1 · PCR
buffer consisting of 0.6 U of Taq polymerase, 2 lm of each
primer (for both b-actin and RFC) and 200 lm of each
dNTP. In optimized PCR, the initial denaturation step was
carried out for 2 min at 95 °C. The denaturation, annealing
A. Hamid et al. Intestinal folate malabsorption in alcoholism
FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS 6325
and elongation steps were carried out for 1 min at 94 °C,
1 min at 68 °C and 1 min at 72 °C, respectively, for 35
cycles. The final extension step was carried out for 10 min
at 72 °C. The primers designed using primer3 input
(version 0.3.0) were as follows: RFC: forward, 5¢GA-
ACGTCCGGCAACCACAG3¢; reverse, 5¢GATGGACTT-
GGAGGCCCAG3¢; b-actin: forward, 5¢CACTGTGCCCA-
TCTATGAGGG3¢; reverse, 5¢TCCACATCTGCTGGAA-
GGTGG3¢. The expected PCR products of 489 and 588 bp
were obtained for RFC and b-actin, respectively, when
electrophoresed on a 1.2% agarose gel. The densitometric
analyses of the products were determined using scion
image software (Scion Image Corporation, Frederick, MD,
USA).
Western blot analysis

polysaccharides, and eosin, which is an acidic dye and
stains the basic components present in cytoplasmic
proteins.
Estimation of folate by microbiological assay
Folate estimations were determined by micotitre plate assay
using Lactobacillus casei [39]. All steps were carried out in
aseptic conditions.
Genomic DNA methylation studies
DNA isolation was performed by the conventional method
using a lysis buffer containing proteinase K, as described
previously [29]. The methylation status of CpG sites in
genomic DNA was determined by the in vitro methyl accep-
tance capacity of DNA using S-adenosyl-[methyl-
3
H]methi-
onine as a methyl donor and a prokaryotic CpG DNA
methyltransferase [40].
Statistics
Each uptake assay was performed three times with eight
independent preparations from each group. The data were
computed as the mean ± standard deviation. Group means
were compared using Student’s t-test, and analysis of
variance was used when necessary. The acceptable level of
significance was P < 0.05 for each analysis.
Acknowledgements
Financial assistance by the Council of Scientific and
Industrial Research (CSIR), New Delhi, India is grate-
fully acknowledged.
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