Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion.
ABSTRACT The bioavailability of α-linolenic acid (ALA) from flaxseed oil in an emulsified form v. a non-emulsified form was investigated by using two complementary approaches: the first one dealt with the characterisation of the flaxseed oil emulsion in in vitro gastrointestinal-like conditions; the second one compared the intestinal absorption of ALA in rats fed the two forms of the oil. The in vitro study on emulsified flaxseed oil showed that decreasing the pH from 7·3 to 1·5 at the physiological temperature (37°C) induced instantaneous oil globule coalescence. Some phase separation was observed under acidic conditions that vanished after further neutralisation. The lecithin used to stabilise the emulsions inhibited TAG hydrolysis by pancreatic lipase. In contrast, lipid solubilisation by bile salts (after lipase and phospholipase hydrolysis) was favoured by preliminary oil emulsification. The in vivo absorption of ALA in thoracic lymph duct-cannulated rats fed flaxseed oil, emulsified or non-emulsified, was quantified. Oil emulsification significantly favoured the rate and extent of ALA recovery as measured by the maximum ALA concentration in the lymph (Cmax = 14 mg/ml at 3 h in the emulsion group v. 9 mg/ml at 5 h in the oil group; P < 0·05). Likewise, the area under the curve of the kinetics was significantly higher in the emulsion group (48 mg × h/ml for rats fed emulsion v. 26 mg × h/ml for rats fed oil; P < 0·05). On the whole, ALA bioavailability was improved with flaxseed oil ingested in an emulsified state. Data obtained from the in vitro studies helped to partly interpret the physiological results.
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ABSTRACT: α-Linolenic acid (ALA), a carboxylic acid with 18 carbons and three cis double bonds, is an essential fatty acid needed for human health and can be acquired via regular dietary intake of foods that contain ALA or dietary supplementation of foods high in ALA, for example flaxseed. ALA has been reported to have cardiovascular-protective, anti-cancer, neuro-protective, anti-osteoporotic, anti-inflammatory, and antioxidative effects. ALA is the precursor of longer chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but its beneficial effects on risk factors for cardiovascular diseases are still inconclusive. The recommended intake of ALA for cardiovascular health is reported to be 1.1–2.2 g/day. Although there are limited toxicological data for ALA, no serious adverse effects have been reported. The evidence on an increased prostate cancer risk in association with dietary ALA is not conclusive. Based on the limited data currently available, it may be concluded that ALA may be beneficial as a nutraceutical/pharmaceutical candidate and is safe for use as a food ingredient.Food and Chemical Toxicology 08/2014; 70:163–178. · 2.61 Impact Factor
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ABSTRACT: The purpose of this study was to quantitatively clarify the effect of alky-chain length of a triglyceride in an emulsion on the partitioning of highly lipophilic compounds into the lymph fluid after their oral administration. Highly lipophilic anthraquinone derivatives were orally administered in emulsions to rats. Emulsions composed of long-, medium-, and short-chain triglycerides (LCT, MCT, and SCT emulsions, respectively) were used. The concentrations of the compounds in plasma and lymph fluid were periodically determined and their partitioning to the lymph was calculated using a mathematical model. Intestinal absorption of all compounds was enhanced and the plasma concentrations of the compounds were found to be in the following order: LCT emulsion > MCT emulsion > SCT emulsion. The amounts of each compound recovered in the lymph were not in agreement with their lipophilicity. Quantitative analysis revealed that the partitioning of the compounds to the lymph may be determined by the solubility of the compound in the triglyceride in the form of an emulsion and the amount of triglyceride transferred to the lymph fluid. These results suggest a possibility that the amount of a compound absorbed via the lymph route after oral administration can be quantitatively controlled by the formulations.Archives of Pharmacal Research 10/2013; · 1.75 Impact Factor
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ABSTRACT: BACKGROUND: Prolonged postprandial hypertriglyceridemia is a potential risk factor for cardiovascular diseases. In the context of obesity, this is associated with a chronic imbalance of lipid partitioning oriented toward storage and not toward β-oxidation. OBJECTIVE: We tested the hypothesis that the physical structure of fat in a meal can modify the absorption, chylomicron transport, and further metabolic handling of dietary fatty acids. DESIGN: Nine normal-weight and 9 obese subjects were fed 40 g milk fat (+[(13)C]triacylglycerols), either emulsified or nonemulsified, in breakfasts of identical composition. We measured the postprandial triacylglycerol content and size of the chylomicron-rich fraction, plasma kinetics of [(13)C]fatty acids, exogenous lipid oxidation with breath-test/indirect calorimetry, and fecal excretion. RESULTS: The emulsified fat resulted in earlier (>1 h) and sharper chylomicron and [(13)C]fatty acid peaks in plasma than in spread fat in both groups (P < 0.0001). After 2 h, the emulsified fat resulted in greater apolipoprotein B-48 concentrations (9.7 ± 0.7 compared with 7.1 ± 0.9 mg/L; P < 0.05) in the normal-weight subjects than did the spread fat. In the obese subjects, emulsified fat resulted in a 3-fold greater chylomicron size (218 ± 24 nm) compared with the spread fat (P < 0.05). The emulsified fat induced higher dietary fatty acid spillover in plasma and a sharper (13)CO(2) appearance, which provoked increased exogenous lipid oxidation in each group: from 45% to 52% in normal-weight subjects (P < 0.05) and from 40% to 57% in obese subjects (P < 0.01). CONCLUSION: This study supports a new concept of "slow vs fast fat," whereby intestinal absorption can be modulated by structuring dietary fat to modulate postprandial lipemia and lipid β-oxidation in humans with different BMIs. This trial was registered at clinicaltrials.gov as NCT01249378.American Journal of Clinical Nutrition 12/2012; · 6.50 Impact Factor
Lymphatic absorption of a-linolenic acid in rats fed flaxseed oil-based
Leslie Coue ¨delo1,2, Carole Boue ´-Vaysse2, Laurence Fonseca2, Emeline Montesinos2,
Sandrine Djoukitch2, Nicole Combe2and Maud Cansell1*
1Universite ´ Bordeaux 1, Laboratoire CBMN UMR 5248, F-33405 Talence Cedex, France
2ITERG, Unite ´ de Nutrition & Sante ´, F-33405 Talence Cedex, France
(Received 8 June 2010 – Revised 22 September 2010 – Accepted 8 October 2010)
The bioavailability of a-linolenic acid (ALA) from flaxseed oil in an emulsified form v. a non-emulsified form was investigated by using two
complementary approaches: the first one dealt with the characterisation of the flaxseed oil emulsion in in vitro gastrointestinal-like
conditions; the second one compared the intestinal absorption of ALA in rats fed the two forms of the oil. The in vitro study on emulsified
flaxseed oil showed that decreasing the pH from 7·3 to 1·5 at the physiological temperature (378C) induced instantaneous oil globule
coalescence. Some phase separation was observed under acidic conditions that vanished after further neutralisation. The lecithin used
to stabilise the emulsions inhibited TAG hydrolysis by pancreatic lipase. In contrast, lipid solubilisation by bile salts (after lipase and phos-
pholipase hydrolysis) was favoured by preliminary oil emulsification. The in vivo absorption of ALA in thoracic lymph duct-cannulated rats
fed flaxseed oil, emulsified or non-emulsified, was quantified. Oil emulsification significantly favoured the rate and extent of ALA recovery
as measured by the maximum ALA concentration in the lymph (Cmax¼ 14mg/ml at 3h in the emulsion group v. 9mg/ml at 5h in the
oil group; P,0·05). Likewise, the area under the curve of the kinetics was significantly higher in the emulsion group (48mg £ h/ml for
rats fed emulsion v. 26mg £ h/ml for rats fed oil; P,0·05). On the whole, ALA bioavailability was improved with flaxseed oil ingested
in an emulsified state. Data obtained from the in vitro studies helped to partly interpret the physiological results.
Key words: Emulsion: Intestinal lipid absorption: Lymphatic lipids: Flaxseed oil: a-Linolenic acid bioavailability
The bioavailability of a lipid nutrient present in food
depends on complex physico-chemical and enzymatic
processes including digestion, intestinal absorption and
delivery to all organs for cell lipid requirements(1–3). Several
parameters affect the lipid digestion and absorption
steps. The influence of the lipid nature(4–8)results, at least
partly, from the pancreatic lipase activity due to the TAG
structure(7), the inherent resistance of very long PUFA to
hydrolysis(4)and the more efficient hydrolysis of short-
and medium-chain TAG(8,9). Fat digestion also depends
on the supramolecular lipid structures ingested(10), i.e.
impact of interfacial composition of the emulsion(13,17,18),
as well as the influence of the droplet size of the emulsi-
fied lipid phase(12–15), should be taken into account.
More recently, it has been demonstrated that the physical
state of the TAG also influences the rate and extent of lipid
In this context, the aim of the present study was to
measure the intestinal absorption of a-linolenic acid
(ALA) in rats after the administration of flaxseed oil in
both emulsified and non-emulsified states. Flaxseed oil
was chosen because of its high ALA content. The current
French dietary guidelines recommend a daily intake of at
least 1g of n-3 ALA for adults, while keeping the linoleic
acid:ALA ratio lower than 5(20). By comparison, the WHO
advises an n-3 PUFA intake between 0·8 and 1·1g of
ALA/d, and the US Dietary Guidelines recommend 1·1g
for women and 1·6g for men. Linoleic acid and ALA
are usually consumed in a ratio ranging from 10 to 15 in
France, thus making ALA supplementation necessary(21,22).
Among various vegetable sources, flaxseed oil nowadays
provides the highest amount of ALA (40–45wt% of total
fatty acids). Several studies have shown the potential to
increase the ALA plasma level through flaxseed oil inges-
tion(23–26). As a result, in July 2006, the French agency
*Corresponding author: Professor M. Cansell, fax þ33 5 40 00 68 08, email firstname.lastname@example.org
Abbreviations: ALA, a-linolenic acid; BS, bile salt; MAG, monoacylglycerol; OD, optical density; O/W, oil-in-water; PL, phospholipid.
British Journal of Nutrition (2010), page 1 of 10
q The Authors 2010
British Journal of Nutrition
for food, environmental and occupational health safety
authorised the introduction of flaxseed oil in foods(27).
Recently, flaxseed oil has been marketed as virgin oil(28).
With the aim of probing how lipid bioavailability depends
on its dispersion state, rats were fed flaxseed oil either in
a bulk phase or an oil-in-water (O/W) emulsion. Soya
lecithin was used to stabilise the oil interface. The present
study was carried out to determine whether the emulsified
state of the lipids influenced ALA lymphatic absorption
and, more generally, its bioavailability. In addition, the
emulsion stability was characterised under conditions that
mimicked those of the gastrointestinal tract in terms of pH
variation, enzyme lipolysis and bile salt (BS) solubilisation.
Flaxseed oil was supplied by ITERG (Pessac, France). Soya
lecithin (Lecimulthin) was kindly provided by Cargill
(Baupte, France). The lecithin composition was determined
two-dimensional TLC using the following solvents: in the
acid–water (50:20:10:10:5, by vol.), and in the second
vol.)(29). Various diacyl PL and lysophospholipid classes
were visualised by exposing the plates to iodine vapour.
Different spots were scraped and analysed for total P
determination according to the method used by Ames(30).
PL composition of the lecithin is reported in Table 1.
Phospholipase A2 from Naja mossambica mossambica,
lipase from porcine pancreas, sodium deoxycholate and
porcine bile extract were obtained from Sigma (Sigma,
St Louis MO, USA). Porcine bile extract was purified from
coloured pigments on activated charcoal, as described
previously(31). The solvents were of analytical grade.
Emulsion preparation and characterisation
O/W emulsions were prepared at room temperature.
The oil phase contained 1g of sodium deoxycholate and
20g of lecithin per 100g of oil. This oil phase was manually
dispersed into the aqueous phase to an oil fraction of
45g/100g. The coarse O/W emulsions obtained were
then sheared using an Ultraturax apparatus (IKA, Staufen,
Germany), equipped with a generator axis (10mm S25-
N-10G; IKA, Staufen, Germany) under a nitrogen flux to
prevent lipid oxidation. Direct visualisation of the oil
droplets just after preparation and under acid stress was
carried out using a phase-contrast microscope (Axiovert
135 with a water immersion, 100 £
Germany). Mean particle diameter (as evaluated by the
volume-weighted average diameter d4,3) and particle size
distribution were determined by static light scattering
using a Coulter LS 230 apparatus (Brea, CA, USA).
O/W emulsions were exposed to acidic conditions
(pH 1·5) using 10 M-HCl. The emulsions were incubated
at acid pH, from 0 to 3h, at 378C and analysed regularly.
Neutralisation was carried out by the addition of small
volumes of 10 M-NaOH solution in emulsions previously
stored 2h under acidic conditions at 378C. All experiments
were performed under stirring.
Lipid hydrolysis by pancreatic lipase
The time course of lipase-catalysed hydrolysis of both flax-
seed oil and O/Wemulsions was monitored using a pH-stat
(Titroline alpha plus TA10 plus; SCHOTT Instrument,
Mainz, Germany) on the basis of protocols described
in Hope & Theimer(32). Experiments were performed in a
thermostated bath at 378C, and at a pH of 8, using
a 0·5 M-NaOH solution for automatic titration. In the case
of flaxseed oil, an emulsion was prepared by mixing
40ml of methocel (3·5%), 0·6 M-NaCl solution and 25ml
of oil, under stirring. The hydrolysis reaction was initiated
by mixing 10ml of the previous emulsion with 10mg of
pancreatic lipase in the presence of 2 M-CaCl2. In the case
of O/W emulsions, the lipase solution was directly
added. In another set of experiments, a colipase solution
(1mg/ml) was also added to the reaction medium. Lipase
activity was calculated from the initial slope of the titration
curve and expressed as milliequivalents of fatty acids
released per min and per g of enzyme extract (mEq/min
per g of enzyme extract). The amount of enzyme and
the oil and emulsion concentrations used were chosen to
ensure substrate-saturating conditions. In both cases,
assays were performed in triplicate.
Lipid solubilisation by bile salts
Lipid micellisation was performed by BS addition to the
hydrolysis products obtained from flaxseed oil in both
emulsified and non-emulsified (bulk) states. In the case
of bulk flaxseed oil, NEFA and monoacylglycerols (MAG)
resulting from pancreatic lipase hydrolysis were mixed
with BS. In the case of emulsified flaxseed oil, the PL
used to stabilise the emulsion were first subjected to phos-
pholipase A2hydrolysis (19units/ml of emulsion) before
pancreatic lipase addition. In this instance, NEFA and
MAG from lipase hydrolysis, and NEFA and lysophospholi-
pids from phospholipase A2hydrolysis were solubilised.
The lipid–BS mixtures were prepared by adding 10ml
of a concentrated BS solution (concentrations ranging
Table 1. Main phospholipid species of the lecithin
Phospholipids (%, w/w)Soya lecithin
L. Coue ¨delo et al.2
British Journal of Nutrition
from 5 to 70mM, considering an average molecular weight
for BS of 480g/mol) to the hydrolysed lipids (ranging from
3 to 50mg, i.e. concentrations ranging from 0·5 to 5mM,
considering an average molecular weight for both TAG
and PL of 775g/mol). Micellisation by BS was followed
by measuring the turbidity at 400nm. Turbidity measure-
ments (optical density (OD) at 400nm) were performed
in a thermostated cell support using a Perkin Elmer
lambda Bio 20 spectrophotometer (Waltham, MA, USA).
After stirring, the mixtures were allowed to equilibrate
until stable turbidity values were obtained. For each lipid
concentration ([lip]tot), a solubilisation curve was obtained
by plotting the evolution of OD as a function of total BS con-
centration ([BS]tot). The solubilisation point, corresponding
to theBS amount required to completelysolubilise thelipids
into mixed micelles, was determined as the point at which
further addition of BS only slightly affected the suspension
turbidity. In practice, this point corresponded to an OD
approximately equal to zero, as in Lichtenberg(33). At this
solubilisation point, the concentration of BS molecules not
associated with the lipids ([BS]bulk) and the molecular
ratio of BS to lipid in the mixed micelles ([BS:lip]mic) were
determined by the following equation(34):
½BS?tot ¼ ½BS?bulk þ ½BS:lip?mic £ ½lip?tot:
Using linear regression analysis, [BS]bulk was deduced
from the intercept of the extrapolated curve with the ordi-
nate axis, and [BS:lip]mic was deduced from the slope.
Solubilisation was performed at 25 and 378C on hydrolysis
products derived from flaxseed oil in the bulk and emulsi-
Animals, surgical procedures and lymph analysis
Male Wistar rats (8 weeks old, body weight 300–350g)
were obtained from Elevage Janvier (St-Berthevin, France)
and were randomly assigned to one of the dietary groups.
The study was conducted in accordance with European
Community Council Directives (861609/EEC). All experi-
ments conformed to the Guidelines for the Handling and
Training of Laboratory Animals. Rats were housed for at
least 3d before the experiment in a controlled environ-
ment, with constant temperature and humidity, and with
free water and food access. Two lipid formulas based
on flaxseed oil were used for the experiments (Table 2).
Rats were fed a fat-free diet (Epinay, France) and had
free access to water 24h before the surgery. For collecting
the lymph, a polyethylene catheter (inner diameter
0·86mm, outer diameter 1·27mm; Biotrol, Paris, France)
was inserted into the main mesenteric lymph duct of
each rat placed under ketamine/xylazine anaesthesia (100
and 10mg/g of body weight), as described in Bollman
et al.(35)and Combe et al.(36). After the surgery, rats were
placed in individual restraining cages, in a warm environ-
ment, with tap water freely available. A few hours later,
0·3g of flaxseed oil, either as bulk oil or O/W emulsion,
were administered through a gastric feeding tube, followed
by 0·5ml of water. The amounts of total fatty acids and ALA
per rat were, for the bulk-phase group, 230 and 103mg,
respectively, and, for the emulsion group, 260 and
104mg, respectively. Lymph was collected for 24h with
fractionation in tared tubes maintained in an ice bath.
During the collection period, the lymph flow averaged
0·30 (SEM 0·03)ml/h. The total lipid contents of chy-
lomicrons were immediately analysed. At least eight cannu-
lated rats were used for each studied lipid ingestion
according to the method of Lepage & Roy(37). Trimyristo-
lein was added as an internal standard for TAG fatty acid
quantification. Fatty acid methyl esters were analysed by
GC on a BPX 70 capillary column (60m long, 0·25mm
film, 0·25mm inner diameter (SGE, Victoria, Australia), H2
as a carrier gas and split ratio of 1:80). The GC system con-
sisted of a gas chromatograph (HP 4890; Hewlett Packard,
Palo Alto, CA, USA) equipped with a flame ionisation
detector maintained at 2808C. The injector temperature
was 2508C. The column temperature was increased from
150 to 2008C (1·38C/min), maintained at 2008C for 20min,
increased from 200 to 2358C (108C/min) and held at
2358C for 20min. Data were collected and integrated by
a Chromjet SP 4400 integration system (Spectra-Physics,
Irvine, CA, USA). Fatty acids from Sigma France (St Quentin
Fallavier, France) and natural extracts of known compo-
sition were used as standards for column calibration.
The variation in peak area between injections was less
Table 2. Main fatty acid profile of the dietary lipids and their intramole-
cular distribution in TAG
Flaxseed oil Flaxseed oil emulsion†
sn-2 MAG, sn-2 monoacylglycerol; TFA, trans-fatty acid.
* Fatty acid composition represents the mean of two measurements.
† Flaxseed oil was emulsified using 8% soya lecithin.
‡ The fatty acid composition of sn-2 MAG of flaxseed oil TAG was determined
after pancreatic lipase hydrolysis followed by isolation and analysis of monoacyl-
glycerols. Results are expressed in mol% corresponding to the distribution of
each fatty acid in the internal position of TAG.
§ Others represent the sum of the fatty acids that each contributes to ,1g/100g.
Flaxseed oil emulsion absorption in rats3
British Journal of Nutrition
The intramolecular fatty acid distribution in TAG of
dietary flaxseed oil and lymph chylomicrons was deter-
mined through lipase hydrolysis according to Desnuelle(38)
and Entressangles et al.(39). The resulting 2-MAG and
1,3-diacylglycerols were separated by TLC using hexane–
diethyl ether–formic acid (70:30:1, by vol.) as a developing
solvent(39). Respective fractions were transmethylated,
and fatty acid methyl esters were analysed by GC, as
described previously. The proportion of ALA in the sn-2
position of TAG was obtained by the following equation:
%ALAðsn ? 2Þ ¼%ALAð2 ? MAGÞ £ 100
%ALAðTAGÞ £ 3
Data are expressed as means with their standard errors.
When only two independent groups of data were com-
pared (solubilisation data and kinetic study), the parametric
Student’s t test was used. The area under the curve was
calculated according to the trapezoidal method, and the
data were compared with the Mann–Whitney test. Differ-
ences were considered significant at P,0·05. The statistical
significance of differences in the fatty acid compositions
of chylomicron TAG between the three dietary conditions
(fasted, bulk flaxseed oil and emulsion) was analysed
by one-way ANOVA. Vmaxvalues obtained with different
hydrolysis conditions were analysed by two-way ANOVA.
These analyses included Dunnett’s multiple comparison
procedure and Tukey’s honestly significant difference
procedure. Only when two of the above tests showed
significance at the P,0·05 level were the differences
judged to be significant.
Emulsion behaviour under acid conditions
The O/W emulsions were characterised in terms of droplet
size distribution. The mean diameter of the droplets was
found to be 4·9mm, with a distribution ranging from 0·8
to 15mm as confirmed by optical microscopy (Fig. 1(a)).
When the medium was acidified (pH 1·5) in order to
mimic gastrointestinal tract conditions, the mean diameter
increased up to 12·2mm because of droplet coalescence
(Fig. 1(b)). Some phase separation was observed under this
acidic condition during a 3h storage period. In the physio-
logical digestion process, emulsions stayed for approxi-
mately 2–3h under acidic conditions (comparable to the
human stomach) before returning to a neutral environment
(comparable to the human intestine). To mimic this shift in
pH, an emulsion sample previously stored at pH 1·5 for 2h
at 378C was further neutralised (pH 7·3). This resulted
in a slight increase in the mean diameter, although no
phase separation was observed in the sample (Fig. 1(c)).
The size distribution did not vary for at least 24h (results
Lipid solubilisation by bile salts
To mimic lipid transfer from the stomach to the intestine,
bulk oil and emulsions were exposed to pancreatic lipase
with or without phospholipase A2 before BS addition.
The solubilisation process was followed by variations in
turbidity measured as a function of BS addition. Almost
zero turbidity levels corresponding to mixed micelles at
equilibrium were obtained after 36h (results not shown),
indicating that equilibrium was slowly reached when BS
were added to the digestion products of either bulk oil
or emulsions. Fig. 2 presents typical solubilisation curves
obtained for various lipid concentrations in emulsions.
Lipid micellisation was achieved when a drastic decrease
in OD was observed. The complex evolutions of the OD
suggested that the lipid–surfactant structures underwent
Fig. 1. Microscopy observation (100 £) of flaxseed emulsions stabilised
by soya lecithin: (a) just after preparation; (b) in acid conditions (pH 1·5,
10min, 378C); (c) in neutral conditions (pH 7·3) after an incubation of 2h at
pH 1·5 (378C).
L. Coue ¨delo et al.4
British Journal of Nutrition
size and/or shape variations, as already reported for the
solubilisation of other colloidal structures such as lipo-
somes(34,40,41). Varying the lipid concentration made it
possible to calculate two parameters that fully describe
the solubilisation process, i.e. [BS]bulk and [BS:lip]mic.
The results for mixed micelles at equilibrium are reported
in Table 3. Irrespective of the initial lipid system and/or
of the temperature, [BS]bulk values were well above
the critical micellar concentration of the main bile acids
present in bile (11mM for cholic acid and 3mM for deoxy-
cholic acid)(42), suggesting that, at the micellisation point
and thereafter, mixed lipid–BS micelles and pure BS
micelles coexisted. The composition of mixed micelles
consisting of fatty acids, MAG and BS was hardly influ-
enced by an increase in temperature up to 378C. This may
be related to the fact that, at 258C, acyl chains were already
in the liquid state. In the present study, increasing the
temperature from 25 to 378C may only modify the
solubility of BS in the aqueous phase and/or the partition
of the surfactant between the aqueous phase and the
micelles. In the case of flaxseed emulsions, the resulting
mixed micelles contained lysophospholipids in addition
to fatty acids, MAG and BS. At 258C, lysophospholipids
significantly decreased the [BS:lip]mic value due to their
surface-active properties (Table 3). Increasing the tempera-
ture from 25 to 378C led to a significant increase in
the [BS:lip]mic value (Table 3). This suggested a lower
solubility of BS monomers at the physiological tempera-
ture. No phase transition of lysophospholipids occurred
because they were already in a liquid-crystalline phase
at 258C. Increasing the temperature may lead to the coex-
istence of different types of micelles. Indeed, with regard
to lecithin/BS-mixed micelles, simple BS micelles may
coexist in varying proportions with mixed micelles,
depending on the type of BS, the [lecithin]:[BS] ratio and
Oil and emulsion hydrolysis by pancreatic lipase
Pancreatic lipase catalyses the hydrolysis of sn-1 and sn-3
fatty acyl ester bonds of TAG to produce 2-MAG and
fatty acids. The catalytic behaviour was studied on bulk
oil and emulsions stabilised by lecithin. All experiments
were performed with a lipid:enzyme ratio greater than
105that ensured a saturating concentration of substrate
(results not shown). Under these conditions, the initial
rate per g of enzyme extract corresponding to the maximal
activity of the enzyme (Vmax) was calculated from curves
(Fig. 3, inset). The Vmax value obtained with bulk oil
(2·2 (SEM 0·7)mEq/min per g of enzyme extract) was sig-
nificantly higher than that measured in the emulsion
(0·7 (SEM 0·2)mEq/min per g of enzyme extract; P,0·05).
0 10 203040 5060
Bile salts (mM)
OD (400 nm)
Fig. 2. Dependence of turbidity on the bile salt concentration in equilibrated
NEFA–lysophospholipid–monoacylglycerol–bile salt-mixed dispersions con-
taining constant lipid levels and varying levels of bile salts. The samples
were made by a series of dilutions of the lipid aggregates after phospholipase
A2followed by pancreatic lipase hydrolysis with various bile salt solutions
([lip]tot ¼ 4·2 (V), 2·1 (A), 1·0 (O) and 0·5 (W)mM). [lip]tot, lipid concen-
tration. OD, optical density.
Table 3. Bile salt:lipid molecular ratio in mixed micelles ([BS:lip]mic) at
equilibrium and the corresponding bile salt concentration in the conti-
nuum medium ([BS]bulk) as a function of temperature and the initial
lipid system (oil and emulsion)
(Mean values with their standard errors)*
[BS:lip]mic [BS]bulk (mM)
Lipid systemTemperature (8C) Mean
* Values represent the mean of at least five independent experiments for each lipid
system (oil and emulsion) and temperature (25 and 378C).
† Oil was subjected to pancreatic lipase before bile salt addition.
‡ Mean values were significantly different from those of the emulsion system
§ Emulsion was subjected to phospholipase A2and pancreatic lipase before bile
k Mean values were significantly different from the experiment performed at 378C
Hydrolysed substrate (mmol)
Fig. 3. Time course for a typical hydrolysis of oil by pancreatic lipase (—),
of oil-in-water emulsion stabilised by lecithin by pancreatic lipase (– –) and
by pancreatic lipase and colipase (······). Inset: the total velocity (Vmax) was
determined as extrapolation of the linear line to zero abscissa. All experi-
ments were performed at least five times. *Mean values were significantly
different at a time point (P,0·05).
Flaxseed oil emulsion absorption in rats5
British Journal of Nutrition
The addition of colipase to this emulsified system did not
increase lipase activity, suggesting that, in vitro, colipase
could not prevent pancreatic lipase inhibition by PL (Fig. 3).
Lymphatic recovery of a-linolenic acid
Because lymph flow did not show any significant difference
between rats throughout the kinetic study, the two treated
groups were compared with regard to fatty acid concen-
tration in the lymph. At 24h after feeding, the total fatty
acid concentration in the lymph was twice as high in the
emulsion group as in the oil group (32·5 v. 14·5mg/ml of
lymph; P,0·001). This tendency was also observed for
ALA (8·0mg/ml of lymph in the emulsion group, compared
with 3·5mg/ml of lymph in the oil group; P,0·05). The rate
and total extent of ALA absorption at the intestinal site are
illustrated in Fig. 4 for up to 6h after feeding. The rate of
ALA absorption was similar for both groups in the first 2h.
After 3h, ALA recovery in the lymph of the emulsion
group was significantly higher than that of the bulk-phase
group (14·0 v. 6·5mg/ml, respectively). Moreover, the
maximum ALA concentration (Cmax) was obtained sooner,
and to a significantly greater extent in the emulsion group
compared with the bulk-phase group (Table 4). Likewise,
the area under the curve that estimates the intestinal
bioavailability of ALA was significantly higher for rats fed
emulsified flaxseed oil (Fig. 4, inset; Table 4).
Fatty acid composition of flaxseed oil and the intramol-
ecular fatty acid distribution in TAG are shown in Table 2.
In bulk flaxseed oil, ALA, the major fatty acid, was evenly
distributed between the three positions of the TAG mol-
ecules. The two other main fatty acids, oleic acid (18:1)
and linoleic acid (18:2), were mainly esterified in the
internalposition. Thefattyacid compositionof
lymphatic chylomicron TAG and their intramolecular fatty
acid distribution following administration of flaxseed oil
as a bulk phase or as an emulsion were examined
(Table 5). The fatty acid composition of chylomicron
TAG of both rat groups mainly reflected the fatty acid pro-
file of the dietary lipid source. As expected, feeding rats
with bulk flaxseed oil or with an emulsion increased the
proportion of ALA in the lymph compared with fasted
rats. A significant decrease in the proportion of endogen-
ous fatty acids, palmitic acid (16:0) and arachidonic acid
(20:4), was observed in the lymph, mainly due to a dilution
of these fatty acids in TAG as a consequence of their low
proportions of dietary lipids. The proportion of ALA in
the sn-2 position of chylomicron TAG was similar in the
three dietary groups (Table 5).
Among various vegetable oils, flaxseed oil is one of the
richest sources of ALA. Several studies in human subjects
have already demonstrated that flaxseed oil intake leads
to an increase in ALA levels in the plasma(23,25)and an
enrichment in ALA of erythrocyte total PL(23,44). Moreover,
flaxseed oil consumption has been associated with signifi-
cant health benefits thanks to an improvement of
biomarkers of cardiovascular risk(23,26,45)and of inflam-
mation(24,26). On the one hand, basic information about
ALA bioavailability from flaxseed oil is still unavailable in
order to understand its biological efficiency. On the other
hand, improving the bioaccessibility of ALA from the
food matrix is a potential strategy for providing additional
In the present study, we compared the metabolic fate of
flaxseed oil delivered either as a bulk phase or as an emul-
sion on lymphatic absorption. We clearly demonstrated
that TAG were more efficiently absorbed when provided
as an emulsion rather than as a bulk phase. As a result,
fatty acid and ALA enrichment in chylomicrons was greater
(Cmax) and faster (Tmax) in rats fed emulsified oil than in
rats fed bulk oil. These results are in agreement with
others studies performed either on animal models(13,46)
or on human subjects(16). These studies have suggested
AUC (mg × h/ml)
ALA (mg/ml lymph)
Fig. 4. Time-course concentration of a-linolenic acid (ALA) in chylomicrons
for the oil (V) and emulsion (W) rat groups. Values are means of at least
eight rats for each lipid system, at each time point. *Mean values were
significantly different at a time point (P,0·05). Inset represents the area
under the curve (AUC) for the two rat groups.
Table 4. Maximum a-linolenic acid concentration (Cmax), time required
to reach Cmax(Tmax) and area under the curve (AUC) in the lymph of
rats fed flaxseed oil in the bulk phase or in the oil-in-water emulsion
(Mean values with their standard errors, n 8)
AUC (mg £ h/ml)
* Lipid ingestion by rats corresponded to 0·3g in the form of flaxseed oil in the bulk
phase or emulsified. Chylomicrons were collected for 24h from eight animals,
and the lipid fractions were extracted. Following separation by TLC, TAG was
analysed for fatty acid composition by GC.
† Mean values were significantly different (P,0·05).
L. Coue ¨delo et al.6
British Journal of Nutrition
that PL from egg phosphatidylcholine(13,47)or soya phos-
phatidylcholine(13)could enhance lipid intestinal absorp-
tion in rats. Nishimukai et al.(13)have also shown that the
enhancement of lipid output in the lymph was highly
related to the TAG:PL ratio used in the dietary formulation.
The digestion process implies a hydrolysis step by lipo-
lytic enzymes including pancreatic lipase that operates
at the oil–water interface. Both the nature of the lipids
present at this interface and the curvature of the interface
could modulate enzyme activity. Indeed, studies based
on in vitro digestion of emulsified lipids coated with
various emulsifiers have shown that PL facilitated access
to emulsified fats compared with other non-ionic surfac-
tants(2,17). Above and beyond the interfacial composition,
the average oil droplet size also influences lipase activity.
Studies have demonstrated that fine emulsions (,1mm)
were hydrolysed by pancreatic lipase faster than coarse
emulsions (.20mm) in vitro(48)in both animals(49)and
human subjects(12,50,51). In the present in vitro study, we
first observed the behaviour of the emulsion provided to
rats in gastrointestinal-like conditions. Results showed
that the emulsified state was maintained even with large
variations in pH (Fig. 1), suggesting that in vivo, the
lipid–water interface may be preserved up to the intestine
level. Thus, TAG of the emulsified oil may be hydrolysed
faster by pancreatic lipase, resulting in an increased
absorption of hydrolysed products. In contrast, in the
case of flaxseed oil in the bulk phase, the interface must
be created by the mechanical mixing in the stomach and
intestine. Nevertheless, the in vitro hydrolysis of emulsified
oil by pancreatic lipase showed that lipase activity was
lower than that in the oil as a bulk phase (Fig. 3). This is
in agreement with previous in vitro studies showing that
long-chain TAG emulsified with PL were not hydrolysed
by pancreatic lipase, even in the presence of BS and
colipase(52–55). However, it has been reported that this
inhibition by PL disappeared due to the presence of
NEFA generated by TAG hydrolysis(54,55)that modified
the interface properties. The efficiency of intestinal absorp-
tion is also determined by the solubilisation of hydrolysis
products into BS-mixed micelles. In vitro micellisation
experiments involving flaxseed oil (both emulsified and
non-emulsified) demonstrated that a lower amount of
surfactants was required to produce mixed micelles with
the emulsion system (Table 3). In the case of rats that
were fed emulsion, the additional amount of lysophospho-
lipids indirectly provided by the dietary lipid formulation
may facilitate the transport of hydrolysis products through
the unstirred water layer of enterocytes. The present
in vivo results suggested that the emulsification of flaxseed
oil enhances its digestibility, due to the faster hydrolysis of
TAG because of the pre-existing oil–water interface and
a better solubilisation of hydrolysis products in mixed
micelles. Consequently, emulsions may be less prone to
Table 5. Main fatty acid composition and distribution of chylomicron TAG in the rat lymph resulting from oil in the bulk phase or oil-in-water (O/W)
emulsion ingestion 24h after feeding
(Mean values with their standard errors, n 8)
Fasted ratsOil O/W emulsion
sn-2 MAG‡ (mol%)
sn-2 MAG (mol%)
sn-2 MAG (mol%)
sn-2 MAG, sn-2 monoacylglycerol; TFA, trans-fatty acid.
a,bMean values within a row with unlike superscript letters were significantly different for lymphatic fatty acid wt% (P,0·05).
* Lipid ingestion by rats corresponded to 0·3g in the form of flaxseed oil in the bulk phase or emulsified. Chylomicrons were collected for 24h from eight animals, and the lipid
fractions were extracted. Following separation by TLC, TAG was analysed for fatty acid composition by GC.
† The data represent the average of three different determinations.
‡ The fatty acid composition of sn-2 MAG of flaxseed oil and chylomicron TAG was determined after pancreatic lipase hydrolysis followed by isolation and analysis of MAG.
The sn-2 MAG analysis was performed on pooled samples of the lymph of eight rats. Results are expressed in mol% corresponding to the distribution of each fatty acid in
the internal position of TAG.
Flaxseed oil emulsion absorption in rats7
British Journal of Nutrition
oxidative degradation and may reside less long in the
intestinal lumen, leading to a reduction of the extent to
which they are conveyed to faeces. Besides parameters
influencing lipid bioavailability in the intestinal lumen,
it is worth noting that lipid recovery in the lymph may
also be affected by the processes occurring in the entero-
cytes, i.e. uptake into the mucosal cells, as well as the
packaging and secretion of chylomicrons. The supply of
dietary phosphatidylcholine may favour the formation
of chylomicrons(56)and/or be involved in the regulation
of jejuna apo A-I synthesis in animals(57). Indeed,
amount of TAG in the rat lymph increased twofold in the
presence of soya lecithin.
Several studies have pointed out that the fatty acid
composition of chylomicrons and fatty acid distribution
in TAG reflected that of the dietary oil(58–60). It is well
established that fatty acids esterified at the sn-2 position
of dietary TAG are mainly retained during the absorption
process(61)due to the positional specificity of pancreatic
lipase. After incorporation of these hydrolysis products
into the mucosal pool, most of the 2-MAG are reacylated
to TAG that are incorporated into chylomicrons secreted
into the lymph. To our knowledge, no similar studies
have been performed with flaxseed oil. ALA enrichment
in chylomicrons was observed irrespective of the dietary
form of flaxseed oil (Table 5). However, the percentage
of ALA esterified at the sn-2 position of chylomicron TAG
was slightly lower (18 and 23% for oil and emulsion,
respectively) compared with that in dietary flaxseed oil
(28%). This may be attributed to a degradation of some
2-monolinolenate glycerols(62). Indeed, chylomicron TAG
contained a high percentage of endogenous fatty acids,
which were supplied by bile lipids, especially 16:0, 18:1
and 18:2 fatty acids(63,64).
On the whole, the present results showed that the extent
of fatty acid absorption, and especially of ALA, was signifi-
cantly higher in the rat group ingesting emulsified oil com-
pared with the group given oil in the non-emulsified state.
Moreover, the results of the in vitro studies dealing with
emulsion stability, lipid hydrolysis and solubilisation were
used to interpret, at least partly, the increased lymphatic
concentration in ALA in the newly synthesised TAG. Never-
theless, basic information on ALA bioavailability from
flaxseed oil is still necessary to understand its biological
efficiency. In particular, because the intramolecular fatty
acid distribution in chylomicron TAG did not exactly reflect
that of the dietary oil, the metabolic pathway of ALA during
the digestion process remains to be further explored.
The authors acknowledge the National Association of
Technical Research and the Aquitaine Regional Council
for their financial support through a PhD research grant
for L. C. The authors state that there are no conflicts of
interest. Contribution made by each author to the research
is as follows: L. C. is a PhD student who contributed to the
design of the in vitro and in vivo experiments. C. B.-V. is
the industrial PhD supervisor, specialist in lipid metab-
olism. L. F., E. M. and S. D. provided technical assistance
for the lipid analysis. N. C. is an expert in lipid metabolism
who helped to interpret the in vivo results. M. C. is the
institutional PhD supervisor and is an expert in the formu-
lation of colloidal systems for nutritional applications and
in lipid bioavailability.
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