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Keywords:

  • Blood-brain barrier;
  • Lipoprotein receptors;
  • α-Tocopherol;
  • High density lipoprotein

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Abstract: From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with α-tocopherol (αTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of αTocH in cells constituting the blood-brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of αTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein-associated αTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4°C, pBCECs bound LDL with high affinity (KD = 6 nM) and apolipoprotein E-free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. αTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]αTocH-labeled lipoprotein preparations were used. The majority of HDL3-associated αTocH was taken up in a lipoprotein particle-independent manner, exceeding HDL3 holoparticle uptake 8- to 20-fold. This uptake route is less important for LDL-associated αTocH (αTocH uptake ∼1.5-fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that αTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic αTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with αTocH in vivo.

In recent years, it has become evident that vitamin E is essential for normal neurological function as chronic deficiency results in characteristic and severe neurological symptoms (Muller and Goss-Sampson, 1990). Vitamin E is a generic term for eight naturally occurring compounds, the tocopherols (TocHs; α-, β-, γ-, and δ-) and tocotrienols (α-, β-, γ-, and δ-), with αTocH having the highest biological activity among the TocHs (Weiser and Vecchi, 1982). To function effectively, αTocH must be absorbed, transported, and delivered to cells after assembly with newly synthesized very low density lipoprotein (VLDL) and exchange to other major lipoprotein classes (Kayden and Traber, 1993).

As no specific transport proteins exist for circulating αTocH, it is transported in association with lipoproteins, the physiological carriers in vivo. Therefore, the mechanisms for the delivery of lipoprotein-associated lipids to tissues are also the major mechanisms for αTocH delivery. Uptake of αTocH can take place via low density lipoprotein (LDL) receptor-mediated pathways (Traber and Kayden, 1984). The importance of this lipoprotein class for αTocH delivery is further substantiated by the fact that apolipoprotein (apo) B-knockout animals have low circulating αTocH levels (Homanics et al., 1993). However, normal tissue supply with αTocH was demonstrated in Watanabe heritable hyperlipidemic rabbits lacking the LDL receptor (Cohn et al., 1992), and from these findings, it appears that uptake mechanisms are redundant. In contrast to LDL, high density lipoprotein (HDL) has received much less attention as a potential αTocH delivery system. Regardless of the precise uptake mechanism, it is evident that effective αTocH supply to tissues and organs in vivo is intimately coupled to functional lipoprotein metabolism.

Clinical and neuropathological evidence for the necessity of normal αTocH concentrations comes from studies with patients with either lipid malabsorption, abetalipoproteinemia, or genetic defects in the α-tocopherol transfer protein (αTTP) (Kayden and Traber, 1993; Muller, 1995). During the second decade of life, patients with lipid malabsorption or abetalipoproteinemia develop a characteristic neurological syndrome comprising loss of reflexes, loss of balance, deformed feet, loss of vibrational sense, a pigmentary muscle weakness, and pigmentary retinopathy (Muller et al., 1983). Patients with familial isolated vitamin E (FIVE) deficiency have no defects in lipid absorption or de novo lipoprotein synthesis, although they suffer the same neurological symptoms as observed for the patients described above. FIVE deficiency is an autosomal recessive disease that closely resembles Friedreich's ataxia (Ouahchi et al., 1995). Studies with deuterated TocHs have revealed a much faster removal of αTocH from the circulation of FIVE patients than from healthy controls (Traber et al., 1990). It has been shown that this higher turnover is caused by a genetic defect in the αTTP (Ouahchi et al., 1995), confirming that FIVE deficiency is due to a liver defect of αTocH assembly with newly synthesized VLDL. One of the cardinal syndromes of this disorder is ataxia, indicative for the particular vulnerability of the cerebellum to αTocH deficiency (Ben Hamida et al., 1993).

The findings mentioned above underline that the central nervous system is dependent on a constant and adequate supply of αTocH. As a lipid-soluble antioxidant, αTocH is carried in association with lipoproteins in the peripheral circulation, with almost similar distribution between the LDL and HDL fraction, and it is conceivable that these lipoprotein classes will provide the majority of αTocH taken up across the blood-brain barrier (BBB). For these reasons, we have attempted to characterize potential lipoprotein binding sites on porcine brain capillary endothelial cells (pBCECs), characterized the interaction of LDL and HDL3 with these cells, and finally compared the uptake of LDL- and HDL3-associated αTocH by monolayers of pBCECs. We have also assessed whether pBCECs are able to discriminate in favor of one of the eight αTocH isomers, which are identical with regard to antioxidative capacity but differ with respect to their biological activity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Materials

Earle's medium M199, Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium (1:1, vol/vol), penicillin/streptomycin (P/S), gentamicin, L-glutamine, and trypsin were obtained from Biochrom (Berlin, Germany); dextran (molecular mass ∼ 162 kDa), Percoll, protease (dispase from Bacillus polymyxa), and 3-amino-9-ethylcarbazole were purchased from Sigma (Vienna, Austria). Ox serum was from PAA Laboratories (Linz, Austria). Collagenase/dispase was from Boehringer Mannheim (Vienna, Austria), nylon sieve from ZBF (Zurich, Switzerland), and Na125I (specific activity 629 GBq/mg) from DuPont NEN (Vienna, Austria). Plasticware for cell culture was from Costar (Vienna, Austria). PD10 size exclusion columns were obtained from Pharmacia (Uppsala, Sweden). Mayer's hemalum and Kaiser's glycerol gelatin were from Merck (Vienna, Austria), and tissue Tec OCT compound (tissue freezing medium) was from Miles (Elkhard, IN, U.S.A.). Antibodies used for immunohistochemistry (von Willebrand factor polyclonal rabbit IgG, desmin murine monoclonal antibody IgG1, rabbit and mouse nonimmune serum, horseradish peroxidase-labeled streptavidin-biotin kit) were purchased from Dako (Vienna, Austria). The rabbit anti-ZO-1 antibody was obtained from Zymed (San Francisco, CA, U.S.A.). All solvents were purchased in the highest available quality from Sigma and all other chemicals were from Boehringer-Mannheim. LC-18 reversed phase HPLC columns were obtained from Supelco (250 × 4.6-mm i.d.; Vienna, Austria) or from Chrompack (200 × 2-mm i.d.; Vienna, Austria). The chiral phase (Chiralcel OD; 250 × 4.6-mm i.d.) used for the separation of 2R- and 2S-αTocH isomers as their methyl ether derivatives was obtained from Stehelin (Basel, Switzerland). Ready Safe scintillation cocktail was obtained from Beckman (Vienna, Austria). [14C]all-rac-αTocH (termed [14C]αTocH throughout this article) was a gift from Henkel (specific activity 2.07 GBq/mmol).

Isolation and culture of pBCECs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Porcine brains were obtained from freshly slaughtered pigs in the local slaughterhouse. pBCECs were isolated by several enzymatic digestion and centrifugation steps according to Tewes et al. (1997). In brief, after removal of the meninges and the secretory areas, the gray and white matter of the brain cortex were minced using a sterile cutter with staggered rolling blades. This material was suspended in preparation medium [M199 containing 1% P/S, 1% gentamicin, and 0.35% glutamine (vol/vol)] and incubated with solid dispase (1% wt/vol) for 2 h at 37°C. A dextran solution (16% wt/vol) was added to obtain a final 10% (wt/vol) suspension followed by centrifugation at 6,800 g (10 min, 4°C). To separate larger vessels, the pellet was resuspended and filtered through a 180-μm nylon sieve. The capillaries were incubated with 0.08% (wt/vol) collagenase/dispase at 37°C (40 min, gentle stirring). After collection of the released cell aggregates by low spin centrifugation (140 g, 10 min, 20°C), they were further purified by density gradient centrifugation: The yield of one brain was resuspended in 5 ml of preparation medium and centrifuged on a discontinuous Percoll gradient (15 ml of 1.07% g/ml; 20 ml of 1.03 g/ml) at 1,300 g for 10 min in a swinging bucket rotor. The cell clusters of the pBCECs gathered at the interface were washed in preparation medium and centrifuged at 140 g for 10 min. After this 5-h preparation procedure, cell clusters from one brain were seeded in M199 [containing 10% ox serum, 1% P/S, 1% gentamicin, and 0.35% glutamine (vol/vol)] on eight 75-cm2 (total) rat tail collagen (40 μg/ml)-coated flasks. After 1 day in vitro (DIV), cells were washed with phosphate-buffered saline (PBS), and gentamicin-free medium was added.

After 2 DIV, cells were seeded on collagen-coated multiwell cell culture clusters or 10-cm diameter Petri dishes or multiwell plates for the experiment at a minimum density of 30,000 cells/cm2. Prior to the experiments (which were performed after 4 or 5 DIV with confluent dishes), the cells were preincubated with serum-free medium [DMEM/Ham's F12 medium (1:1 vol/vol) containing 1% P/S and 0.35% glutamine] overnight.

Immunohistochemical characterization

Fresh porcine brain specimens (0.7 cm3) were either snapfrozen in liquid nitrogen for later processing or immediately frozen in a cryostat (Microm HM 500 OM; Microm, Walldorf, Germany) supported by Tissue Tec OCT compound. Serial cryosections (5 μm) were collected on glass slides, air dried for 2 h at 22°C (room temperature), fixed in acetone for 5 min at 22°C, and stored at -40°C until required. Glass chamberslides (Lab-Tek; Miles Scientific, U.S.A.) were washed with PBS and dried at 22°C for 3 h. Afterward, the slides were fixed in acetone for 5 min at 22°C and stored at -40°C until required. Before staining, sections or chamberslides were thawed, fixed once more in acetone for 5 min at 22°C, and rehydrated in PBS for 5 min. Immunostaining was performed as described (Hammer et al., 1999). All incubations were performed in a moist chamber at 22°C, using PBS for washes between the incubation steps. In brief, after 10-min incubation with blocking solution, sections were incubated with the primary antibodies (30 min) followed by sequential 15-min incubations with a biotinylated link antibody and peroxidase-labeled streptavidin. The reaction was developed with 3-amino-9-ethylcarbazole as peroxidase substrate solution and terminated by washing with distilled water. Counterstaining of the sections was performed with Mayer's hemalum, and the slides were mounted with Kaiser's glycerol gelatin. Control experiments for all immunohistochemical assays encompassed immunohistochemistry with nonimmune mouse or rabbit IgG and without primary and without secondary antibodies. Pictures were taken with an Axiophot microscope (Zeiss, Oberkochen, Germany).

Lipoprotein preparation

Human LDL and apoE-free HDL3 were prepared by density gradient ultracentrifugation of plasma obtained from normolipidemic human volunteers in a TL120 tabletop ultracentrifuge (at rav = 350,000 g; Beckman) (Sattler et al., 1994). Lipoproteins were recovered by direct aspiration and desalted by size exclusion chromatography on PD-10 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of apoA-I as the major apolipoprotein in HDL3 and apoB-100 in LDL. Molar concentrations were calculated from total lipoprotein mass using molecular masses of 2,500 kDa (LDL) and 175 kDa (HDL3) (Chapman, 1986).

Labeling procedures

HDL3 (1.5 mg of protein/2 ml of PBS) or LDL (0.6 mg of protein/2 ml of PBS) was labeled by addition of an ethanolic [14C]αTocH solution (max. 25 μl) and incubation at 37°C (3 h under argon in a shaking water bath) and resulted in specific activities of 5-10 dpm/ng of protein. Non-lipoprotein-associated [14C]αTocH was removed by size-exclusion chromatography on PD-10 columns. Lipids were extracted and analyzed by radio-HPLC as described below. To allow simultaneous quantification of tracer and endogenous αTocH, the radiometric detector was preceded by a fluorescence detector (see below).

HDL3 and LDL were iodinated using N-bromosuccinimide as the oxidizing agent (Goti et al., 1998). Routinely, 1 mCi of Na125I was used to label either 5 mg of HDL3 protein or 2 mg of LDL protein. This procedure resulted in specific activities of between 300 and 450 dpm/ng of protein. Lipid-associated radioactivity was always <3% of total radioactivity. No cross-linking or fragmentation of apoA-I or apoB-100 due to the iodination procedure was detected by SDS-PAGE and subsequent autoradiography.

SDS-PAGE, ligand blots, and immunoblots

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

SDS-PAGE of pBCEC membrane protein fractions was performed on 3.5-5 or 5-15% gradient gels with electrophoresis at 150 V for 90 min in a Bio-Rad Miniprotean chamber. Samples for SDS-PAGE (50 μg of protein/lane) were treated with sample buffer (0.1 M Tris-HCl, pH 6.8, 4% SDS, 15% glycerol, and 1% mercaptoethanol) at a ratio of 1:1 (vol/vol). For ligand and western blots, proteins were electrophoretically transferred to nitrocellulose membranes (150 mA, 4°C, 90 min).

For ligand blotting experiments, the nitrocellulose membranes were incubated with blocking buffer containing either LDL or HDL3 (50 μg/ml of protein, 24°C, 90 min). After washing, the membranes were incubated with rabbit anti-human apoB-100 or rabbit anti-human apoA-I antiserum (1:1,000) overnight. Visualization of bound lipoproteins was performed with peroxidase-conjugated goat anti-rabbit IgG (1: 2,000) and subsequent enhanced chemiluminescence (ECL) development.

Immunochemical detection of the LDL receptor in immunoblots was performed using a monoclonal anti-LDL receptor antibody (clone C7, dilution 1:1,000). Detection was performed with peroxidase-conjugated rabbit anti-mouse IgG (dilution 1:2,000) and subsequent ECL development.

Cell culture studies

Mass transfer of αTocH. pBCECs were plated on 10-cm Petri dishes. Freshly prepared HDL3 and LDL were added to DMEM/Ham's F12 medium (1:1, vol/vol) at a final concentration of 167 ng of αTocH/ml. After incubation, the cells were washed twice with bovine serum albumin-containing PBS (10 mg/ml) and twice with PBS prior to lipid extraction and αTocH analysis.

[14C]αTocH uptake. Cells were plated on 6- or 12-well cluster plates. [14C]αTocH-labeled HDL3 or LDL (specific activities between 5 and 10 cpm/ng of protein) was added to DMEM/Ham's F12 medium at a final concentration of 10 μg/ml of lipoprotein protein. At the end of the incubation, the cells were washed twice as described above. Cells were lysed in NaOH (0.3 M). An aliquot of the cell lysate was used to determine the cellular protein content as described (Goti et al., 1998), and the remaining lysate was mixed with scintillation cocktail to determine the radioactivity. [14C]αTocH-lipoprotein uptake is expressed in terms of apparent particle uptake. Based on the specific activity of the 14C-labeled lipoprotein preparations, the amount of lipoprotein that would be necessary to deliver the observed amount of tracer was calculated. This is necessary to allow quantitative comparison of the data and is a result of lipoprotein particle-independent (selective) lipid uptake by nonendocytotic mechanisms (Pittman et al., 1987).

125I-Lipoprotein uptake. Cells were plated on 12-well cluster plates. 125I-HDL3 and 125I-HDL (specific activities between 400 and 800 cpm/ng of lipoprotein protein) were added to DMEM/Ham's F12 medium at the concentrations indicated. After the incubation, cells were washed and lysed as described above. All incubations were performed in the absence or presence of a 20-fold excess of the corresponding competitor to differentiate between total and nonspecific uptake. The equilibrium dissociation constant (KD) and maximal binding (Bmax) were determined from experiments performed at 4°C. Specific binding was obtained by subtraction of nonspecific binding from total binding. The binding isotherms were fitted according to a single-site displacement model using a computerized non-linear fitting program (GraphPad Prism). Degradation of 125I-HDL3 or 125I-LDL was estimated by measuring the non-trichloroacetic acid-precipitable radioactivity in the medium after precipitation of free iodine with AgNO3 (Jessup et al., 1992).

αTocH analysis

The cellular lipids were extracted with hexane/propan-2-ol (3:2, vol/vol) at 25°C (30 min on a plateau shaker). The lipid extracts were dried under nitrogen, redissolved in 200 μl of methanol, and analyzed by reversed-phase HPLC on an LC-18 column with methanol as mobile phase (1 ml/min) and fluorescence detection (excitation and emission wavelengths 292 and 335 nm, respectively). Quantitation was performed by peak area comparison with external standards of known concentrations. The concentration of freshly prepared αTocH standards in hexane was estimated photometrically (ε292 = 3,467 M-1· cm-1).

Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

The cellular lipid extracts were dried under argon and converted to the corresponding methyl ether derivatives as described (Riss et al., 1994). In brief, to the dry extracts (kept under argon), 50 μl of monoglyme, 25 μl of KOH (25% wt/vol, dropwise addition, vortex after each addition), and 30 μl of dimethyl sulfate (dropwise addition, vortex after each addition) were added. The incubation mixture was then kept at 50°C for 30 min. The ether phase was removed with nitrogen, and to the remaining mixture 100 μl of water and 500 μl of hexane were added and vortexed for 1 min. To achieve phase separation, the samples were centrifuged at 3,000 rpm (4°C, 10 min). The hexane phase was removed, the samples were extracted a second time, and the combined hexane layers containing the αToc-ME derivatives were dried under argon, resuspended in 150 μl of hexane, and analyzed by chiral-phase HPLC with radiometric detection as described below.

Chiral-phase HPLC of αToc-ME derivatives

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

αToc-ME derivatives were separated on a 250 × 4.6-mm Chiralcel OD column. This chiral phase separates all-rac-αToc-ME derivatives into five peaks, the first peak containing the four 2S isomers and separating the four 2R isomers (RSS, RRS, RRR, and RSR) (Riss et al., 1994). Forty microliters of the cellular lipid extracts in hexane was injected into the chromatographic system, which consisted of a Waters 510 pump, a Waters 717 plus autosampler, and a Packard/Canberra radiometric FlowOne beta detector that was preceded by a Waters 490E programmable multiwavelength UV-Vis detector set at 283 nm to allow simultaneous detection of labeled and unlabeled αToc-ME isomers. The mobile phase was hexane at 1 ml/min, and the scintillation fluid flow was set at 2 ml/min. Total analysis time for isomer separation was 60 min.

Characterization of pBCECs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Von Willebrand factor (factor VIII), widely used as the most reliable marker for cells of endothelial origin (Jaffe, 1977), showed a strong punctate staining pattern on cryosections of porcine brain (Fig. 1A). The endothelial nature of the purified pBCECs used during the present study was confirmed by their positive granular staining for the factor VIII antigen (Fig. 1B), in a similar manner as observed in the brain cryosections. The antibody directed against the tight junction-associated protein ZO-1 revealed a strong staining of the tight junction complexes (a typical feature of the BBB) of microvessels in the brain specimens (Fig. 1C). In primary cultures of pBCECs this antibody displayed a continuous network at sites of cell-cell contact between endothelial cells, suggesting the presence of functional tight junctions after 5 DIV (Fig. 1D). pBCECs were not contaminated with pericytes, as judged from immunohistochemistry using antidesmin antibodies (not shown).

image

Figure 1. Characterization of brain tissue slices and capillary endothelial cell monolayers isolated therefrom. Localization of von Willebrand factor (arrows) in cryosections of porcine brain (A) and pBCECs (B) and of tight junction-associated protein ZO-1 (arrows) in cryosections of porcine brain (C) and pBCECs (D) is shown. ×350.

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Identification of possible binding proteins for HDL3 and LDL

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

As αTocH is transported in association with lipoproteins and possibly also transcytosed in lipoprotein-associated form across the BBB, we have tried to identify possible binding proteins by immunoblotting and ligand blotting techniques. Immunoblots revealed the presence of the LDL receptor (Fig. 2A) with an apparent molecular mass of 163 kDa [reducing conditions during SDS-PAGE, in line with Schneider et al. (1985)]. Ligand blotting experiments with pBCEC membrane protein fractions revealed one major binding protein for HDL3 (Fig. 2B) with a molecular mass of ≈100 kDa. This experimental approach was unsuitable to reveal LDL binding proteins on nitrocellulose-immobilized membrane proteins of pBCECs.

image

Figure 2. Identification of the LDL receptor and a putative HDL3 binding protein on pBCECs. A: Membrane proteins were separated on 3.5-5% SDS-PAGE gradient gels (reducing conditions) and transferred to nitrocellulose. The LDL receptor protein was identified using monoclonal antibody C7 (dilution 1:1,000). After addition of peroxidase-conjugated rabbit anti-mouse IgG (dilution 1:2,000), immunoreactive bands were visualized with the ECL system. B: Membrane proteins were separated on 5-15% gradient gels and transferred to nitrocellulose. Ligand blotting experiments were performed by incubating the nitrocellulose strips with 50 μg of HDL3, followed by a rabbit anti-human apoA-I antiserum as primary antibody (lane 1). Another nitrocellulose strip (lane 2) was incubated in the presence of HDL3 (200 μg) but with the absence of the primary antibody as a control. Peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody (1:2,000). Immunoreactive bands were visualized with the ECL system. The molecular masses of the marker proteins are indicated. Arrows indicate the LDL receptor (A) and the 100-kDa HDL3 binding protein (B).

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Binding of LDL and HDL3 to pBCECs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

To determine the equilibrium binding constants (KD and Bmax) for 125I-LDL and 125I-HDL3, pBCECs were incubated at 4°C in the presence of the corresponding tracer. Total, nonspecific, and specific binding was measured and calculated as described in Materials and Methods. pBCECs were incubated in the presence of increasing lipoprotein concentrations (1-70 μg of 125I-LDL and 1-50 μg/ml 125I-HDL3) for 2 h at 4°C. The corresponding specific binding isotherms (calculated as described in Materials and Methods) are shown in Fig. 3. The kinetic constants calculated by nonlinear regression analysis were 5.5 and 98 nM (KD, LDL vs. HDL3) and 316 and 254 ng of total lipoprotein mass/mg of cell protein (Bmax, LDL vs. HDL3). On the basis that 1 mg of cell protein corresponds to ∼4 × 106 cells, it can be calculated that pBCECs contain ∼20,000 and 200,000 binding sites for LDL and HDL3, respectively.

image

Figure 3. Binding of LDL (A) and HDL3 (B) to pBCECs. Confluent 12-well cluster plates were incubated at 4°C (2 h) in the presence of the indicated 125I-labeled lipoprotein concentrations. Binding experiments were performed in the absence or presence of a 20-fold excess of the corresponding competitor to differentiate between specific and nonspecific binding. Cells were washed and lysed in NaOH, and the radioactivity was counted. An aliquot was used to determine the cellular protein content. The specific binding data (calculated by subtraction of nonspecific binding and total binding as described in Materials and Methods) were fitted according to a single-displacement site model, and the KD and Bmax values were calculated by nonlinear regression analysis. Results shown represent mean values from one representative experiment (of four) performed in triplicate dishes.

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Uptake of LDL- and HDL3-associated αTocH by pBCECs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

The following experiments were performed to clarify uptake mechanisms of LDL- and HDL3-associated αTocH and to compare the efficacy of αTocH delivery from LDL or HDL3 to pBCECs. The lipoproteins were labeled in the protein (125I) and in the lipid ([14C]αTocH) moiety. Data of these experiments are shown in Fig. 4 and are expressed in terms of apparent (the amount of lipoprotein that would be necessary to deliver the observed transfer of lipid tracer) lipoprotein particle uptake, as suggested by Pittman and colleagues (1987). At all time points investigated, LDL holoparticle uptake exceeded HDL3 holoparticle uptake by almost fourfold (1,840 vs. 440 ng/mg of cell protein at 24 h, LDL vs. HDL3, respectively).

image

Figure 4. Time-dependent lipoprotein and lipoprotein-associated αTocH uptake by pBCECs. Cells were cultivated in 12-well cluster plates and incubated at 37°C in 1 ml of medium containing 10 μg of 125I- or [14C]αTocH-labeled LDL or HDL3. At the indicated times, the cells were washed as described in Materials and Methods and lysed in NaOH, and the radioactivity in the cellular lysate was determined. An aliquot of the lysate was used to determine the cellular protein content. Results shown are expressed in terms of apparent particle uptake and represent means ± SD from one representative experiment (of four) performed in triplicate dishes.

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However, the situation was different when αTocH uptake from LDL or HDL3 was compared: Whereas the uptake of LDL-associated αTocH showed at least a tendency for saturation kinetics, uptake of HDL3-associated αTocH increased in a nearly linear manner over the 24-h incubation time. In addition, uptake of HDL-associated αTocH was significantly higher than observed for LDL-associated αTocH (8,540 vs. 3,390 ng/mg of cell protein, HDL3 vs. LDL, respectively, at 24 h). It is of importance to note that the uptake of HDL-associated αTocH exceeded HDL3 holoparticle uptake between 8-fold (2 h) and 20-fold (24 h). These findings clearly indicate that the uptake of HDL-associated αTocH occurs in a holoparticle-independent manner. The same—although to a much lesser extent—is true for LDL-associated αTocH where αTocH uptake exceeded holoparticle uptake between 1.5-fold (2 h) and 1.8-fold (24 h).

The accumulation of cellular αTocH can be attributed to lipoprotein binding, internalization, degradation, and/or lipoprotein-independent αTocH uptake. To clarify which one of these processes contributes to the majority of αTocH uptake from LDL or HDL3 in our in vitro system, pBCECs were incubated in the presence of 125I-and [14C]αTocH-labeled LDL and HDL3, respectively, and the amount of total αTocH uptake was compared with the amount of cell-associated and degraded 125I-lipoprotein particles (Fig. 5). In line with data shown in Fig. 4, HDL3-associated αTocH (Fig. 5B) was taken up more efficiently than LDL-associated αTocH (Fig. 5A, e.g., 2,730 vs. 1,580 ng/mg of cell protein at 6 h). In contrast, holoparticle uptake of HDL3 was less efficient (121 and 73 ng/mg of cell protein, cell association and degradation, respectively) than of LDL (221 and 697 ng/mg of cell protein, cell association and degradation, respectively). It is also important to note that the degradation rates of HDL are lower than those of LDL.

image

Figure 5. Comparison of metabolic pathways contributing to total αTocH uptake by pBCECs. Cells were cultivated in 12-well cluster plates and incubated at 37°C in the presence of 10μg of 125I- or [14C]αTocH-labeled LDL (A) or HDL3 (B). To determine the amount of lipoprotein degradation, the medium was removed at the indicated time points and the non-trichloroacetic acid-precipitable radioactivity (degradation) was determined as described in Materials and Methods. The remaining cells were washed, and the cell-associated 125I radioactivity was determined (cell association). In parallel incubations, [14C]αTocH-labeled lipoproteins were used to determine total αTocH uptake. Results shown represent mean values from one representative experiment (of three) performed in triplicate dishes.

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A major question arising from the results shown in Figs. 4 and 5 is whether uptake of [14C]αTocH occurs by mass transfer or merely tracer exchange. To clarify this issue, pBCECs were incubated in the presence of LDL or HDL3 adjusted for equal concentrations of radioactive and/or unlabeled (endogenous) αTocH. Lipoproteins were analyzed for their αTocH content immediately after isolation and revealed a content of 1.8 and 2.6 nmol of αTocH/mg of HDL3 or LDL (total lipoprotein mass), respectively. This corresponds to a molar ratio of 0.3 and 6.5 mol of αTocH/mol of HDL3 and LDL. At the indicated time points, cells were washed and analyzed for their αTocH content by liquid scintillation counting or HPLC analysis of the cellular lipid extracts. From data shown in Fig. 6, it is evident that the amount of [14C]αTocH delivered by HDL3 exceeded that transferred via LDL at all the time points analyzed. This effect was most pronounced after 24 h when HDL3-mediated [14C]αTocH transfer was ∼2.5-fold higher than that mediated by LDL (apparent particle uptake 8,540 vs. 3,390 ng/mg of cell protein at 24 h, HDL3 vs. LDL). The same effect was observed when pBCECs were incubated in the presence of unlabeled LDL or HDL3. After a 24-h incubation period, cells acquired 48 ng of αTocH/mg of cell protein during an incubation in the presence of 167 ng/ml HDL3-associated αTocH. In contrast, when cells were incubated in the presence of 167 ng/ml LDL-associated αTocH, the corresponding value was 8.8 ng of αTocH/mg of cell protein, indicating that HDL3-associated αTocH is transferred with higher efficacy than LDL-associated αTocH. In addition, these experiments show that tracer uptake closely reflects uptake of endogenous lipoprotein-associated αTocH and not merely exchange of the [14C]αTocH tracer to the cells.

image

Figure 6. Mass transfer of lipoprotein-associated αTocH to pBCECs. Mass transfer of LDL- and HDL3-associated αTocH was determined in 10-cm Petri dishes. The lipoprotein concentrations were normalized to contain equal amounts of endogenous αTocH (167 ng/ml). At the indicated times, the cellular lipids were extracted (hexane/propan-2-ol 3:2, vol/vol), and the αTocH content was analyzed by HPLC with fluorescence detection as described in Materials and Methods. Data shown represent means ± SD from one representative experiment (of two) performed in triplicate dishes. Alternatively, pBCECs were incubated in the presence of [14C]αTocH-labeled LDL or HDL3 adjusted to contain equal radioactivity. At the indicated times, the cells were washed and lysed, and the radioactivity was counted. An aliquot of the lysate was used to determine the cellular protein content. Data shown represent means ± SD from one representative experiment (of three) performed on triplicate dishes.

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pBCECs do not discriminate between different αTocH isomers

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Natural vitamin E is in the 2,4′,8′-RRR configuration, whereas synthetic vitamin E preparations contain all of the eight possible isomers (Traber and Packer, 1995). It has been demonstrated that the brain has an exceptional capacity to discriminate in favor of RRR-αTocH (Ingold et al., 1987). As the BBB would be the first system involved in such a biodiscrimination step during uptake of lipoprotein-associated αTocH from the peripheral circulation into the central nervous system, we have investigated whether pBCECs are able to discriminate between different isoforms of αTocH. pBCECs were incubated in the presence of [14C]αTocH for 6 h, the cellular lipids were extracted, and the TocHs were converted into their corresponding αToc-ME derivatives (see Materials and Methods). The αToc-ME derivatives were then separated by chiral HPLC, which allows the separation of all R isomers from the remaining S isomers. The chromatogram obtained after separation of the αToc-ME derivatives prepared from an authentic [14C]αTocH standard is shown in Fig. 7A. Within the 6-h incubation period, pBCECs accumulated 5,025 ± 416 cpm of the 2S isomers and 5,277 ± 141 cpm of the 2R isomers, indicating that pBCECs are not able to discriminate in favor of the 2R isomers (Fig. 7B). This was also evident for the four members of the 2R isomers, which accumulated in comparable concentrations (1,256, 1,448, 1,365, and 1,209 cpm of the RSS, RRS, RRR, and RSR isomers, respectively). These findings indicate that the pronounced capacity of the brain for biodiscrimination in favor of the 2,4′,8′-RRR isomer does not occur at the BBB level, at least in the in vitro model used during the present study.

image

Figure 7. Isomer uptake from αTocH by pBCECs. pBCECs were cultured on 10-cm Petri dishes and received medium containing 500,000 dpm/dish [14C]αTocH. After a 6-h incubation, the cellular lipids were extracted, converted to the corresponding αToc-ME derivatives, and analyzed by chiral radio-HPLC as described in Materials and Methods. A: A representative chromatogram of the αToc-ME derivatives prepared from an authentic [14C]αTocH standard (≈1,000 cpm injected) is shown (1 = 2S isomers; 2 = RSS, 3 = RRS, 4 = RRR, and 5 = RSR; peak assignment as in Riss et al., 1994). B: The intracellular αTocH isomer distribution after a 6-h incubation of pBCECs under the conditions described above is shown. Data represent means ± SD from one representative experiment (of two) performed on triplicate dishes.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Clinical evidence for the necessity of an optimal supply of the brain with αTocH is coming from three major patient groups, with either deteriorations in lipoprotein metabolism (Traber et al., 1994; Muller, 1995), lipid malabsorption (Harries and Muller, 1971), or mutations in αTTP (Traber et al., 1993; Ouahchi et al., 1995). All of the three patient groups suffer a severe αTocH deficiency accompanied by severe neurological syndromes. If recognized early enough, supplementation with αTocH can halt or sometimes reverse the symptoms (Muller, 1995). These observations provide clear-cut evidence that an adequate and sufficient supply to the nervous system of αTocH is essential for normal neurological functions. As αTocH is a lipid-soluble compound/antioxidant, it is transported in association with lipoproteins in the peripheral circulation, with the LDL and the HDL fraction being the major carriers (Kostner et al., 1995; Sattler et al., 1996). Therefore, it seems reasonable to assume that one of these—or even both lipoprotein species together—act to maintain sufficiently high concentrations of αTocH in the central nervous system.

As αTocH is transported in association with circulating lipoproteins, lipoprotein receptors and/or binding proteins present at the BBB conceivably mediate the uptake of lipoprotein-associated αTocH into the brain endothelial cell layer. In line with previous reports (e.g., Dehouck et al., 1994, 1997) demonstrating LDL receptor expression at the BBB, we could identify the LDL receptor in the pBCEC model used during the present study. The LDL receptor is most probably the single high affinity binding site for LDL observed in the present study and by others (Dehouck et al., 1994). Although it was suggested that the LDL receptor that is expressed at the BBB might be a transcytotic rather than a recycling receptor (Dehouck et al., 1997)—a function very different from the classic role of the LDL receptor—we have observed pronounced LDL degradation by pBCECs grown in monolayers.

With regard to potential HDL binding proteins, the situation is less clear. During the present study, we could identify a putative 100-kDa HDL3 binding protein in ligand blots. However, whether this protein is identical to HB2 (an HDL binding protein with an apparent molecular mass of 100 kDa), which is expressed in brain (besides lung, liver, and kidney) (Matsumoto et al., 1997; Fidge, 1999), remains to be elucidated. It is, however, noteworthy that HB2 mediates low affinity (KD≈ 300 nM), high capacity binding of HDL (Matsumoto et al., 1997), compatible with findings of the present study. The KD and Bmax values for HDL binding to pBCECs observed during the present study are in the same range as reported for bovine cerebral endothelial cells (de Vries et al., 1995).

One of the key observations during the present study was the pronounced difference in αTocH uptake when either LDL or HDL3 was used as donor vehicle in vitro. This is not only true for uptake efficacy but also for the mode of uptake. With regard to uptake efficacy, HDL is a better αTocH donor than LDL, a finding compatible with one of our previous reports (Goti et al., 1998). However, the uptake mechanisms also appear to be markedly different: Whereas LDL-associated αTocH is taken up in ∼1.5-fold excess of holoparticles, HDL-associated αTocH is taken up in 8- to 20-fold excess of HDL3 lipoprotein particles. Lipoprotein particle-independent lipid uptake is commonly termed “selective uptake” and is most pronounced for HDL-associated cholesteryl esters. Selective HDL cholesteryl ester uptake is mediated by a scavenger receptor of the B class (SR-BI, the murine HDL receptor; Acton et al., 1996) or its human homologue (CLA-1; Murao et al., 1997). Selective uptake was also observed for LDL-associated lipids, however, to a much lesser extent than HDL (Rinninger et al., 1995), compatible with findings obtained during the present study. Currently, it is not clear whether the lipoprotein particle-independent uptake of lipoprotein-associated αTocH as observed during the present study is a receptor-mediated effect and whether SR-BI could be involved. As αTocH uptake into brain is a tightly regulated process, we would consider free diffusion of αTocH across the endothelial cell layer of the BBB of minor importance. The underlying tight regulatory mechanisms of αTocH uptake across the BBB are reflected by either negligible (Pappert et al., 1996) or marginal (Vatassery et al., 1998) increases of αTocH concentrations in cerebrospinal fluid after supplementation with very high doses of αTocH for up to 1 year.

Different uptake mechanisms of LDL- and HDL3-associated αTocH are also reflected by the markedly different degradation rates of the two lipoprotein classes: Whereas in the case of LDL, ∼80% of the lipoprotein particles were degraded within a 6-h incubation, only 40% of HDL3 particles (in line with data presented by de Vries et al., 1995) were subjected to intracellular degradation. This is indicative of pronounced differences in the intracellular handling of the two lipoprotein classes by pBCECs. The different intracellular routing of αTocH taken up in conjunction with lipoprotein particles could have bearings for transcytosis of αTocH across the BBB and subsequent delivery to astrocytes, glial cells, and neurons. Oxidative stress is one of the factors implicated in the pathogenesis of neurodegenerative diseases, and severe neurodegeneration was observed in experimentally induced vitamin E-deficient animal models (for review, see Muller and Goss-Sampson, 1990) and vitamin E-deficient patients (Sung et al., 1980). A number of reports have shown that αTocH can rescue neurons from the assault of a variety of different agents inducing oxidative stress. Among these agents are peroxynitrite (Vatassery, 1996), β-amyloid (Koppal et al., 1998), glutamate (Ciani et al., 1996), transition metals (Cardoso et al., 1998), and hydroperoxides (Amano et al., 1994). On the other hand, two major in vivo intervention trials have shown that supplementation with αTocH only moderately slowed the progression of moderately severe Alzheimer's disease (Sano et al., 1997), whereas it was without benefits (at the doses used) in Parkinson's disease (Shoulson, 1998). This could be a reflection of the rather different effects displayed by αTocH in vitro. Among effects of αTocH that are beyond antioxidant capacity are, for example, ramification of microglia (Heppner et al., 1998), differential gene expression (Houglum et al., 1991; Aratri et al., 1999), and effects on interleukin-1β and O2 [UNK] production (Deveraj et al., 1996).

Another important finding arising from the present study is the lack of biodiscrimination between different αTocH isomers at the BBB level. This was also observed when the pBCECs were cultured in the Transwell system (D. Goti and W. Sattler, unpublished observations). Although the eight different αTocH stereoisomers are identical with respect to their antioxidative capacity, they have remarkably different biopotencies in the rat (an “HDL animal”) resorption and gestation assay (Weiser and Vecchi, 1982), which might also be of relevance for the function of the different isomers in brain. This is corroborated by the exceptional ability of brain to discriminate in favor of the RRR isomer of αTocH (Ingold et al., 1987). However, the failure of pBCECs to discriminate between the different isomers might be related to the finding that strongest expression of the αTTP message in brain was observed in Bergmann glial cells (Hosomi et al., 1998) rather than at the BBB. On these grounds, it was suggested that Bergmann glial cells might supply Purkinje and possibly other cerebellar cells via αTTP (Hosomi et al., 1998) in a manner comparable with the sorting step mediated by hepatic αTTP. However, the physiological acceptor and carrier of αTTP-sorted αTocH in brain remain to be identified.

In summary, the present study has demonstrated that (1) the LDL receptor and a 100-kDa HDL3 binding protein are expressed by pBCECs, (2) HDL3 is a better αTocH donor to these cells than LDL, and (3) the majority of HDL3-associated αTocH is taken up in a lipoprotein particle-independent manner. This could indicate that in vivo the HDL fraction may provide a major contribution to the supply of the central nervous system with this essential vitamin, a fact that should be considered during future supplementation studies.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of pBCECs
  5. SDS-PAGE, ligand blots, and immunoblots
  6. Preparation of αTocH methyl ether (αToc-ME) derivatives for chiral HPLC
  7. Chiral-phase HPLC of αToc-ME derivatives
  8. RESULTS
  9. Characterization of pBCECs
  10. Identification of possible binding proteins for HDL3 and LDL
  11. Binding of LDL and HDL3 to pBCECs
  12. Uptake of LDL- and HDL3-associated αTocH by pBCECs
  13. pBCECs do not discriminate between different αTocH isomers
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES
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