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*
Instituto de la Grasa, Consejo Superior de Investigaciones Cientificas, 41012 Sevilla, Spain and
Hospital Universitario Virgen del Rocío, 41013 Sevilla, Spain
2To whom correspondence should be addressed at Avda. Padre García Tejero, 4. 41012 Sevilla, Spain. E-mail: valruiz{at}cica.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • olive oil • triacylglycerol molecular species • triacylglycerol-rich lipoproteins • postprandial period • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). VLDL are synthesized in the liver
and contain a molecule of apolipoprotein B-100. The actual importance
of these lipoproteins lies in the accumulated evidence, suggesting that
the rate of clearance of TRL may be an independent risk factor for
atherosclerotic cardiovascular disease (Groot et al. 1992
, Havel 1994
, Simons et al. 1987
, Zilversmit 1979
). Furthermore, in humans,
delayed removal of chylomicron remnants was documented in diabetes
(Chen et al. 1993
), renal failure
(Weintraub et al. 1992
), familial combined
hyperlipidemia (Cabezas et al. 1993
) and obesity
(Mekki et al. 1999
).
Considerable data are available concerning the effect of different
diets on TRL clearance during the postprandial period. The overall
conclusion which can be drawn is that diets containing polyunsaturated
fatty acids (PUFA), particularly (n-3) PUFA, result in reduced
concentrations of chylomicrons and VLDL due to an increased rate of
lipolysis and to a more rapid removal of remnants by the liver
(Botham et al. 1997
, Bravo et al. 1995
,
Lambert et al. 1995
, Zampelas et al. 1994
). Monounsaturated fatty acid (MUFA) diets were less
studied, however, MUFA in the background diet seems to lead to faster
rates of triacylglycerol entry and clearance (van Heek and Zilversmit, 1990
). Among dietary fats, olive oil intake was
associated with a low incidence in coronary heart diseases by improving
the atherogenic plasma lipid profile in both healthy subjects and
hyperlipidemic patients (Mata et al. 1992
,
Ruiz-Gutierrez et al. 1996
). Furthermore, diets rich in
olive oil resulted in an increased resistance of LDL to oxidation and a
lower rate of monocyte adhesion to endothelial cells in vitro when
compared to saturated fatty acids (SFA) and (n-3) and (n-6) and
PUFA-rich diets (Mata et al. 1996
). Recent studies
in vitro also suggested that lipoproteins enriched with oleic acid are
less susceptible to oxidation than those enriched with linoleic or
arachidonic acid and may have less proinflammatory activity when
exposed to oxidizing conditions (Lee et al. 1998
).
Chylomicrons and VLDL function as carriers of triacylglycerol-fatty
acids from the intestine and liver to the peripheral tissues,
respectively. The obligatory step in the transport of
triacylglycerol-fatty acid is the hydrolysis of the triacylglycerol
core of the lipoprotein, which takes place under the influence of
lipases (Pedersen and Schotz 1980
). This is followed by
possible internalization of the lipoprotein remnants’ particles by the
peripheral tissues (Karpe et al. 1997
) and by the liver
(Cooper 1997
). Little information is available on the
importance of the molecular species of triacylglycerols as a regulating
factor of TRL clearance, probably due to the difficulty in the
identification of natural triacylglycerols. It was reported for
lipoproteins (Hauton et al. 1987
) and for adipose tissue
(Raclot 1997
) that the accessibility of the enzyme to
its lipid substrate may depend, among other factors, on physicochemical
properties of the triacylglycerol molecules such as its polarity.
We recently suggested that the triacylglycerol composition of vegetable
oils with a similar content of MUFA (olive and high oleic sunflower
oils) may be an important factor to take into account when studying
human VLDL profile (Ruiz-Gutierrez et al. 1996b
). This
also explains different nutritional and clinical effects, including the
benefits for membrane homeostasis of healthy subjects and patients with
cardiovascular risk factors (Muriana et al. 1997a
and
1997b
, Ruiz-Gutierrez et al. 1997
).
Based on the above observations, experiments were designed to study the effect of a meal rich in olive oil on triacylglycerol composition of human postprandial TRL (fraction Sf > 400) and the effect of the polarity and concentration of the triacylglycerol molecular species on lipoprotein clearance. For this purpose, eight healthy normolipidemic subjects given medical examination were chosen for the study. Postprandial blood samples were collected hourly for 7 h after the ingestion of a meal rich in olive oil. As far as we know, this is the first study showing changes in the type of triacylglycerol in TRL during the postprandial period in humans. This information could contribute to a better understanding of the beneficial effects of olive oil on the prevention of atherosclerosis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Eight volunteers (one female, seven males) aged 27 ± 7 y and body mass index 22.7 ± 1.7 kg/m2 participated in the study. Plasma chemistry and hematological indices were within the normal range for all subjects. The fasting blood lipid concentrations were as follows: plasma total cholesterol, 3.85 ± 0.91 mmol/L; LDL cholesterol, 2.48 ± 0.78 mmol/L; HDL cholesterol, 1.60 ± 0.23 mmol/L; triacylglycerols, 0.79 ± 0.17 mmol/L. The subjects did not suffer from any digestive or metabolic disease as verified by medical history. The subjects gave written, informed consent to a protocol approved by the Institutional Committee on Investigation in Humans (Hospital Universitario Virgen del Rocío, Sevilla, Spain).
Participants were asked to refrain from smoking and drinking alcohol
during the preceding day of the study given their influence on lipid
metabolism. After an overnight fast (12 h), a cubital vein was
catheterized with a small bore extension set with
SMARTSITETM needle-less valve port equipped with a
disposable vacutainer (Vacutainer® Meylen, Cedex,
France). A baseline fasting blood sample was collected into
precooled vacutainer tubes (1 g/L EDTA-K3) immediately
before the subjects ate the test meal. Blood samples were drawn hourly
during a 7-h postprandial period. Blood samples were placed into ice
water and plasma recovered rapidly by centrifugation (1,750 x g, 20 min, 1°C). NaAzide (1 mol/L), PMSF (10 mmol/L in
isopropanol) and aprotinin (1400 mg/L) were added to the plasma to a
final concentration of 1 mmol/L, 10 µmol/L and 28 mg/L, respectively
(Karpe et al. 1993
). Plasma was kept at 4°C for
12 h until lipoprotein fractionation.
The test meal consisted of one slice of brown bread (28 g); 100 g
of plain pasta (cooked with 200 mL of water); 130 g of tomato
sauce and one skimmed yogurt, providing 1936 kJ of energy. The
virgin olive oil (70 g) was supplied mixed with the tomato sauce.
(Table 1
). During the course of the study, the participants were allowed to
drink water and undertook only light activities. The composition of the
saponifiable fraction of the virgin olive oil (v. cornicabra) (Aceites
Toledo SA, Los Yébenes, Toledo, Spain) used for the meal is shown
in Tables 2
and 3
.
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Isolation of TRL.
TRL [Sf > 400, density >0.93 kg/L ] were isolated from 4 mL of
plasma layered with 6 mL of a NaCl solution (density 1.006 kg/L) by a
single ultracentrifugation (95,000 x g, 42 min,
15°C) (Berr and Kern 1984
, Mills et al. 1989
). Ultracentrifugation was performed using an SW 41 Ti
rotor in a Beckman L8–70M preparative ultracentrifuge (Beckman
Instruments, Inc., Palo Alto, CA).
Triacylglycerol concentrations were measured in plasma and in the Sf > 400 lipoprotein fraction in the control and test samples by a colorimetric enzymatic method (Peridocrom Triglycerides GPO-PAP kit; Boehringer Mannheim, Meylen, France)
Identification of apolipoproteins in the TRL fraction.
The structural apolipoproteins B-100 and B-48 were taken as an indicator of the presence of VLDL and chylomicrons, respectively, in the isolated TRL fraction.
Apolipoproteins B-100 and B-48 were identified by the Laemmli SDS-PAGE system (7.5% SDS-PAGE slab gels, 1.5 mm thick). The gel electrophoresis was carried out at 30 mA/gel for 120 min.
TRL were isolated at 2 and 6 h as above from plasma containing benzamidine (0.3 g/L) to prevent scission of apolipoprotein B. After isolation, the lipoproteins were recentrifuged in an SW 41 Ti rotor at 93,000 x g at 12°C for 18 h to remove albumin and other proteins.
TRL were delipidated and the apolipoproteins precipitated as follows.
Samples containing ~70 µg of total protein was diluted to a volume
of 2 mL with 0.12 mol/L of NaCl. Because the concentration of
apolipoprotein B-48 in chylomicrons was very low, we added 60 µg of
apotransferrin before delipidation to facilitate protein precipitation.
The mixture was delipidated with 20 mL of ice-cold
ethanol-diethylether (3:1, vol/vol) at -20°C overnight. The samples
were centrifuged at -10°C for 20 min at 720 x g,
the supernatant removed and the pellet washed twice with
ethanol-diethylether (3:1, vol/vol) and cold anhydrous diethylether
(Kotite et al. 1995
).
Perfect ProteinTM Markers (MW 10–225 kDa) (Calbiochem-Novabiochem,
Schwalbach, Germany) were used as standards, and LDL as a B-100
standard. LDL were isolated by cumulative rate centrifugation in a
density gradient using an SW 41 Ti rotor in a Beckman L8–70M
(Mills et al. 1989
, Redgrave et al. 1975
).
Analysis of fatty acid methyl esters (FAME).
Triacylglycerols were isolated by solid-phase extraction diol columns (SupelcleanTM LC-Diol; Supelco, Bellefonte, PA) using hexane/methylene chloride (9:1, vol/vol) as eluent. An aliquot was taken for analysis of total fatty acids by GLC. A second aliquot was stored at -80°C for further analysis of the triacylglycerol molecular species by HPLC.
Triacylglycerols were transmethylated and the resulting fatty acid
methyl esters (FAME) analyzed by GLC as described by Ruiz-Gutierrez et al. (1993)
using a model 5890 series II gas chromatograph
(Hewlett-Packard, Avondale, PA) equipped with a flame-ionization
detector and a capillary silica column Supelcowax 10 (Supelco) of
60 m length and 0.25 mm internal diameter.
Analysis of triacylglycerol molecular species.
Triacylglycerols were evaporated completely in a vacuum, redissolved in n-hexane and passed through a filter with a pore size of 0.2 µm (Millipore, Bedford, MA). The chromatographic system consisted of a model 2690 Alliance liquid chromatograph (Waters Co., Milford, MA), provided with a Spherisorb ODS-2 column (250 x 4.6 mm) of 3 µm particle size (Waters). The liquid chromatograph was coupled to a light-scattering detector model DDL31 (Eurosep, Instruments, Cergy-Pontoise, France). The system was controlled by computer through Millenium System (Waters). The mobile phase consisted on an initial elution gradient of 20% of acetone in acetonitrile raising the percentage of acetone to 45% in 12 min and then to 80% after 65 min, this percentage was held upto the end of the analysis. The flow rate was 1 mL/min. Quintupled analyses of 10 µL of n-hexane solution containing 0.5 g/L of pure triacylglycerol (Sigma Grade, 99% pure; Sigma Chemical Co., St. Louis, MO): tritridecanoin, 1,3-dioleoyl-2-palmitoyl-glycerol, trimyristin, 1,3-dioleoyl-2-stearoyl-glycerol, 1,3-dioleoyl-2-linoleoyl-glycerol, tripentadecanoin, tripalmitin, triolein and trilinolein, were injected in order to establish the capacity factor (k') of the system.
The triacylglycerol composition was predicted by means of relationships
between k' and molecular variables of the pure triacylglycerols. The
values of the equivalent carbon number (ECN) were calculated as ECN
= CN - 2ND - 2 NUFA (where CN is the carbon number, ND the number
of double bonds and NUFA the number of unsaturated fatty acids attached
to the glycerol molecule). A linear regression analysis was applied to
relate the ECN with log k'of the pure triacylglycerols. The
triacylglycerol composition in samples was calculated by comparison
between the ECN of the chromatographic peaks and theoretical ECN of all
possible triacylglycerols. Fatty acids, obtained by GLC analysis, were
combined in threes to calculate all possible triacylglycerols. We
considered all the stereospecific positions in the glycerol molecule as
equivalents since HPLC can not separate positional isomers
(Perona et al. 1998a
and 1998b
).
Triacylglycerols were quantified using tridecanoin as internal
standard.
Statistical analysis.
Results are presented as means ± SD Time-course
data of total triacylglycerol concentations in plasma (Fig. 1
) and in the TRL fraction (Fig. 2
) after the test meal wasre analyzed for significant difference from
data obtained after the control meal using a paired
t-test. The effect of olive oil intake on
triacylglycerol (Table 2)
and fatty acid composition (Table 3)
of TRL
was assessed by one-way (repeated measured) ANOVA with Tukey’s
post-hoc comparison of the means. When necessary, values were
transformed reciprocally before statistical analysis to compensate for
unequal variance. One-way ANOVA was also used to compare the
different profiles of clearance of the major triacylgycerol molecular
species from plasma (Fig. 2A)
. The relationship between either, ECN or
percentage of triacylglycerol, and rate of clearance (Fig. 3
) was assessed using the t-test and linear regression
components of Microsoft Excel 97. Other analyses were done with the
GraphPAD InStat (GraphPAD Software, San Diego, CA) and CoStat (CoHort
Software, Berkeley, CA) statistical packages. Differences of
P < 0.05 were considered statistically
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Detection of apolipoprotein B in the Sf > 400 TRL fraction.
The presence of both apolipoprotein-B isoproteins within the TRL
fraction at 2 and 6 h after the ingestion of the meal was detected
after Comassie blue protein stain. ApoB-48 was the principal form
identified; as apolipoprotein B-48 can serve as a marker for
intestinally derived lipoproteins (Kane 1983
), we can
therefore assume that the TRL of Sf > 400 fraction chosen for the
study was composed mostly of chylomicrons and their remnants.
Apolipoprotein B-100 also could have been synthesized and secreted by
the human intestine (Hoeg et al. 1990
) or be associated
with hepatic VLDL.
Triacylglycerol concentration in plasma and in the TRL fraction.
Mean triacylglycerol concentration in plasma and in TRL increased
quickly over fasting value (0 h) and peaked twice during the 7-h
postprandial period following the ingestion of the olive oil meal (Fig. 1A)
. Six of the subjects demonstrated a
biphasic response and two a monophasic response over the studied
period. On average, plasma triacylglycerol concentration rose to give a
peak concentration at 2 h. Triacylglycerols were then cleared from
the circulation and reached a smaller second peak at 6 h. The
triacylglycerol profile in TRL closely mirrored the plasma
triacylglycerol pattern (Fig. 1B)
. The concentrations at 2 and 6 h
accounted for about 40 and 20%, respectively, of the total
concentration of triacylglycerol present in plasma. The ingestion of
the control meal (without olive oil) showed no early peak in either the
plasma or lipoprotein fractions.
Triacylglycerol molecular species composition in the TRL fraction.
The triacylglycerol molecular species were determined in the TRL
fraction at 2 and 6 h after ingestion of the meal. Using a
PR-HPLC technique, we identified an extensive profile of
triacylglycerols from lipoprotein samples. At the 2-h peak time, the
principal molecular species detected were triolein (OOO),
palmitoyl-dioleoyl-glycerol (POO), linoleoyl-dioleoyl-glycerol (LOO),
palmitoyl-oleoyl-linoleoyl-glycerol (POL) and
stearoyl-dioleoyl-glycerol (SOO) and were followed, in order of
concentration by dipalmitoyl-oleoylglycerol (PPO),
dipalmitoyl-linoleoyl-glycerol (PPL),
palmitoyl-dilinoleoyl-glycerol/plamitoyl-oleoyl-linolenoyl-glycerol
(PLL/POLn),
palmitoyl-oleoyl-stearoyl-glycerol/distearoyl-linoleoyl-glycerol
(POS/SSL) and
oleoyl-dilinoleoyl-glycerol/dioleoyl-linolenoylglycerol
(OLL/OOLn) (Table 2)
. The same molecular species were found in the
olive oil; however, the percentage of OOO was lower, POO, SOO and POS
were maintained and the rest of the triacylglycerols were greater in
the lipoproteins compared with the composition in the parent oil.
Smaller amounts of trilinolein (LLL), trimyristin (MMM),
dipalmitoyl-linolenoyl-glycerol (PPLn),
linoleoyl-palmitoyl-mirystoyl-glycerol (LPM),
oleoyl-dimyristoyl-glycerol (OMM), stearoyl-dilinoleoyl-glycerol (SLL),
mirystoyl-dipalmitoyl-glycerol (MPP), distearoyl-palmitoyl-glycerol
(SSP) and tristearin (SSS) were only identified in the lipoprotein
particles.
There were no significant differences in the triacylglycerol composition of TRL between 2 and 6 h; however, there was a tendency for triacylglycerols rich in PUFA and SFA to increase and of OOO to decrease at the 6-h peak time (P = 0.061).
Fatty acid composition of triacylglycerols in the TRL fraction.
The fatty acid composition of the olive oil and TRL during the
postprandial period is shown in Table 3
. In olive oil, 72% of the
total fatty acid composition was identified as oleic acid
[18:1(n-9)]; the remainder was mainly palmitic (16:0, 13.0%),
linoleic [18:2(n-6), 5.5%] and stearic (18:0, 2.7%) acids. The
formation of the TRL fraction at the 2-h peak time produced a
significant decrease in the proportion of oleic acid (P
< 0.05), an increase in SFA [myristic (14:0), palmitic and
stearic (P < 0.05) acids] and the appearance of PUFA
of the (n-6) family arachidonic acid [20:4(n-6)] and of the (n-3)
family docosapentaenoic [22:5(n-3)] and docosahexaenoic [22:6(n-3)]
acids (P < 0.01).
The metabolism of TRL significantly increased the proportions of both SFA and PUFA (P < 0.005) and decreased MUFA (P < 0.01) from 2 to 4 h. The period of maximal clearance of triacylglycerols (2 to 4 h) was associated with a small but significant decrease in the proportion of oleic acid (18%) and parallel increases in myristic (156%), stearic (95%) and palmitic (13%) acids. There were also increases in linoleic (30%) and arachidonic (132%), docosapentaenoic (150%) and docosahexaenoic (105%) acids (P < 0.05). The fatty acid composition of TRL at the 6-h peak time during the late postprandial period showed a significant decrease in oleic acid (P < 0.05) and increases in palmitic, stearic (P < 0.005) and arachidonic (P < 0.05) acids compared with the fatty acid composition at 2 h.
Determination of the triacylglycerol molecular species clearance during the postprandial period.
The rate of removal of triacylglycerols in TRL during the postprandial
period is shown in Figures 2A
and 2B
.
Subjects had low fasting plasma concentrations. POL was found in large
concentrations (4.20 µmol/L of plasma) when compared to the rest of
the triacylglycerols, in which baseline plasma values ranged from 0.11
µmol/L of plasma for OOO to 0.03 µmol/L for PPL.
All the molecular species had similar patterns of clearance; they showed a biphasic profile with peak concentrations at 2 and 6 h after the ingestion of the meal. PPO, POS, and OLL had the first peak at 1 h. The fastest clearance of the molecular species from the particles occurred from 2 to 4 h and was different among the triacylglycerols (P < 0.05).
To determine the possible relationship between the triacylglycerol
polarity and concentration, and their rate of clearance in blood, we
plotted the rate of disappearance of the triacylglycerols between 2 and
4 as a function of both, the ECN and the concentration of each
triacylglycerol at 2 h (Fig. 3)
. The
ECN is an index of polarity, since triacylglycerol molecules elute from
the HPLC column in growing order of ECN in a reversed-phase system.
Thus, the lowest ECN values correspond to the most polar molecules and
the highest to the most apolar ones.
Figure 3
shows a strong positive correlation between the amount of
triacylglycerol in the TRL at 2 h (see Table 2
) and the rate of
disappearance of these triacylglycerols in blood (r = 0.99, P < 0.05). No correlation was found between the
ECN of the triacylglycerol and its clearance (r = 0.17,
P > 0.1). OOO had the fastest rate of disappearance
[100 ± 13 µmol/(L plasma.h), ECN = 47.4] followed by POO
[79 ± 9 µmol/(L plasma.h), ECN = 47.6], OOL [35 ± 8 µmol/(L plasma.h), ECN = 45.4], POL [14 ± 4 µmol/(L
plasma.h), ECN = 45.6], PPO [8.6 ± 2.1 µmol/(L
plasma.h), ECN = 47.8], SOO [8.3 ± 2.0 µmol/(L
plasma.h), ECN = 49.6]; the lowest values [2 ± 0.6
µmol/(L plasma.h)] were shown by PLL (ECN = 43.6), POS (ECN
= 49.8) and OLL (ECN = 43.4). The rates of clearance of the
major triacylglycerols (OOO, POO, OOL and POL) differed (P
< 0.05) as did the later ones and PLL, POS and OLL (P
< 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The mean concentration of triacylglycerols in plasma (Fig. 1A)
and in
the lipoprotein fraction (Fig. 1B)
indicated the presence of two
postprandial peaks during the 7-h follow-up period. The magnitude
and the pattern of triglyceridemia varied among subjects; a biphasic
triacylglycerol response was more common than a monophasic one. Plasma
triacylglycerol concentration after a fat-rich meal can often have
multiple postprandial peaks, including a biphasic and monophasic
response (Cohn et al. 1989
, Heller et al. 1993
, Kashyap et al. 1983
). Furthermore, some
studies found a correlation between the magnitude of postprandial
triglyceridemia and the age and gender of the subjects (Cohn et al. 1988
), and the type of triacylglycerols in dietary oil
(Barr et al. 1985
).
The triacylglycerol peaks in the TRL fraction occurred at 2 and 6 h after the ingestion of the meal and corresponded exactly with the
rise of total triacylglycerols in plasma. Both of them were triggered
by the olive oil meal, as suggested by the lack of triacylglycerols in
the lipoprotein fraction of Sf > 400 in the subjects after the
control meal (Fig. 1B)
. The triacylglycerols at 2 h reflected the
molecular species of the dietary olive oil from which they were
derived. In this respect, there is substantial evidence that the
distribution and composition of fatty acids in dietary triacylglycerols
are important determinants of fat absorption and digestion; thus, the
dietary triacylglycerol sn-2 position fatty acids are used
as substrate for reesterification of triacylglycerols in the enterocyte
(Aoyama et al. 1996
, Innis and Dyer 1997
). We observed a better balanced composition of
triacylglycerols in the lipoproteins, achieved by a lower incorporation
of OOO and a concomitant increase of the triacylglycerols present in
smaller concentrations in the parent oil such as OOL, POL and OLL
(Table 2)
. This probably occurred during the synthesis of chylomicrons
in the enterocytes to compensate for an extreme composition of
triacylglycerols in the ingested olive oil. Similarly, ~0.8% of the
triacylglycerols, mainly formed by SFA, were only detected in the
lipoprotein fraction and probably originated endogenously during the
processes leading to the formation of chylomicrons, or of VLDL in the
liver. The determination of the fatty acid profile of the
triacylglycerols showed an increase in the proportions of linoleic,
myristic and palmitic acids and a decrease in MUFA, due to a reduction
in oleic acid, whereas the percentage of palmitoleic acid was
maintained (Table 3)
. We could not identify all the triacylglycerol
molecular species present in TRL. However, the fatty acid analysis
suggested the formation of triacylglycerols constituted of the
long-chain PUFA of the (n-3) family (docosapentaenoic and
docosahexaenoic acids) and of the (n-6) family (arachidonic acid). In
agreement with these results, Lambert et al. (1996)
pointed out that an
endogenous contribution of linoleic and docosahexaenoic acids may be
obligatory in the formation of chylomicrons. This mechanism of fatty
acid selection in the enterocyte could protect the tissues against
sudden changes in the composition of dietary fat and supply them with
essential fatty acids (EFA) when there is a deficiency in the diet.
The triacylglycerol composition of the TRL fraction at the maximal
point of the late postprandial peak (6 h) did not differ from the
composition at the early peak (2 h). However, when we analyzed the
fatty acid profile, we detected a significant decrease in oleic acid
along with significant increases in palmitic, stearic and arachidonic
acids, which denotes a difference in composition and probably of
origin. There is not a clear explanation for the increase in
triacylglycerols during the late postprandial period although some
authors attributed this to the release of preformed chylomicrons which
could have been stored at their site of synthesis in the enterocyte, or
perhaps in the lymphatics and be released later when needed by the body
(Cohn et al. 1989
, Fielding et al. 1996
).
In our study, the presence of apo B-100 at 6 h may suggest the
release of VLDL from the liver.
Several factors control triacylglycerol removal postprandially,
including the hydrolysis of triacylglycerol by lipoprotein lipase (LPL)
and hepatic lipase, the uptake of chylomicrons by receptor- and
nonreceptor-mediated processes and transfer of lipids with other
lipoproteins (De Bruin et al. 1993
, Mortimer et al. 1995
, Skottova et al. 1995
). To the best of
our knowledge there is little information regarding the removal of the
different triacylglycerol molecular species of the lipoprotein
particles and its regulating factors in humans. In these particles, all
the lipid classes are complex mixtures due to the processes of
intestinal absorption and formation of the triacylglycerols in the
mucosal cells. Lack of information may be due to the difficulty in
measuring them. In our study, the molecular species identified
in the TRL fraction had similar patterns but different rates of
clearance from blood (clearance should be understood as the sum of two
processes, lipolysis and particle uptake). OOO had the fastest rate of
disappearance followed by POO, OOL, POL, PPO, SOO, PLL, POS and OLL
(Figs. 2A
and 2B)
. Different rates of clearance of lipids of dietary
origins were also equally found in the rat (Bravo et al. 1995
); further in vitro studies (Botham et al. 1997
) related this to differential rates of lipolysis of
chylomicrons of different fatty acid composition by lipoprotein
lipase. LPL is interfacially activated; thus binding to a
lipid/water interface is a prerequisite for enzyme catalysis
(Brockman 1984
). It was reported for lipoproteins that
polar lipids, mainly triacylglycerols enriched in PUFA, have a higher
partition coefficient than triacylglycerols enriched in SFA. This leads
to a selective accessibility of the enzyme to the substrate, which
could, at least in part, explain the preferential release of PUFA from
triacylglycerols found in several studies (Zampelas et al. 1994
). Based on the above hypothesis, in a heterogeneous
triacylglycerol mixture, lipolytic enzymes would be more efficient with
polar triacylglycerols. In our study, we did not find a correlation
between the rate of clearance of the triacylglycerols in blood and its
polarity. The clearance of TRL was positively correlated with the
concentration of the triacylglycerol in the lipoprotein particle. Not
all the triacylglycerols were cleared at the same rate; thus, POO was
cleared twice as fast as OOL even though the POO was present at a
threefold higher concentration. This implies that changes in
triacylglycerol composition over time could be related to varying rates
of lipolysis by LPL and agrees with studies undertaken by Wang et al. (1992)
in which LPL functions preferentially in the sequence 18:1
> 18:3 > 18:2 > 16:0 > 18:0. On the contrary,
Sato et al. (1999)
suggested that the lipoprotein catalysis by LPL is
modulated by the palmitic acid content of the lipoprotein
triacylglycerol, which affects the fluidity of the lipoprotein. The
explanation for these differences is currently lacking. Differences may
not depend on the fatty acids themselves but on the nature of the
triacylglycerol molecule. The increase over time of arachidonic acid
[20:4(n-6)] found in our study could also be explained by the fatty
acid specificity of LPL; Chen et al. (1987)
found that during lipolysis
of chylomicron triacylglycerols, the arachidonic acid ester bond may be
resistant to LPL.
In summary, feeding healthy subjects a meal rich in olive oil produced a biphasic pattern of postprandial triglyceridemia in the fraction of TRL of Sf > 400. The processes leading to the formation of the chylomicrons in the enterocytes seemed to have balanced the composition of the parent olive oil through a decrease in the proportion of OOO and an increase in triacylglycerols with linoleic and palmitic acids (OOL, POL, PPL, OLL, LLL). We also detected the formation of endogenous particles either in the enterocytes (chylomicron) and/or in the liver (VLDL) enriched with myristic, palmitic and the long-chain PUFA of the (n-3) family (docosapentaenoic and docosahexaenoic acids) and of the (n-6) family (arachidonic acid). The rates of clearance of the triacylglycerols during the postprandial period were different among the molecular species; thus, OOO was removed faster and was followed by POO, OOL, POL, PPO, SOO, PLL, POS and OLL. The clearance was directly related to the concentration of the triacylglycerol in the lipoprotein particle and not with its polarity. This finding may have important consequences because the fatty acid composition of the principal triacylglycerols might be expected to predict the fatty acids hydrolysed by LPL and therefore available to peripheral tissues. Differential rates of clearance of TRL of different oil sources remain to be determined in humans and are currently under investigation in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ECN, equivalent carbon number; FAME, fatty acid methyl eters; L, linoleic acid [18:2 (n-6)]; LOO,
linoleoyl-dioleoyl-glycerol; LPL, lipoprotein lipase; MUFA, monounsaturated fatty acids; O, oleic acid [18:1(n-9)]; OLL,
oleoyl-dilinoleoyl-glycerol; OOL, dioleoyl-linoleoyl-glycerol; OOLn, dioleoyl-linolenoyl-glycerol; OOO, triolein; P, palmitic acid
(16:0); OOP, dioleoyl-palmitoyl-glycerol; PLL, palmitoyl-dilinoleoyl-glycerol; POL,
palmitoyl-oleoyl-linoleoyl-glycerol; POLn, palmitoyl-oleoyl-linolenoyl-glycerol; POO, palmitoyl-dioleoyl-glycerol;
POS, palmitoyl-oleoyl-stearoyl-glycerol; PPL, dipalmitoyl-linoleoyl-glycerol; PPO, dipalmitoyl-oleoyl-glycerol; PUFA, polyunsaturated fatty acids; S, stearic acid (18:0); SFA, saturated fatty acids; SOO, stearoyl-dioleoyl-glycerol; SSL,
distearoyl-linoleoyl-glycerol; TRL, triacylglycerol-rich lipoproteins.
Manuscript received May 11, 1999. Initial review completed June 14, 1999. Revision accepted August 31, 1999.
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