Vitamin E Transfer from Lipid Emulsions to Plasma Lipoproteins: Mediation by Multiple Mechanisms
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- Hacquebard, M., Vandenbranden, M., Malaisse, W.J. et al. Lipids (2008) 43: 663. doi:10.1007/s11745-008-3184-3
The present study determined alpha-tocopherol mass transfer from an alpha-tocopherol-rich emulsion to LDL and HDL, and assessed the potential of different mechanisms to modulate alpha-tocopherol transfers. Emulsion particles rich in alpha-tocopherol were incubated in vitro with physiological concentrations of LDL or HDL. The influence of plasma proteins was assessed by adding human lipoprotein poor plasma (LPP) fraction with intact vs heat inactivated PLTP, or with a specific cholesteryl ester transfer protein (CETP) inhibitor, or by adding purified PLTP or pig LPP which lacks CETP activity. After 4 h incubation in absence of LPP, alpha-tocopherol content was increased by ~80% in LDL and ~160% in HDL. Addition of LPP markedly enhanced alpha-tocopherol transfer leading to 350–400% enrichment in LDL or HDL at 4 h. Higher (~10 fold) enrichment was achieved after 20 h incubation with LPP. Facilitation of alpha-tocopherol transfer was (i) more than 50% higher with human vs pig LPP (despite similar PLTP phospholipid transfer activity), (ii) reduced by specific CETP activity inhibition, (iii) not fully suppressed by heat inactivation, and (iv) not restored by purified PLTP. In conclusion, alpha-tocopherol content in LDL and HDL can be markedly raised by rapid transfer from an alpha-tocopherol-rich emulsion. Our results indicate that alpha-tocopherol mass transfer between emulsion particles and lipoproteins is mediated by more than one single mechanism and that this transfer may be facilitated not only by PLTP but likely also by other plasma proteins such as CETP.
KeywordsVitamin EAlpha-tocopherol mass transferLipid emulsionsLDLHDLPLTPCETP
Cholesteryl ester transfer protein
High density lipoproteins
Low density lipoproteins
Lipoprotein poor plasma
Phospholipid transfer protein
Although alpha-tocopherol is transported by chylomicrons and very low density lipoproteins in the immediate and late postprandial phases, respectively, low density lipoproteins (LDL) and high density lipoproteins (HDL) are the main carriers of alpha-tocopherol in plasma and largely contribute to its delivery to cells and tissues . Exchanges of alpha-tocopherol molecules occur between plasma lipoproteins and depend on the relative alpha-tocopherol content in each lipoprotein subpopulation [11, 12]. Alpha-tocopherol transfer between lipoproteins as well as between lipoproteins and cells may be facilitated by plasma proteins. Plasma phospholipid transfer protein (PLTP), member of the plasma lipid transfer protein family, has been reported to play a major role in the facilitation of alpha-tocopherol transfer between plasma lipoproteins as well as between lipoproteins and cells [13, 14], whereas the related cholesteryl ester transfer protein (CETP), has been reported not to facilitate alpha-tocopherol transfer .
Alpha-tocopherol exchanges may also occur, in both directions, between lipoproteins and intravenous lipid emulsion particles [16–18]. The relative importance of alpha-tocopherol net mass transfer from alpha-tocopherol rich lipid emulsion particles to LDL and HDL as well as the extent to which plasma factors facilitate these transfers have not been investigated in depth. In the present study, an in vitro model was used in which particles isolated from an alpha-tocopherol-enriched emulsion were incubated with total plasma or physiological concentrations of LDL or HDL in presence of non-lipid plasma fractions or purified transfer proteins. The aims were to determine: (i) the kinetics of alpha-tocopherol transfer from emulsion particles to plasma lipoproteins; (ii) the amount of alpha-tocopherol that can be acquired by LDL and HDL; (iii) the influence of proteins present in non-lipid plasma fractions on such alpha-tocopherol exchanges. Our data indicate that multiple mechanisms are involved in alpha-tocopherol mass transfer from lipid emulsions to plasma lipoproteins.
Material and Methods
A lipid emulsion (100 g triglycerides/l) containing a mixture of medium-chain triglycerides and long-chain triglycerides (1:1; w:w) with an elevated alpha-tocopherol content (10.70 ± 0.90 g/l) was prepared by B. Braun (Melsungen, Germany) for the purpose of this in vitro study. Since lipid emulsions contain a mixture of triglyceride-rich particles (TGRP) together with other particles formed from the excess of phospholipid emulsifier, the TGRP fraction was isolated by flotation in saline solution (NaCl 0.9%, EDTA 0.01%) through 3 × 30 min ultracentrifugation at 70,000 × g using a SW-41Ti rotor in a L8-55 ultracentrifuge (Beckman Instruments, Palo Alto, CA, USA). Approximately 90% of emulsion alpha-tocopherol content was recovered in the TGRP fraction. TGRP were stored overnight under nitrogen at 4 °C. In all incubation assays, TGRP were incubated at a triglyceride (TG) concentration of 100 mg/dL, a level commonly observed in the plasma of patients receiving parenteral nutrition with lipids .
Separation of LDL, HDL and Lipoprotein Poor Plasma Fraction
Lipoproteins (LDL and HDL) were separated from pooled normolipidemic human plasma by sequential ultracentrifugation  using a 50-2Ti rotor (Beckman, Palo Alto, CA, USA) in a L8-55 ultracentrifuge (Beckman). Plasma density was adjusted by addition of solid KBr at density ranges 1.025–1.055 and 1.070–1.18 g/mL for LDL and HDL, respectively. Plasma was centrifuged at 302,000 × g (5 °C) for 20 h for LDL and for another 40 h for HDL separation. The isolated lipoprotein fractions were then extensively dialysed against a saline solution (NaCl 0.9%, EDTA 0.01%, glycerol 10%, pH 7.4) and stored under nitrogen at −70 °C until incubation assays. Glycerol present in LDL and HDL fractions was removed by filtration on PD10 Sephadex columns (Amersham Pharmacia, Uppsala, Sweden) prior to incubation.
The lipoprotein poor plasma (LPP) fraction, containing the majority of plasma proteins, was also separated by sequential ultracentrifugation. Plasma density was adjusted at d > 1.24 g/mL and centrifuged during 40 h at 302,000 × g (5 °C) to remove all lipoproteins. After ultracentrifugation, the LPP fraction was extensively dialysed against a saline solution (NaCl 0.9%, EDTA 0.01%, pH 7.4) and stored under nitrogen at −20 °C until incubation assays.
Alpha-tocopherol Mass Transfer from Lipid Emulsion Particles to LDL and HDL
To determine the kinetics of alpha-tocopherol net mass transfer from emulsion particles to plasma lipoproteins, TGRP (100 mg TG/dL) were incubated at 37 °C in presence of LDL (120 mg LDL-cholesterol/dL) or HDL (50 mg HDL-cholesterol/dL) over increasing time periods (0.5, 1, 2, 4 h) in a gently shaking water bath. Incubations were performed in absence or presence of human LPP (65 g protein/l). Furthermore, a few incubations were performed over 20 h in presence of LPP to determine the sub-maximal amount of alpha-tocopherol that can be acquired by LDL and HDL. Finally, TGRP were incubated in total plasma (with a LDL-cholesterol/HDL-cholesterol ratio of 1.2:1) to determine alpha-tocopherol partitioning between LDL and HDL. At the end of the incubation period, the TGRP fraction was isolated by flotation (ultracentrifugation of 45 min at 80,000 × g at 12 °C) from the infranatant containing the lipoproteins and LPP. After adjusting the density of the infranatant, individual lipoprotein fractions were isolated by sequential ultracentrifugation. Each collected fraction was stored at 4 °C for lipid analyses and aliquots of 300 μl were stored under nitrogen at −70 °C for vitamin E analyses performed within 2 weeks. Vitamin E content in LDL and HDL was expressed relative to particle lipid (total cholesterol + TG) content. Net alpha-tocopherol enrichment in LDL and HDL was calculated by comparison to data from control incubations of lipoproteins over the same time periods but without TGRP. Alpha-tocopherol content in lipoproteins was not significantly modified after control incubations over 4 and 20 h, whether in absence or presence of LPP.
Influence of Non-lipid Plasma Components on Alpha-tocopherol Transfer
To assess the influence of proteins present in the LPP plasma fraction on alpha-tocopherol exchanges, TGRP (100 mg TG/dL) were incubated at 37 °C with LDL (120 mg LDL-cholesterol/dL) or HDL (50 mg HDL-cholesterol/dL) for 2 h in the absence or presence of human LPP (65 g protein/l). To distinguish the effect of PLTP from that of CETP, experiments were carried out with LPP heated for 1 h at 58 °C to inactivate PLTP activity [13, 21, 22], or with addition of a potent CETP inhibitor (synthetised according to the patent application:WO2006013048; F. Hoffmann-La Roche, Basel, Switzerland) to LPP and to total plasma. CETP inhibitor was used at a concentration of 212 nM corresponding to four times the IC50 for a complete inhibition of CETP activity in plasma samples (personal communication, Roche Basel). In parallel, experiments were carried out with LPP (65 g protein/l) isolated from pig plasma, the latter being largely devoid of CETP [23, 24] but not of PLTP activity , Finally, incubations were also performed in absence of LPP but with albumin as the most abundant plasma protein (40 g/l fatty acid-free BSA, Sigma Aldrich, Steinheim, Germany), and with partially purified PLTP (5–10 μg/mL, Cardiovascular Targets Inc., NY, USA). After incubation, lipoproteins were isolated by ultracentrifugation and their alpha-tocopherol content analysed. The enhancing action of non-lipid plasma components upon alpha-tocopherol transfer from emulsion particles to LDL and HDL was obtained by subtracting spontaneous transfer measured in incubations with no proteins.
Total cholesterol (TC) and TG concentrations were measured in LDL and HDL fractions using enzymatic kits CHOD-PAP (Roche Diagnostics GmbH, Mannheim, Germany) and Triglycerides Glycerol blanked (Roche Diagnostics GmbH), respectively. For alpha-tocopherol measurements in LDL and HDL [26, 27], lipoproteins were saponified at 70 °C with alcoholic KOH followed by alpha-tocopherol extraction with hexane. Hexane was dried and the extract was dissolved in methanol:water (95/5, w/w). Alpha-tocopherol content in the extract was then measured by reverse-phase HPLC (Merck-Hitachi Ltd, Tokyo, Japan) using a Lichrospher column 100 RP 18 (125 m2× 4 m3; LxID; Merck, Darmstadt, Germany) and a mobile phase of methanol:water (95/5, w/w) at a flow rate of 1.5 mL/min; alpha-tocopherol was monitored at 292 nm by a UV detector .
Phospholipid Transfer Activity Assay
Phospholipid transfer activity was assessed by measuring the transfer of 1,2 di-[1-14C]palmitoyl phosphatidylcholine (100 mCi/mmol, Amersham Biosciences, Buckinghamshire, UK) from small unilamellar liposomes to HDL particles [29, 30]. In brief, labeled liposomes (125 nmol of phospholipids) were incubated with HDL (250 μg proteins) and LPP (10 μl) or partially purified PLTP (20 μg, Cardiovascular Targets Inc., NY, USA) in a final volume of 400 μl. The percentage of transferred radioactivity to HDL was calculated after correcting for background radioactivity. In this assay, partially purified PLTP induced a 19% transfer of total radiolabelled phospholipids, after correction for spontaneous transfer (7%).
Results are expressed as mean values ± SEM. Statistical analyses were made by using Students’t test. The statistical significance of differences between values was assessed by paired comparison of data recorded within each individual experiment.
Alpha-tocopherol Mass Transfer from Lipid Emulsion Particles to LDL and HDL
Alpha-tocopherol content of LDL and HDL after 20 h incubation
20 h (μmol/mmol)
LDL + LPP
3.18 ± 0.21
27.53 ± 0.68
HDL + LPP
6.72 ± 0.28
47.37 ± 2.69
Finally, when TGRP were incubated in whole plasma for 4 h, a comparable alpha-tocopherol enrichment of ∼75% was observed in both LDL (from 3.50 ± 0.14 to 6.06 ± 0.08 μmol/mmol, n = 4; p < 0.0001) and HDL (from 6.05 ± 0.11 to 10.72 ± 0.23 μmol/mmol, n = 4; p < 0.0001).
Influence of Non-lipid Plasma Components on Alpha-tocopherol Transfer
Since substantial alpha-tocopherol transfers to plasma lipoproteins were observed within 2 h incubation (and since the residence time of TG-rich emulsion particles in the circulation does not exceed 2 h), further experiments to characterise the influence of LPP proteins on alpha-tocopherol transfer were performed over 2 h.
Influence of albumin on alpha-tocopherol transfer
TGRP + albumin (μmol/mmol)
TGRP + LPP (μmol/mmol)
3.44 ± 0.14
5.30 ± 0.27
5.63 ± 0.19
12.20 ± 0.15
7.29 ± 0.07
13.94 ± 0.20
11.19 ± 1.32
18.80 ± 0.70
Phospholipid transfer activity
HDL + human LPP
22.5 ± 0.5
HDL + heated human LPP
5.3 ± 0.4
HDL + pig LPP
26.1 ± 0.2
HDL + heated pig LPP
20.7 ± 0.2
Addition of partially purified PLTP tended to increase alpha-tocopherol net transfer from TGRP to LDL (18.5 ± 8.5%; NS) and HDL (7.3 ± 2.8%; NS).
LPP fraction from pig plasma (devoid of functional CETP) induced comparable transfer of radiolabelled phospholipids as human LPP (Table 3); however, alpha-tocopherol mass transfer to LDL was lower with pig vs human LPP (5.29 ± 0.55 vs 7.94 ± 0.32 μmol/mmol; n = 6; p < 0.001). Hence, the enhancing effect of pig LPP on alpha-tocopherol transfer to LDL represented 55 ± 10% of that induced by human LPP. Surprisingly, heat inactivation had only a limited effect on reducing phospholipid specific transfer activity in pig LPP (Table 3). Consistently, prior heating of pig LPP did not significantly reduce alpha-tocopherol transfer (p > 0.6 by comparison to non-heated pig LPP; n = 6). In incubations of TGRP with HDL, alpha-tocopherol transfer was also much lower in presence of pig vs human LPP (6.44 ± 0.64 vs 13.59 ± 0.46 μmol/mmol; n = 4; p < 0.002). A limited enhancing action of pig LPP on alpha-tocopherol transfer was observed in only 3 out of 4 individual experiments. Even in these 3 experiments, this represented only 13.6 ± 2.9% of the facilitated transfer observed with human LPP within the same experiments (p < 0.005). Prior heating suppressed the modest enhancing action of pig LPP on alpha-tocopherol transfer to HDL particles (p < 0.05).
Previous experiments studying alpha-tocopherol transfer between plasma lipoproteins have mainly used radiolabelled transfer assays and have generally focused on evaluating the effect of single mechanisms susceptible to facilitate alpha-tocopherol transfer [11, 13, 15]. The present study measures mass transfers of alpha-tocopherol from an artificial lipid emulsion to LDL and HDL and investigates the potential for several mechanisms to mediate such transfers. An in vitro model of incubation was used with an emulsion markedly enriched in alpha-tocopherol to avoid a rate-limiting availability of alpha-tocopherol when assessing mass transfers. In the different experiments, the emulsion lipid concentration was maintained at a relatively low level and lipoprotein concentration was comparable to that commonly found in the circulation. The range of incubation periods generally covered the residence time of emulsion particles in plasma.
In the first part of this study, alpha-tocopherol mass transfer from emulsion particles to lipoproteins was measured over increasing time periods. Incubations of alpha-tocopherol-rich emulsion particles together with either LDL or HDL resulted in a rapid net mass transfer of alpha-tocopherol to both plasma lipoprotein fractions. In absence of non-lipid plasma components, spontaneous alpha-tocopherol transfer was already detected after ≤1 h incubation and led to sub-maximal alpha-tocopherol enrichment after 4 h in LDL (+∼80%) and HDL (+∼160%). The higher spontaneous transfer to HDL may be related to the larger surface to volume ratio of HDL compared to LDL particles, which increases the available accepting surface for alpha-tocopherol molecules. To our knowledge, this is the first study to report such significant spontaneous alpha-tocopherol mass transfer from lipid emulsions to plasma lipoproteins. These observations differ from previous data of Desrumaux et al.  who did not detect spontaneous mass transfer between lipoproteins. However, these authors used for their transfer experiments lipoproteins depleted of alpha-tocopherol by oxidation, a procedure that may considerably modify particle surface and capacity for accommodating extra-alpha-tocopherol. Kostner et al.  who reported spontaneous transfer of radiolabelled alpha-tocopherol between plasma lipoproteins, showed that the addition of LPP accelerated alpha-tocopherol transfer without a net increase in lipoprotein alpha-tocopherol enrichment over spontaneous transfer. In our experiments, addition of LPP fraction not only accelerated but also increased the maximal amount of alpha-tocopherol transferred to LDL and HDL. In this condition, enrichment was not sub-maximal after 4 h incubation and alpha-tocopherol content could be further increased to almost 10 fold in both lipoproteins when incubation period was prolonged to 20 h.
Incubations with total plasma (containing LDL and HDL at a 1.2:1 cholesterol ratio) led to a comparable enrichment (∼75% after 4 h) in both lipoprotein fractions, suggesting a competition between LDL and HDL fractions for alpha-tocopherol acquisition. All together, this data indicates that, at a fixed concentration of alpha-tocopherol provided by emulsion particles, the limiting factor for alpha-tocopherol enrichment is not the capacity of plasma lipoproteins to accommodate extra-amounts of alpha-tocopherol but rather the facilitating effect of non-lipid plasma components on alpha-tocopherol transfer, the residence time of emulsion particles and possibly the competition between different acceptor particles.
In the second part of this study the influence of individual or groups of proteins was assessed in relation to facilitating alpha-tocopherol transfer. Results of experiments using addition of heat inactivated LPP, of partially purified PLTP, of pig LPP and of CETP inhibitor suggest that the facilitating effect of LPP on alpha-tocopherol transfer from emulsion particles to plasma lipoproteins is not related to a single protein but rather to several non-lipid components present in the LPP fraction. This observation is at variance with previous papers suggesting an exclusive role for PLTP in enhancing vitamin E exchanges between plasma lipoproteins (together with surface phospholipids) , as well as between lipoproteins and cells . Indeed, inactivation of PLTP activity by heating human LPP for 1 h at 58 °C reduced alpha-tocopherol transfer from emulsion particles by only ∼40% in the case of LDL and ∼30% in the case of HDL by comparison to experiments with non heated LPP. Moreover, addition to the incubation medium of partially purified PLTP (with maintained phospholipid-specific transfer activity) did not substantially increase alpha-tocopherol net mass transfer, although our model does not take into account alpha-tocopherol back transfers. Overall these results confirm a role for PLTP in alpha-tocopherol transfer facilitation but indicate that ≥50% of alpha-tocopherol transfer facilitation by human LPP is due to components other than heat-sensitive PLTP. This leads to question the role of heat-resistant lipid transfer proteins such as CETP. Granot et al.  who evaluated the role of purified CETP in alpha-tocopherol transfer between plasma lipoproteins did not report a facilitating effect of CETP. Nevertheless, this study used radiolabelled alpha-tocopherol which may not have distributed normally throughout the lipoprotein particles, did not assay for mass transfers and measured alpha-tocopherol transfer over relatively long incubation periods which would promote back transfers; these factors could explain the lack of effect of CETP. In our experiments using pig LPP with PLTP activity comparable to human LPP but deficient in CETP, alpha-tocopherol enrichment in LDL and HDL was much lower (∼50%) than that facilitated by human LPP. This data again supports the point that alpha-tocopherol transfer from emulsion particles to plasma lipoproteins is facilitated not only by PLTP but also by other transfer proteins, and possibly CETP. Indeed, addition of a specific CETP inhibitor to incubation assays with LDL or HDL, or with total plasma, significantly reduced alpha-tocopherol transfer facilitated by human LPP. The reduction of alpha-tocopherol facilitated transfer was more marked for HDL when lipoproteins were incubated separately. However, addition of CETP inhibitor to total plasma, reduced by ∼50% alpha-tocopherol transfer to both LDL and HDL particles. Hence, these experiments using a specific CETP inhibitor also support a role for CETP in alpha-tocopherol transfer from emulsion particles to plasma LDL and HDL. In addition to transfer proteins, lipoprotein lipase may facilitate alpha-tocopherol exchange between lipoproteins during the lipolytic process [27, 31]. This was not tested in the in vitro model used in the present study.
The present study demonstrates a very high capacity of LDL and HDL to accommodate extra-amounts of alpha-tocopherol by rapid transfer from an alpha-tocopherol enriched emulsion. Such increased alpha-tocopherol content in LDL and HDL may facilitate subsequent alpha-tocopherol delivery to cells and tissues. The emulsion used in this study was prepared for these in vitro experiments and not as a component of parenteral nutrition. However, similar alpha-tocopherol enriched preparations could be developed in view of intravenous bolus injections in patients with increased alpha-tocopherol requirements associated to acute conditions. If supported by evidence-based data, intravenous administration of emulsions with extra-alpha-tocopherol may find an indication in critically ill patients (injury, trauma, sepsis, ischemia, or severe inflammation) who consistently show reduced numbers of circulating LDL and HDL particles and are at high risk of oxidative tissue injury . Furthermore, using intravenous emulsions as suppliers of alpha-tocopherol allows to bypass intestinal absorption which may be a limiting factor for alpha-tocopherol supplementation particularly in critically ill patients [33–35].
In conclusion, the present study demonstrates rapid and substantial mass transfer of alpha-tocopherol from an alpha-tocopherol rich lipid emulsion to plasma lipoproteins. Our data further indicates that alpha-tocopherol mass transfer is mediated by multiple mechanisms and that this transfer is largely facilitated not only by PLTP but also by other non-lipid factors present in plasma.
M. Hacquebard is recipient of a fellowship from the Danone Institute, Belgium. Prof. R.J. Deckelbaum receives support from NIH grant #HL40404. The authors gratefully thank Dr. E. Niesor and Dr. C. Maugeais (F. Hoffmann-La Roche, Basel, Switzerland) for kindly providing CETP inhibitor and related information.