Biochem. J. (1974) 140, 509-516Printed in Great Britain

Involvement of Binding Lipoproteins in the Absorption andTransport of a-Tocopherol in the Rat

By 0. V. RAJARAM, P. FATTERPAKER and A. SREENIVASANBiochemistry and Food Technology Division, Bhabha Atomic Research Centre,

Bombay-400 085, India

(Received 3 December 1973)

1. Specific lipoproteins binding a-tocopherol but not its known metabolites have beenisolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A time-study of the appearance of the orally administered a-[3H]tocopherol with these lipo-proteins indicates that very-low-density lipoprotein of serum acts as a carrier of thevitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitaminfrom the intestine has been inferred from observations on the amounts of a-tocopherol inserum of orotic acid-fed rats where release of lipoproteins from the liver to serum iscompletely inhibited. A considerable decrease in the association of a-tocopherol withserum very-low-density lipoprotein under this condition is interpreted to mean that serumlipoproteins are limiting factors for the transport of the vitamin across the intestine andthat this is possibly effected by exchange of a-tocopherol between serum very-low-densitylipoprotein and mucosal lipoprotein.

The occurrence of an a-tocopherol-binding lipo-protein in rat liver cytosol, its isolation and purifi-cation have been reported in a previous communi-cation (Rajaram et al., 1973). Since little is knownabout the mechanism of absorption and mode oftransport of this vitamin, the present studies weredesigned to find out whether similar lipoproteins existin the cytosol of intestinal mucosa and in serum and,if so, their possible involvement in the processes ofabsorption and transport of the orally fed vitamin.

Materials and Methods

Chemicals

DL-a-[5-Me-3H]Tocopherol (4.6mCi/mg) was ob-tained from The Radiochemical Centre, Amersham,Bucks., U.K. Acrylamide, NN'-methylenebisacryl-amide and NNN'N'-tetramethylethylenediamine werepurchased from Koch-Light Laboratories Ltd.,Colnbrook, Bucks., U.K. Sephadex G-200 wasobtained from Pharmacia Fine Chemicals, Uppsala,Sweden. PPO (2,5-diphenyloxazole), dimethyl-POPOP [1,4-bis(4-methyl-5-phenyloxazol-2-yl)benz-ene] and orotic acid monohydrate were purchasedfrom Sigma Chemical Co., St. Louis, Mo., U.S.A.All other chemicals used were either AnalaR gradefrom BDH Chemicals Ltd., Poole, Dorset, U.K., orProanalyst quality from E. Merck A.G., Darmstadt,Germany.

Vol. 140

Animals and treatment with orotic acid

Male albino rats of the Wistar strain, housed inindividual cages, were fed normal laboratory stockdiet and used when they attained a weight of about200g. The normal diet contained vitamin-free casein(18 %), glucose (68 Y.), cellulose (5%), peanut oil (5 %.)and salt mixture (USP XIV; 4%) The followingvitamins were added to this diet (mg/kg): thiaminehydrochloride, 2; riboflavin, 4; pyridoxine, 4; cho-line, 1000; inositol, 1000; p-aminobenzoic acid, 300;nicotinamide, 100; folic acid, 2.5; vitamin B12, 0.05;biotin, 0.1; ergocalciferol, 0.042; vitamin K, 10;a-tocopherol, 50; vitamin A, 1.72; calcium panto-thenate, 10 (Roels et al., 1964). Orotic acid, when fed,was mixed into this diet at a concentration of 1%and animals were maintained on this diet for 1 week.

Methods

Oral administration of a-[3H]tocopherol. DL-a-[5-Me-3H]Tocopherol (100,uCi) was mixed with1.2mg of unlabelled DL-a-tocopherol in 95% (v/v)ethanolic solution. The mixture, after evaporation ofthe solvent, was dissolved in 0.5ml of olive oil andadministered orally by stomach tube under lightether anaesthesia to rats weighing 200g that hadbeen starved overnight.

Preparation of liver and small-intestinal mucosalcytosol. Rats under light ether anaesthesia had their

509

0. V. RAJARAM, P. FATTERPAKER AND A. SREENIVASAN

livers perfused via the hepatic portal vein withchilled 0.9% NaCl. After perfusion, which tookabout 5min, the liver was quickly excised, weighedand chilled in 0.25M-sucrose containing lOmM-Tris-HCI buffer, pH7.4, and lmM-EDTA. A 50% liverhomogenate was prepared in the same medium byusing a Potter-Elvehjem-type glass homogenizerwith a Teflon pestle and giving two strokes at a lowspeed of 1000-2000rev./min. The homogenate wascentrifuged at 1050OOg for 60min in a BeckmanL265B ultracentrifuge to give clear cytosol which waswithdrawn with a syringe without disturbing the topfatty layer.The lumen of the entire small intestine was washed

by flushing several times with 0.25M-sucrose contain-ing lOmM-Tris-HCl buffer, pH7.4, and 1mM-EDTAto remove the food particles, and the washings werechecked for counts until free of radioactivity. Theintestine was cut into short lengths, split longitudin-ally to expose the mucosa and scraped. The mucosalscrapings were homogenized in 9vol. of a solutioncontaining 0.25M-sucrose, 1OmM-Tris-HCI (pH7.4),1mM-EDTA and 0.1 % BaSO4 as described by Huibs-cher et al. (1965). After an initial centrifugation at100OOg for lOmin to sediment cell debris, mucus andmitochondria, the supernatant was spun at 105000gfor 60min to obtain clear cytosol. This was concen-trated by reverse dialysis against Polywax (poly-ethylene glycol) 6000 (Koppikar et al., 1971).Serum was obtained from the blood drawn through

the posterior vena cava.Sephadex chromatography. A column (1.8cmx

50cm) uniformly packed with Sephadex G-200 geland equilibrated with 0.05M-sodium phosphate buffer(pH7.4) was employed for the isolation and initialpurification of a-tocopherol-binding protein. Asample of cytosol or serum containing 70-100mg ofprotein was loaded on the gel and elution carried outin the cold (4°C) at a flow rate of 20ml/h with thephosphate buffer mentioned above, and fractions(2.5ml) were collected (Koppikar et al., 1971).Extinction of the various fractions was measured ina Shimadzu QV 50 spectrophotometer at 280nm ina 1cm light-path cell. Suitable dilutions of the sampleswere made wherever necessary to measure E withinthe working range of the instrument.

Thin-layer chromatography. The fractions compris-ing peak 1 from the Sephadex column were extractedthree times with acetone-light petroleum (b.p. 40-60°C) (1:2, v/v) (7.5ml of acetone+15ml of lightpetroleum each time) and the extracts were eva-porated to dryness in the dark under a stream of N2.The residue was dissolved in 0.5ml of ethanol con-taining 300,ug of carrier a-tocopherol. a-Tocopherolwas separated from interfering lipids by t.l.c. onsilica-gel G with benzene-hexane (19:1, v/v) as de-veloping solvent. The completed chromatogram wasviewed under u.v. light to mark the a-tocopherol

spots which were quickly scraped and eluted with2x 1.5ml of absolute ethanol. The u.v.-absorptionspectra of the eluates were recorded in a Perkin-Elmer-Hitachi double beam spectrophotometer.Additionally, the eluates were also tested by theEEmmerie-Engel(1938)reaction.Thechromatographicplate was exposed to 12 vapour to identify otherspots, if any.

Polyacrylamide-gel electrophoresis. Polyacryl-amide-gel electrophoresis was carried out in a3.5 % running gel. The spacer gel of 2.5% polyacryl-amide, as modified by Koppikar et al. (1971), wasused. All other details of the electrophoretic systemwereessentiallyas described byNarayan etal. (1966a).Prestaining of the lipoprotein was carried out withSudan Black B in ethylene glycol as described byMcDonald & Ribiro (1959). Amido Black was usedas a protein stain.

Uptake in vitro of a-[3H]tocopherol by serum frommucosal cells of orotic acid-fed rats. a-[5-Me-3H]-Tocopherol (l00,uCi) was fed orally to orotic acid-fed rats and 4h later the mucosal scrapings werecollected. A portion (1 g) of the mucosal cell sus-pension in 2ml of0.25M-sucrose, buffered with 10mM-Tris-HCl, pH7.4, and lmM-EDTA and containing atotal radioactivity of about 389750c.p.m., was incu-bated with 1 ml of unlabelled serum either fromnormal rat or from orotic acid-fed rat with or without1 ml of lipoprotein fraction I isolated by Sephadexchromatography of normal serum. The incubationswere carried out in test tubes at 37°C for 30min ina Dubonoff metabolic shaker water bath, and thenwere chilled immediately and centrifuged quicklyto sediment the mucosal cells. The radioactivityincorporated into the supernatant was determined.

Analytical methods. Protein determinations werecarried out by the method of Lowry et al. (1951) withcrystalline bovine albumin as standard. Lipids wereextracted by chloroform-methanol (2:1, v/v) andportions were used for lipid analysis by the method ofBragdon (1951).

Radioactivity determinations. Portions ofthevarioussamples were spotted on strips (7cmx 1.8cm) ofWhatman 3MM filter papers, dried and placed incounting vials containing 0.3% PPO and 0.01%dimethyl-POPOP in lOml -of toluene as scintillator(Roodyn et al., 1965). Radioactivity was counted ina Beckman LS-100 liquid-scintillation spectrometerwith 59% instrument efficiency. A comparison of theeffect of filter-paper strips on counting efficiency wasmade by measuring the radioactivity of 0.01 ml ofethanolic DL-a-[5-Me-3H]tocopherol ofknown radio-activity in the presence and absence of Whatman3MM filter-paper strips. In the presence of paperstrips the counting efficiency was 12%. All valuesreported are actual c.p.m. measured without anycorrection being applied. For determining the radio-activity in the polyacrylamide gel, the gels were sliced

1974

510

oa-TOCOPHEROL BINDING TO CELLULAR LIPOPROTEINS

withathinrazor to isolate the individual bands and thesegments placed in loosely capped vials containing0.5ml of 30% H202 and incubated at 60°C for 5has described by Young & Fulhorst (1965). Portionsof this solution were spotted on Whatman 3MMstrips and counted as described above.

Portions of ethanolic extracts from the silica-gelt.l.c. plates were also counted for 3H radioactivity.

ResultsAssociation of a-tocopherol with proteins of mucosaland liver cytosol and serumA possible binding of orally fed a-(3H]tocopherol

with proteins of mucosal cytosol and serum wasinvestigated to determine its significance in theabsorption and transport of the vitamin. Rats fedwith 100,cCi of a-[5-Me-3H]tocopherol were killedafter 4h. This time-interval was chosen becauseit is known that lipids are maximally absorbed at 3-4hafter oral administration (Fatterpaker, 1952). Sepha-dex G-200 gel filtration of mucosal cytosol (Fig. la)gave three distinct protein fractions of which thefirst protein peak, eluting between fractions 15 and20, contained about 87% of the total cytosola-[3H]tocopherol. This particular fraction alsoshowed the presence of lipids when analysed by themethod ofBragdon (1951), indicating that the bindingprotein is probably lipoprotein in nature. Vitamin Ewas not associated with any other protein fractions ofthe cytosol.The serum proteins were separated into five frac-

tions (Fig. lb). Vitamin E was maximally (82%)associated with the first protein fraction which had aspecific radioactivity (i.e. c.p.m. in a portion ofprotein solution that gives E280 = 1 in a 1cm-light-

path cell) of 5080 c.p.m./E. This fraction, on the basisof lipid composition, has been shown to contain amixture of chylomicrons, VLD lipoprotein* and LDlipoprotein (S. V. Koppikar, P. Fatterpaker & A.Sreenivasan, unpublished work). No measurableradioactivity was found in other fractions.A simultaneous study of the liver cytosol proteins

(Fig. 1 c) was done for comparison. As has been shownpreviously (Rajaram et al., 1973), vitaminE is carriedby a lipoprotein fraction of liver cytosol that comesimmediately after the void volume. The lipoproteinfractions of liver and mucosal cytosol that carryvitamin E, as well as that of serum, resemble eachother in their elution pattern.

It was also ascertained that the radioactivity associ-ated with the lipoprotein fractions of mucosa, serumand liver was not due to the metabolic products ofa-tocopherol. For this, the first protein fractionsobtained after Sephadex chromatography of thesesamples were extracted with acetone-light petroleum(b.p. 40-60'C) (1: 2, v/v) and the concentrated extractwas subjected to t.l.c. with benzene-hexane (19:1,v/v) as developing solvent. The completed chromato-gram in each case revealed a major spot with RF value0.71 corresponding to that of standard a-tocopherol;this spot also gave a u.v.-absorption spectrum charac-teristic of a-tocopherol (Fig. 2), besides showing apositive Emmerie-Engel (1938) reaction. In addition,the chromatogram also showed a minor spot withRF value 0.11, which corresponds to ac-tocopheryl-quinone. The distribution of radioactivity amongthese spots is represented in Table 1. About 75-80%of the radioactivity in the various fractions could be

* Abbreviations: VLD lipoprotein, very low-densitylipoprotein; LD lipoprotein, low-density lipoprotein.

3

20

0 20 40 60 0 20 40Fraction number

Fig. 1. Gelfiltration ofintestinal mucosal cytosol (a), serum (b) and liver cytosol (c)A Sephadex G-200 column was equilibrated with 0.05M-sodium phosphate buffer (pH7.4). Portions (2ml) of the respectivesamples were loaded on the columns and 2.5ml fractions collected. Other details are given in the text. - , E280;----, radioactivity.

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(b)

IIIII

4I-

II

It)

'4

3 ,3'Ea

Cu

0

0

6060 0 20 40

511

(a) +

l1

6k-

0. V. RAJARAM, P. FATTERPAKER AND A. SREENIVASAN

260 280 300 320 340 260 280 300 320 340Wavelength (nm)

Fig. 2. U.v.-absorption spectra of a-[3H]tocopherol boundto lipoproteinfractions

U.v. spectra, showing absorption maxima at 292nm ofa-tocopherol separated by t.l.c. from cytosol of mucosa(b), serum (c) and liver cytosol (d) respectively. For detailssee the Materials and Methods section. The spectrum forstandard a-tocopherol (a) is included for comparison.

Table 1. Percentage recovery of ca-[3H]tocopherol fromlipoprotein fractions

The first lipoprotein fractions obtained after Sephadexchromatography of cytosol of mucosa and liver and ofserum samples were each extracted with acetone-lightpetroleum (b.p. 40-60'C) (1:2, v/v) and a-tocopherol wasseparated from the concentrated extract by t.l.c. asdescribed in the text. R, values of a-tocopherol and a-tocopherylquinone were 0.71 and 0.11 respectively.Values in parentheses represent the total radioactivity(c.p.m.) in the sample used for extraction. Results aretypical of three independent experiments.

a-Tocopherol a-Tocopherylquinone

SampleMucosa

(54260)Serum

(13415)Liver

(24590)

(c.p.m.) (%recovery) (c.p.m.) (%recovery)41695 75 780 2.0

10730

19180

80 340 3.0

78 425 2.5

Table 2. Association of a-[H3]tocopherol with serum, liverand mucosal lipoproteins

a-[5-Me-3HJTocopherol (100Ci/200g body wt.) wasadministered orally to rats, and its association with the firstlipoprotein fraction obtained by Sephadex chromato-graphy of mucosal cytosol, serum and liver cytosol wasstudied at various time-intervals. Other details are givenin the text. Values are expressed as specific radioactivities(i.e. c.p.m. present in a portion of a protein solution thatgives E280 = 1 in a 1cm light-path cell). Results are meansof five independent determinations ±S.E.M.

a-[3H]Tocopherol (c.p.m./E)Time(h)3468

18

Mucosal cytosol4160± 32119030± 13659275± 7151240+ 95105± 12

Serum1700+ 1515080± 5401600+ 125845+ 60212+ 20

Liver cytosol

8160+6203388±2801970+ 165479± 30

recovered as a-tocopherol and only 2-3% as a-

tocopherylquinone.In the second experiment (Table 2), the association

of a-[3H]tocopherol with serum, liver and mucosalcytosol lipoproteins was studied at various time-intervals after oral administration of the vitamin.Orally administered vitamin appears in the mucosalcytosol and blood serum only at 3h after adminis-tration and not at earlier time-intervals studied,whereas at 3h the liver cytosol contained no measur-able radioactivity. The association of a-tocopherol

with these lipoprotein fractions increased with time,reaching a maximum at 4h after administration anddeclining thereafter. The serum showed no furtherincrease in radioactivity up to 18h.

Further purification of vitamin E-binding lipo-protein fraction of mucosal and liver cytosol andserum has been achieved by polyacrylamide-gelelectrophoresis. The first protein peak obtained afterSephadex chromatography of these samples wasconcentrated by reverse dialysis against Polywax 6000and used for electrophoresis. The proteins of themucosal cytosol fraction resolved into five bands ofwhich the first protein band was a lipoprotein (Plate

1974

512

The Biochemical Journal, Vol. 140, No. 3

1_ ,~~~~~~~~~1 Olo |:.r-.

13

2- ~ -43-_-5 1- _

_--8_-9

1 :. m -

_ _ ._.

2

's 3-4-

.0) (i (i) (ii)

Plate 1

1-

2-3-4-.5-.

6--

(i) (ii)(a) (b) (c)

EXPLANATION OF PLATE I

Polvacrylamide-gel electrophoretograni ofmucosal cytosol (a), serum (b) and liver cy)tosol (c) protein fractionsThe first protein peak obtained after Sephadex chromatography of these samples was concentrated by reverse dialysis againstPolywax and used for gel electrophoresis. Protein (350,pg) was loaded on the column. For lipoprotein separation a prestainedsample of 1mg equiv. of protein was used. Other details are given in the text. (i) Lipoprotein; (ii) protein.

0. V. RAJARAM, P. FATITERPAKER AND A. SREENIVASAN

.I

(Facing p. 512)

The Biochemical Journal, Vol. 140, No. 3

(a) (b) (c)

EXPLANATION OF PLATE 2

Electrophoretic mobilities ofthe lipoprotein carrying a%-[3H]tocopherol(a) Mucosal cytosol; (b) serum; (c) liver cytosol. For details see the text.

0. V. RAJARAM, P. FATTERPAKER AND A. SREENIVASAN

Plate 2

a-TOCOPHEROL BINDING TO CELLULAR LIPOPROTEINS

Table 3. Relative radioactivity ofthe different lipoprotein/protein bands separable bypolyacrylamide-gel electrophoresis

Details ofelectrophoresis are given in the text and in the legend to Plate 1. The individual bands were sliced, treated with 30°/H202 and radioactivity determinations were made as described in the text. Values in the bands are expressed as percentageof the total radioactive (c.p.m.) of all protein fractions (minus blanks) in a given gel. Remaining bands contain negligibleradioactivity. Results are typical of four independent experiments. Maximum radioactivity was observed in the samefraction in each experiment.

Yo recovery of cx_[3HJtocopherol

Mucosal cytosol

Proteins Lipoproteins Proteins4.2 8.0 7.2

81.5 79.8 9.46.7 - 77.33.2 _

6.1

Serum

Lipoproteins Proteins5.6 6.04.5 82.9

84.0 5.54.6 3.0

Liver cytosol

Lipoproteins8.3

84.5

la), which stained with Sudan Black B. Themaximumrecovery of radioactivity from this lipoprotein band(Table 3) indicated the specific association of vitaminE with this lipoprotein. The proteins of the serumfraction resolved into nine well-defined bands ofwhich three were lipoproteins (Plate lb). Vitamin Ewas associated with the lipoprotein band 2. Narayanet al. (1966b) have measured the electrophoreticmobilities of the lipoproteins of various densityclasses. By using this system it has been shown in ourlaboratory (S. V. Koppikar, P. Fatterpaker & A.Sreenivasan, unpublished work) that fraction I fromSephadex chromatography ofserum could be resolvedinto chylomicrons, VLD lipoprotein and LD lipo-protein. The association of a-tocopherol with thelipoprotein band 2 strongly indicates that VLD lipo-protein is a carrier of orally fed vitamin. The proteinand lipoprotein bands separable from the livercytosol fraction are represented for comparison inPlate 1(c). The presence of a specific a-tocopherol-binding lipoprotein in this fraction has already beendiscussed in our previous communication (Rajaramet al., 1973). Plate 2 compares the electrophoreticmobilities of lipoproteins from mucosal cytosol,serum and liver cytosol carrying a-tocopherol.The radioactivity in the protein and lipoprotein

bands obtained after electrophoresing the mucosaland liver cytosol and serum fractions has been repre-sented in Table 3. In the small-intestinal mucosalsupernatant, protein band I (Table 3) and the corre-sponding lipoprotein band 1 contained about 80%of the total radioactivity as against negligible valuesin the remaining bands. Protein bands 2 and 3 (Table3) of serum together account for about 77% of thetotal radioactivity. These two bands had very similarelectrophoretic mobility, hence they could not beindividually sliced. They correspond to the lipoproteinband 2 from which 84% of the radioactivity couldbe recovered. For the liver cytosol fraction, protein

Vol. 140

band 1 contained the highest radioactivity and thiscorresponds to the lipoprotein band from which 84%radioactivity could be recovered.

Effect of orotic acid feeding on the absorption andbinding of a-tocopherol with serum lipoproteins

To understand the part played by the lipoproteinof intestinal mucosa in the transfer of the vitaminacross the intestine, absorption of cx-[3H]tocopherolwas studied in orotic acid-fed rats. It is known thataddition of as little as 1% orotic acid in the dietcompletely inhibits the production of lipoproteinsby the liver (Windmueller & Levy, 1967). With this asa basis, Windmueller & Levy (1968) have tested thecapacity of the intestine to synthesize lipoproteins.Their results indicate that intestinal lipoproteinsynthesis is not affected by orotic acid feeding. Hence,we have utilized this treatment as a means of ascer-taining whether a-tocopherol is transported directlyinto serum in association with mucosal lipoproteinor not. Orotic acid feeding resulted in a 75-80%

Table 4. Effect of orotic acidfeeding on the association ofat-[3H]tocopherol with lipoprotein fractions of serum and

mucosal cytosol

Orotic acid (1%) in the diet was fed to rats for 7 days.Lipoproteins from serum and intestinal mucosal cytosolwere isolated by Sephadex G-200 gel chromatography as

described in the text. Results are averages of four experi-ments ±S.E.M.

GroupOrotic acid-fedControl

a-[3H]Tocopherol (c.p.m./E)

Mucosal Serumlipoprotein lipoprotein18120+1150 1215±11019030± 1365 5080±540

Bandno.

Origin1234

513

0. V. RAJARAM, P. FAl-TERPAKER AND A. SREENIVASAN

decrease in the radioactivity of serum lipoproteinfraction as compared with controls (Table 4). In thecase of mucosal lipoprotein, however, there was nochange in radioactivity. A very low specific radio-activity of a-[3H]tocopherol in the serum lipoproteinof orotic acid-fed rats with no change in the radio-activity of the mucosal lipoprotein probably indi-cates that a-tocopherol is not carried in toto into serumbound with mucosal lipoprotein and that the con-centration of serum VLD lipoprotein may be impor-tant for optimum transfer of the vitamin across theintestine.Uptake of ac-tocopherol by serum from the mucosal

cells in vitro was examined to gain further informa-tion. Table 5 shows the uptake of a-[3H]tocopherolby serum from the small-intestinal mucosal cells oforotic acid-fed rats. Serum isolated from oroticacid-treated rats takes up 11 % of the label and thisis enhanced to 38% on the addition of VLD lipo-protein-containing fraction of normal serum to theincubation medium. Control serum takes up to 33%of the label which is three times the value for serumfrom orotic acid-fed rats. The results thus indicatethat there is enhancement in the uptake of a-toco-pherol by serum from orotic acid-fed rats uponaddition of VLD lipoprotein fraction of normalserum.

Table 5. Uptake in vitro ofa-[3H]tocopherolby serumfromsmall intestinal mucosal cells of orotic acid-fed rats

Orotic acid-fed rats weighing about 200g received an oraldose of lOO1 Ci of a-[5-Me-3H]tocopherol and were killed4h later. Mucosal cells (1 g) suspended in 2ml of thehomogenizing medium and containing a total radio-activity of 389750c.p.m. were incubated with 1 ml of serumfor 30min at 370C. The first lipoprotein fraction (VLDlipoprotein) isolated by Sephadex chromatography ofnormal serum (Fig. lb) was concentrated by reversedialysis over polyethylene glycol 6000, and 1 ml of concen-trate containing 20-25mg of protein was added to theincubation system. Other details are described in thetext. Results are averages of four experiments each with adifferent batch of mucosal cells ±S.E.M. P values for signi-ficance of increase from A to B and A to C are less than0.01.

Uptake in serum

SystemA. Mucosal cells+serum

from orotic acid-fed ratsB. Mucosal cells+serum

from normal ratsC. Mucosal cells+serum

from orotic acid-fedrats+lipoproteinfraction of normal serun

(total c.p.m.) (%)45500±1960 11.7

128360±9471 33.0

147800+ 12400 38.0

Discussion

Despite the numerous studies on the absorption ofa-tocopherol in animals (Simon et al., 1956a; Krish-namoorthy & Bieri, 1963; Pearson & Barnes, 1968;Kelleher et al., 1969) and a few in man (Klatskin &Molander, 1952; Rosenkrantz et al., 1953; Kelleher& Losowsky, 1970), the mechanism of absorption isstill obscure. Unlike vitamin A, as reported byGanguly (1969), vitamin E is not re-esterified duringabsorption. This is inferred from several studies(Simon et al., 1956a,b; Wiss et al., 1962) that haveshown an increase in the amount of free a-tocopherolin plasma even after oral dosing with a-tocopherolacetate or a-tocopheryl phosphate. Blomstrand &Forsgren (1968) have suggested that the route ofabsorption is lymphatic and that most ofthe absorbedvitamin is probably located in the chylomicrons(Johnson & Pover, 1962). It is known that orallyadministered a-tocopherol is absorbed to the extentof about 20-25% only, the remainder being excretedin faeces (Simon et al., 1956a).

In our preliminary experiments on the intracellulardistribution of the vitamin, we observed that thesoluble portion of the small-intestinal mucosa con-tained appreciable radioactivity 4h after oral ad-ministration of a-[3H]tocopherol. About 40% ofthe total radioactivity in the mucosa was associatedwith cytosol and the remainder with the particulatematerial. The results of the present experimentsindicate that the absorbed a-tocopherol is partlyassociated with a lipoprotein fraction of the intestinalmucosal cytosol separable by Sephadex gel filtrationand polyacrylamide-disc electrophoresis. This isaccompanied by a simultaneous appearance of thevitamin in the VLD lipoprotein of serum and a lipo-protein fraction of liver cytosol. The association ofthe vitamin with these lipoproteins reaches a maxi-mum at 4h after administration and declines there-after. Unlike vitamin A, there is no further increase inthe vitamin content of serum implying that the trans-ported vitamin is not released back from the liverinto the serum. This suggests that VLD lipoproteinofserum may be the carrier of orally fed vitamin.A variety of metabolic products, chiefly a-toco-

pherylquinone, dimers and trimers, are known to beformed after administration of the vitamin (Csallanyet al., 1962; Csallany & Draper, 1963; Draper et al.,1967). Csallany et al. (1970) have successfully sepa-rated these products by t.l.c. with benzene-hexane(19:1, v/v) as developing solvent. In the presentinvestigation, the same solvent system has beenemployed for the purification of a-tocopherol.The results indicate that over 75% of radioactivityassociated with the lipoprotein fraction is due toa-tocopherol alone. The recovery of radioactivityas a-tocopherylquinone was negligible and it ispossible that this product could have been formed

1974

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a-TOCOPHEROL BINDING TO CELLULAR LIPOPROTEINS 515

during the isolation procedures. Dimers or trimers,detectable by this procedure, were absent in theprotein-bound fractions. The results thus rule out thepossibility of any of these metabolic products beingassociated with the lipoprotein fraction.The lipoproteins of the mucosal and liver cytosol

are identical with VLD lipoprotein of serum in theirelution pattern from the Sephadex column (Fig. 1)and mobility on polyacrylamide gel (Plate 2). Theelectrophoretic mobility of these lipoproteins ina sufficiently large-pore gel of 3.5% polyacrylamidewas rather slow and at higher concentrations (5%and 7.5% polyacrylamide) these bands could notpenetrate the running gel. The Sephadex gel-filtrationpattern and the electrophoretic behaviour of theselipoproteins of mucosal and liver cytosol that carrya-tocopherol strongly indicate that the molecularsize of the binding protein is very high. Of the severalprotein bands obtained, vitamin E is associated witha lipoprotein band in all the cases as seen by the re-covery of 80% radioactivity in these bands. Theresults incidentally give a direct prooffor the presenceof VLD lipoprotein-like lipoprotein in the mucosalcells of small intestine, though its presence had beenimplicated by other workers (Windmueller & Levy,1968; Kessler et al., 1970). The observed associationof a-tocopherol with the soluble lipoproteins of theintestinal mucosa is of significance from the pointof view of its possible involvement in the transfer ofa-tocopherol across the intestinal wall. The resultsof orotic acid feeding experiments suggests that a-tocopherol is not secreted directly into serum inassociation with mucosal lipoproteins. These studiesalso suggested that the concentration of serum VLDlipoprotein is a limiting factor for optimum transferof the vitamin across the intestine, and that this ispossibly effected by an exchange of ac-tocopherolbetween mucosal cytosol lipoprotein and serum VLDlipoprotein, analogous to the exchange of phospho-lipids among the various plasma lipoproteins (Minari& Zilversmit, 1963). No increase in the radioactivityof the mucosal lipoprotein was observed (Table 4) aswould be expected for a feedback mechanism. It isprobable that such an increase may occur in themucosal subcellular fractions which may reflect anincrease, perhaps, in the free a-tocopherol. Thispossibility needs further investigation. The presentobservations are in accordance with the suggestionsof Ganguly et al. (1959) and Glover & Green (1957)regarding occurrence of specific lipoproteins in theintestinal mucosa, which help in the transport offat-soluble substances like carotenoids, vitamin A,sterols etc. across the intestine. A probable involve-ment of a-tocopherol-carrying lipoprotein of livercell-sap in the intracellular transport and distributionof the vitamin has been reported in our previouscommunication (Rajaram et al., 1973).Thus the present observations point to the exis-

tence of specific lipoproteins in rat tissues, i.e. intes-tinal mucosa, blood and liver cells. The mucosallipoprotein that carries the vitamin probably aids inthe absorption of a-tocopherol. The serum VLDlipoprotein, which represents the transport form isinvolved in the carriage of the vitamin to the varioustissues in addition to its important role in the transferof the absorbed vitamin across the intestine. Lastly,the transported vitamin is distributed among theintracellular organelles, in association with a lipo-protein of the cytosol probably specific to variousorgans.

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