Alcohol 41 (2007) 441e446

Long-term alcohol ingestion alters the folate-binding kinetics inintestinal brush border membrane in experimental alcoholism

Abid Hamid, Jyotdeep Kaur*Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160 012, India

Received 16 March 2007; received in revised form 30 April 2007; accepted 2 May 2007

Abstract

The folic acid transport across epithelial cell membrane of the intestine is an essential step for its absorption, conservation, and homeo-stasis in the body. In this study, we sought to examine the kinetics of binding to intestinal brush border membrane (BBM) consideringintestinal malabsorption as the major contributing factor to alcohol-induced folate deficiency. Male Wistar rats were fed 1 g/kg bodyweight/day ethanol (20% solution) orally for 3 months. We studied [3H]-folic acid binding to the intestinal BBM and acidic pH-dependentbinding was observed to be associated with reduced maximal binding (Bmax) in chronic ethanol-fed group. However, under such conditions,there was no significant effect of ethanol ingestion on Kd and pH optimum of the binding process. Increasing the osmolarity at pH 5.5 hadno effect on the binding of folate to BBM, thus confirming that the observed changes in Bmax values were due to site-specific binding to theextravesicular sites. Importantly, ethanol ingestion disturbs the SeS status at the binding site besides interfering with the Naþ and divalentcation dependency of the binding process. These results highlight the possible mechanism of folate malabsorption at primary absorptive siteduring alcoholism. � 2007 Elsevier Inc. All rights reserved.

Keywords: Brush border membrane; Reduced folate carrier; Chronic alcoholism; Binding; Folic acid; Intestine

Introduction

The mechanism of folate transport is of considerableimportance because mammals require the ingestion and ab-sorption of preformed folates to meet their needs for one-carbon moieties to sustain key biosynthetic reactions (Suhet al., 2001), for example, the de novo synthesis of purinesand thymidylate. The most well-characterized folate trans-porter, the reduced folate carrier (RFC), is an integral mem-brane protein that mediates cellular uptake of reducedfolates and antifolates and is ubiquitously expressed in tis-sues (Sirotnak and Tolner, 1999; Zhao and Goldman, 2003).It has been demonstrated that the characteristics displayedby RFC depend on the cell context, which could be attrib-uted to differences in membrane composition, cell-specificposttranslational modifications, and/or involvement of cell-specific accessory protein that modulate RFC activity in thedifferent cell types (Said, 2004). Moreover, epithelial folatetransport is essential for normal folate homeostasis regulat-ing intestinal uptake, renal tubular reabsorption, and tissuedistribution of folate.

* Corresponding author. Tel.: þ91-172-2755181; fax: þ91-172-

2744401/2745078.

E-mail address: jyotdeep2001@yahoo.co.in (J. Kaur).

0741-8329/07/$ e see front matter � 2007 Elsevier Inc. All rights reserved.

doi: 10.1016/j.alcohol.2007.05.002

Deficiency of folate is highly prevalent throughout theworld (Novakovic et al., 2006; Olivares et al., 2005).Importantly, the association of chronic alcoholism withthe development of folate deficiency is also well known andethanol is considered as one of the most important toxinsconsumed regularly and in large quantities by humans(Poschl and Seitz, 2004; Salaspuro, 2003). It is of greatconcern in view of the fact that incidence of folate defi-ciency associated with anemia, leukopenia, and thrombocy-topenia is more common among alcoholics (Villanuevaet al., 2001). The folate deficiency can develop becauseof dietary inadequacy, intestinal malabsorption, alteredhepatobiliary metabolism, and increased renal excretion(Schalinske and Nieman, 2005; Villanueva et al., 1994).However, it is evident from the literature that most of theethanolic effects on folate metabolism are reflected in its ef-fect on intestinal absorption (Halsted et al., 2002; Masonand Choi, 2005; Villanueva et al., 1994). Earlier studiesfrom our laboratory revealed reduced binding and uptakeof folate in renal brush border membrane (BBM) whichcan contribute to urinary folate loss during alcoholism (Ha-mid and Kaur, 2005, 2006). Though the presence of folatebinding protein has not been established in intestinal BBM,earlier reports demonstrate a pH-dependent, specific, andsaturable binding of folate to the intestinal BBM

442 A. Hamid, J. Kaur / Alcohol 41 (2007) 441e446

(Reisenauer et al., 1986; Shoda et al., 1990). So, the presentstudy was planned to elucidate kinetic characterization offolate binding to intestinal BBM because the intestinal ep-ithelial surface constitutes the primary absorptive site forthe exogenous folate and intestinal folate malabsorption isan established fact associated with alcoholism.

Materials and methods

Animals

Young adult male albino rats (Wistar strain) weighing100e150 g were obtained from the Institute’s Central Ani-mal House. The rats were housed in clean wire mesh cageswith controlled temperature (23 6 1�C) and humidity(45e55%) and had 12 h dark/light cycle throughout thestudy. The rats were randomized into 2 groups of 10 animalseach, such that the mean body weights and the range of bodyweights for each group of animals were similar. The ratsin group I were given 1 g ethanol (20% solution)/kg bodyweight/day and those in group II received isocaloricamount of sucrose (36% solution) orally by Ryle’s tubedaily for 3 months. The rats were fed commercially avail-able pellet diet (Ashirwad Industries, India) and water adlibitum. The body weights of rats were recorded twiceweekly. Animals from both the groups were sacrificed un-der anesthesia using sodium pentothal. Starting from theligament of Trietz, 2/3rd of the small intestine was re-moved, flushed with saline, and used for various studies.The blood was collected for alcohol estimation at theend of 3 months (Burtis and Ashwood, 1994).

The protocol of the study was approved by InstitutionalAnimal Ethical Committee and Institutional BiosafetyCommittee.

Chemicals

Radiolabeled [30,50,7,9-3H]-folic acid, potassium saltwith specific activity 24.0 Ci/mmol was purchased fromAmersham Pharmacia Biotech (Hong Kong). D-[U-14C]-glucose with specific activity 140 mCi/mmol was providedby Radioisotope Division, Bhabha Atomic Research Cen-tre, Mumbai, India. Methotrexate, bovine serum albumin,and DL-dithiothreitol (DTT) or Cleland’s reagent were pur-chased from Sigma Aldrich Co., St. Louis, MO, USA. Cel-lulose nitrate membrane filters (0.45 mm) were obtainedfrom Millipore Corporation (Bedford, MA, USA). All otherchemicals and reagents used in this study were of analyticalgrade.

Isolation of intestinal epithelial cells

The intestinal epithelial cells were isolated (Weiser,1973) with some modifications. Upper 2/3rd of the smallintestine was removed, flushed with 0.9% saline 2e3 timesusing the blunt needle and syringe until the intestine wascleared. One end of the intestine was tied with thread and

filled with rinsing buffer containing 1 mM DTT in normalsaline. The rinsing buffer filled in the intestine was then re-placed with a solution consisting of 1.5 mM KCl, 96 mMNaCl, 27 mM sodium citrate, 8 mM KH2PO4, and 8 mMNa2HPO4 and kept at 37�C for 15 min in a beaker contain-ing phosphate buffer saline (PBS). The solution in the intes-tine was discarded and the intestine was filled witha solution containing 1.5 mM EDTA and 0.5 mM DTT inPBS and kept at 37�C in a shaker at 100 rpm for 30 min.The cells were collected in the centrifuge tube and centri-fuged at 3,000 rpm for 15 min. The supernatant was dis-carded and 5 ml of the cold PBS was added to thepelleted cells. The pellet contents were mixed with Pasteurpipette and centrifuged at 3,000 rpm for 10 min. After twomore PBS washings, the pellets containing intestinal epi-thelial cells were collected.

Preparation of brush border membrane vesicles (BBMV)from isolated intestinal epithelial cells

Brush border membrane vesicles were prepared from theisolated cells at 4�C by the method of Kessler et al. (1978)with some modifications. The final pellet containing cellswas homogenized by adding 2 mM Trise50 mM mannitolbuffer; 0.5 ml of the homogenate was stored at 4�C forfurther use. To the major portion of the homogenate wasadded MgCl2 (10 mM final concentration) in 2 mM Trise50 mM mannitol buffer followed by intermittent gentle shak-ing for 10 min. The contents were centrifuged at 3,000� gfor 15 min. The supernatant was then run at 27,000� g for30 min. The pellet thus obtained was mixed in a smallamount of loading buffer containing 280 mM mannitol,20 mM HEPES (N-[2-Hydroxyethyl] piperazine-N0-[2-ethanesulfonic acid])eTris, pH 7.4 and homogenized byhand homogenizer and centrifuged at 27,000� g for30 min. The final pellet obtained was suspended in loadingbuffer to achieve a final protein concentration of approxi-mately 5 mg/ml.

Purity of membrane vesicles

Purity of the membrane preparations was checked bymeasuring the specific activities of alkaline phosphataseand Naþ, Kþ-ATPase in BBMs and in the original homog-enate by the method of Bergmeyer (1974) and Quigley andGotterer (1969), respectively. The vesicle preparations fromboth the groups showed enrichment of 10e12-fold withrespect to alkaline phosphatase and showed negligibleamount of Naþ, Kþ-ATPase activity. The method of Lowryet al. (1951) was used for the determination of protein con-centration using bovine serum albumin as a standard. Thevesicles used in the study were intact and stable as theyshowed the properties of typical BBM as revealed by a tran-sient overshoot of the intravesicular glucose concentrationover its equilibrium uptake in the presence of sodiumgradient.

443A. Hamid, J. Kaur / Alcohol 41 (2007) 441e446

Binding of [3H]-folic acid

Ten microliters of BBMV (50 mg protein) were incu-bated at 4�C in 37.5 ml of the binding buffer consisting of100 mM NaCl, 80 mM mannitol, 10 mM HEPES, 10 mMMES (2-morpholinoethanesulfate acid), pH 5.5 containing[3H]-folic acid (0.5 mM) unless otherwise specified. Aftera specified time, the reaction was stopped with the ice-coldstop solution and bound folate was separated from unboundfolate by vacuum filtration (Hamid and Kaur, 2006). Theradioactivity retained on the filters was determined by liq-uid scintillation counting (Beckman Coulter LS 6500).The binding phenomena were characterized in intestinalBBM under different experimental conditions as describedpreviously (Hamid and Kaur, 2005).

Statistics

Each uptake assay was performed thrice with three inde-pendent pooled preparations of BBMV from each group.The data were computed as mean 6 S.D. Group meanswere compared by using the Student’s t-test and analysisof variance was used wherever necessary. The acceptablelevel of significance is P ! .05 for each analysis.

Results

Estimation of blood alcohol levels

The alcohol level was 88% higher (P ! .001) in chronicethanol-fed group in comparison with the control group(Fig. 1).

Kinetics of folate binding

The folate-binding kinetics was characterized in vesicu-lar membrane preparations at 4�C. Fig. 2 depicts the bind-ing of folic acid to intestinal BBMV from control andethanol-fed rats at various time intervals. There was20e45% decrease in binding component in ethanol-fedgroup at different time intervals studied (P ! .01,P ! .001). It was observed that maximum folate was bound

Fig. 1. Blood alcohol levels (mg/dl) in control and ethanol-fed rats.

Values are mean 6 S.D. ***P ! .001 versus control.

to BBMV at 30 s and it decreased, when the binding wasmeasured for 60 s. Hence, further binding experiments werecarried out at 30 s intervals for the determination of the ini-tial rate of binding. The effect of increasing osmolarity inthe extravesicular compartment was studied by increasingthe mannitol concentration (30e380 mM) in the incubationbuffer (Fig. 3). In the osmolarity range from 300 to600 mOsm, no considerable difference in binding was de-tected in the two groups. The observation confirmed thatbinding to the extravesicular sites and not the transport in-side the vesicles was taking place under these conditions.The effect of extravesicular pH revealed that in the controlgroup, binding of folate was maximum at pH 5.0(21.51 6 0.11 pmol/30 s/mg protein) and it reduced withfurther increase in pH, and at pH 8.0, the binding was2.98 6 0.04 pmol/30 s/mg protein. In the ethanol-fedgroup, the maximum binding observed at pH 5.0(13.41 6 0.07 pmol/30 s/mg protein) decreased sharply

Fig. 2. [3H]-Folic acid binding in intestinal BBMV as a function of time.

Each data point is mean 6 S.D. of three separate binding determinations

carried out in duplicate. **P ! .01, ***P ! .001 versus control.

Fig. 3. [3H]-Folic acid binding in intestinal BBMV as a function of osmo-

larity. Each data point is mean 6 S.D. of three separate binding determina-

tions carried out in duplicate.

444 A. Hamid, J. Kaur / Alcohol 41 (2007) 441e446

thereafter (Fig. 4). In more acidic pH range, the decrease inbinding in the ethanol-fed group was in the range of25e47% (P ! .001) and such a decreased binding couldnot be observed when the extravesicular pH was increasedabove 6.0. To determine the binding constants, the bindingof folate was studied by varying the folic acid concentrationfrom 0.125 to 1.5 mM (Fig. 5). There was 29e69% reducedbinding (P ! .001) in the ethanol-fed group at various sub-strate concentrations studied. The data were then repre-sented in the Scatchard plot in which bound folate/freefolate (V/[S]) was plotted against bound folate (V). Theslope and intercept were used for the determination of Kd

(binding constant) and Bmax (maximal binding), respec-tively (Fig. 5 inset). The observed Kd values did not changesignificantly in the two groups of rats (0.97 6 0.21 mM vs.0.95 6 0.24 mM). Importantly, the Bmax values were90 6 10 and 44 6 6 pmol/30 s/mg protein (P ! .001) incontrol and ethanol-fed groups, respectively.

Further, the presence of DTT in reaction mixture led toa 73% (P ! .001) increase in the binding of folate toBBMV in control group (Fig. 6). However, the binding offolate in the ethanol-fed group was irresponsive to the pres-ence of DTT in the medium. In addition, binding of folatewas studied in the presence of 1 mM concentration of Zn2þ,Mn2þ, or Mg2þ ions to elucidate the masking effect ofpositive charge on binding of negatively charged folate sub-strate. In the control group, the binding of folate to BBMVwas not significantly affected except that Zn2þ resulted ina significant 23% (P ! .05) increase in binding (Table 1).However, the binding of folic acid to BBMV from etha-nol-fed group remained unaltered when any of the three di-valent cations was present in the incubation medium. Folatebinding to BBMV was also studied in the absence of Naþ inincubation medium and binding decreased significantly(P ! .01) by 26% in the control group, whereas in the eth-anol-fed group, Naþ replacement by Kþ in the incubationmedium had no significant effect (Table 1).

Fig. 4. [3H]-Folic acid binding in intestinal BBMV as a function of pH

optimum. Each data point is mean 6 S.D. of three separate binding deter-

minations carried out in duplicate. *P ! .05, ***P ! .001 versus control.

Discussion

The folate transport system activities could become dis-tinct in response to external stimuli such as folate availabil-ity and exposure to chemotherapeutic agents (Agnieszkaet al., 2000). In the present study, ethanol feeding at 1 g/kgbody weight/day ethanol (20%) did not show any clinicalsigns of intoxication similar to our earlier study (Hamidand Kaur, 2005). The blood alcohol concentration observedin the chronic ethanol-fed group showed that a significantlevel of blood alcohol concentration was maintained duringthe 3-month course of ethanol administration. The dose waschosen as per earlier studies (Persson, 1991) which sug-gested that the ethanol concentration of the jejunal contentshould not exceed 6% in animal experiments to have rele-vance for the human intestine. In the present study, devel-oped nontoxic values of blood alcohol concentrations inrats did not show any clinical signs of intoxication similar

Fig. 5. [3H]-Folic acid binding in intestinal BBMV as a function of sub-

strate concentration (inset Scatchard plot). Each data point is mean 6 S.D.

of three separate binding determinations carried out in duplicate.

***P ! .001 versus control.

Fig. 6. [3H]-Folic acid binding in intestinal BBMV as a function of SeS

group reacting reagent (DTT). Each data point is mean 6 S.D. of three sep-

arate binding determinations carried out in duplicate. ###P ! .001 versus

�DTT.

445A. Hamid, J. Kaur / Alcohol 41 (2007) 441e446

to an earlier study (Muldoon and McMartin, 1994). Suchresults validate the suitability of the present model forrelevant mammalian studies.

The transport of folic acid using intestinal BBMV re-vealed an associated binding component (unpublisheddata); we sought to characterize the binding kinetics of fo-late in these membrane preparations at 4�C. Folate bindingby BBMV carried out at 4�C at different time intervals re-vealed maximum binding at 30 s in both the control andethanol-fed groups. It was significantly less at all time inter-vals studied in the ethanol-fed group. In an earlier study(Ross and McMartin, 1996), the authors have reported nochange in binding of folate to renal BBM when the vesicleswere incubated in the presence of ethanol. However, ourstudy suggests that ethanol when given orally in chronicdose might affect the binding process at metabolic or ge-netic level and this effect is different from its in vitro effect.The observed reduced binding to BBM will lead to lessabsorption at the folate entry site of the intestine and maycontribute to the folate malabsorption during alcoholism.By varying the medium osmolarities, it was confirmed thatthe binding was to the extravesicular sites and did not rep-resent the intravesicular accumulation of the folate underthese conditions. Although folate-binding protein has notbeen shown to be present in the intestine (Villanuevaet al., 2001), some earlier studies proposed the existenceof similar binding proteins and sites at the intestinal brushborder surface (Elsborg et al., 1980; Shoda et al., 1990).Moreover, the intestinal absorption of dietary folate hasbeen shown to involve distinct binding and transport activ-ities at the brush border surface of the enterocyte (Reisena-uer et al., 1986). The observed reduction of binding offolate to intestinal BBM might contribute to low folate up-take and hence folate malabsorption observed in alcoholics(Villanueva et al., 2001). Because the folate transport sys-tems are highly sensitive to change in proton concentrationat the site and respond to tissue microenvironment (Said,

Table 1

Effect of different divalent cations and Naþ absence on [3H]-folic acid

binding in BBMV of intestine

Ion

V (pmol/30 s/mg protein)

Control Ethanol

None 11.74 6 0.88 8.59 6 0.94*

Zn2þ 13.79 6 1.49# 10.79 6 2.43

Mn2þ 13.29 6 1.41 10.98 6 2.64

Mg2þ 10.78 6 1.63 10.08 6 1.54

�Naþ 7.89 6 0.51## 11.00 6 2.19**

Each value is mean 6 S.D. of three separate binding determinations

carried out in duplicate. The binding was carried out by using binding

buffer consisting of 100 mM NaCl, 80 mM mannitol, 10 mM (N-[2-

Hydroxyethyl] piperazine-N0-[2-ethanesulfonic acid]), 10 mM 2-morpholi-

noethanesulfonic acid, pH 5.5 containing [3H]-folic acid (0.5 mM) in the

presence of 1 mM concentrations of either of the divalent cations. In

Naþ free (�Naþ) medium, 100 mM KCl replaced the NaCl in the incuba-

tion medium. *P ! .05, **P ! .01 versus control. #P ! .05, ##P ! .01

versus none within the group.

2004), it was of interest to examine the effect of pH varia-tions on folate-binding process. The maximum binding offolic acid in both the groups was in acidic pH; this sug-gested that the acidic microclimate at the surface of enter-ocytes (Kumar et al., 1998) provides an appropriatemicroenvironment for maximal binding of folate to thesesites. In addition, ethanol ingestion resulted in reduced fo-late binding in optimum pH range, similar to that for thefolic acid binding in renal BBM (Hamid and Kaur, 2005).Overall, such an observation proposes that acidic pH isan important factor for efficient binding of folic acid viaspecific receptors to intestinal BBM surface.

Similar values of the binding constants (Kd) in controland ethanol-fed groups suggested that the binding affinityof the receptor does not change during chronic alcoholism.However, the decreased value of Bmax suggested that inchronic alcoholism, the numbers of binding sites decreasedat the intestinal absorptive surface. The Kd values were sim-ilar to that observed in renal BBM earlier (Hamid and Kaur,2005), suggesting that binding affinity of receptors to thefolate is similar in intestine and renal tissues. The bindingprocess in intestinal BBM seems to be independent of mostof the divalent cations. However, an increase in binding inthe presence of Zn2þ under physiological conditions can beattributed either to its masking effect of negative charge onthe substrate molecule or the conformational change im-parted to the binding site on the intestinal surface. The roleof Zn2þ in folic acid binding was in agreement with an ear-lier study by Reisenauer et al. (1986), where it was shownto enhance the folic acid binding in pig intestine. However,in the same study, the authors have reported differential be-havior of divalent cations on folate hydrolysis. Importantly,absence of Naþ ions in the medium could not influencefolate binding during chronic alcoholism, suggesting thatNaþ-independent folate binding do exist in chronic alcohol-ism. Thus, it seems that in ethanol ingestion, the Naþ con-centration is not a prerequisite factor for folate binding toBBM. A similar effect of alcohol ingestion was observedin renal BBM earlier by us (Hamid and Kaur, 2005). How-ever, both Hþ/folate and Naþ/folate seem to be importantunder physiological conditions for folate binding in thetwo tissues. Moreover, an increased binding in the presenceof DTT proposes that eSH group(s) status on the carrier is/are crucial for the binding and chronic alcoholism resultedin the decreased responsiveness to such treatment. This maysuggest that the orientation of SeS groups may not be ac-cessible to DTT presumably due to change in conformationof the transporter protein during alcoholism or these groupswere present in reduced form during ethanol ingestion.

In conclusion, the reduced folate binding to the intestinalBBM in chronic alcoholism is associated with decreasedBmax of binding; eSH group status, divalent cation depen-dency, and shifting the process to Naþ-independent one.The perturbed folate-binding kinetics at intestinal surfacemay form the primary step in intestinal folate malabsorp-tion during alcoholism.

446 A. Hamid, J. Kaur / Alcohol 41 (2007) 441e446

Acknowledgments

Financial assistance by the Council of Scientific and In-dustrial Research, New Delhi, India is highly acknowledged.

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