1 Unité Maladies Métaboliques et Micronutriments, Institut National de la Recherche Agronomique, Clermont-Ferrand/Theix, 63122 Saint-Genès-Champanelle; 3 Unité d'Exploration en Nutrition, Laboratoire de Nutrition Humaine du Centre de Recherche en Nutrition Humaine d'Auvergne, 63000 Clermont-Ferrand; and 2 Unité INSERM 476, Faculté de Médecine, 13385 Marseille Cedex 05, France
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ABSTRACT |
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Carotenoids are thought to diminish the
incidence of certain degenerative diseases, but the mechanisms involved
in their intestinal absorption are poorly understood. Our aim was to
obtain basic data on the fate of carotenoids in the human stomach and
duodenum. Ten healthy men were intragastrically fed three liquid test
meals differing only in the vegetable added 3 wk apart and in a random order. They contained 40 g sunflower oil and mashed vegetables as
the sole source of carotenoids. Tomato purée provided 10 mg lycopene as the main carotenoid, chopped spinach (10 mg lutein), and
carrot purée (10 mg -carotene). Samples of stomach and
duodenal contents and blood samples were collected at regular time
intervals after meal intake. all-trans and cis
carotenoids were assayed in stomach and duodenal contents, in the fat
and aqueous phases of those contents, and in chylomicrons. The
cis-trans
-carotene and lycopene ratios did not
significantly vary in the stomach during digestion. Carotenoids were
recovered in the fat phase present in the stomach during digestion. The
proportion of all-trans carotenoids found in the micellar
phase of the duodenum was as follows (means ± SE): lutein
(5.6 ± 0.4%),
-carotene (4.7 ± 0.3%), lycopene
(2.0 ± 0.2%). The proportion of 13-cis
-carotene
in the micellar phase was significantly higher (14.8 ± 1.6%)
than that of the all-trans isomer (4.7 ± 0.3%). There
was no significant variation in chylomicron lycopene after the tomato
meal, whereas there was significant increase in chylomicron
-carotene and lutein after the carrot and the spinach meals,
respectively. There is no significant cis-trans
isomerization of
-carotene and lycopene in the human stomach. The
stomach initiates the transfer of carotenoids from the vegetable matrix
to the fat phase of the meal. Lycopene is less efficiently transferred
to micelles than
-carotene and lutein. The very small transfer of
carotenoids from their vegetable matrices to micelles explains the poor
bioavailability of these phytomicroconstituents.
-carotene; lycopene; lutein; postprandial; absorption; bioavailability
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INTRODUCTION |
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EPIDEMIOLOGICAL STUDIES CONSISTENTLY associate diets rich in fruits and vegetables with a lower incidence of several diseases (22). The plant pigments, carotenoids, are assumed to be involved in this effect because of their antioxidant properties (20, 21).
In common foods, carotenoids are mostly found as all-trans
isomers (25), but significant amounts of geometrical
cis isomers can be produced during processing (36,
37). The cis isomers of -carotene and lycopene
have attracted attention, because several studies have suggested that
their bioavailabilities are different from those of their corresponding
trans isomers (2, 15, 18, 39) and that they may
possess specific functions (16, 23, 26, 28).
The absorption efficiency of all-trans -carotene, the
most extensively studied carotenoid, is generally poor but widely
variable (4, 11), ranging between 3.5 and 90%, depending
on the dose, the matrix in which it is incorporated, and the method
used to estimate absorption (8, 12, 31, 47). Data on the
absorption efficiency of other carotenoids are scant, although such
information is needed for dietary recommendations, supplement
formulation, and the design of intervention studies involving
carotenoids (33). Although a number of factors is thought
to affect the bioavailability of carotenoids (46), much
work remains to be done to identify the main factors among all those
proposed. The mechanisms that might explain the low and variable
absorption of carotenoids are largely unknown, because the fate of
these micronutrients in the human upper gastrointestinal (GI) tract is
still obscure. Three assumptions are currently made about carotenoid
metabolism in the lumen of the human upper GI tract. First, carotenoids
cannot be absorbed while they remain embedded in their original
vegetable matrices. Second, carotenoids have to be solubilized in mixed micelles to be absorbed. Third, carotenoids are absorbed by passive diffusion [as suggested for
-carotene (17)]. These
assumptions are not sufficient to understand the factors limiting the
absorption of natural carotenoids. Further data are required on the
role of the stomach in carotenoid absorption, possible
cis-trans isomerization of carotenoids in the acidic
environment of the stomach, the extent of release of carotenoids from
their vegetable matrices, and the relative availabilities for
absorption of cis and trans isomers of carotenoids.
The aim of this study was to ascertain the fate of the main natural
human dietary carotenoids, i.e., -carotene, lycopene, and lutein in
their natural vegetable matrices, in the lumen of the human upper GI
tract, and to determine whether this fate affects their
bioavailability. For this purpose, we adapted a model, previously used
by us to study lipids (1) and vitamins A and E
(5) metabolism in the human GI tract, to study the
metabolism of carotenoids supplied in their natural vegetable matrices.
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MATERIALS AND METHODS |
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Subjects. Ten healthy male volunteers (24.2 ± 1.0 yr, 1.80 ± 0.02 m, and 74.7 ± 3.5 kg) were enrolled in the study after giving their written, informed consent. The study was approved by the Regional Ethical Committee on Human Experimentation of Auvergne (France). No volunteer had any history of GI disease or lipid metabolic disorders according to clinical examination, disease history, and fasting plasma lipid parameters [plasma total triacylglycerols (0.53 ± 0.03 mM) and plasma total cholesterol (3.80 ± 0.24 mM) concentrations were in the normal range]. No volunteer was taking any drugs known to affect GI function or lipid metabolism.
The subjects' usual diet was monitored by means of a 5-day food recall. This dietary recall was analyzed for nutrient composition using diet analysis software (GENI; Micro 6, Nancy, France) completed for carotenoids by a carotenoid food-composition database (6). The subjects consumed a typical Western diet, with 11.88 ± 0.90 MJ/day, 14.52 ± 0.78% of energy as proteins, 33.82 ± 1.90% as fat, 48.03 ± 2.58% as carbohydrates, and 3.63 ± 1.45% as alcohol. Carotenoid intake was as follows (mg/day):Test meals.
Each subject was given three test meals differing only in the vegetable
added, 3 wk apart and in a random order. The test meal compositions are
given in Table 1. Note that vegetables were the sole source of carotenoids. Tomato purée was used as the
source of lycopene, carrot purée as the source of -carotene, and chopped spinach as the source of lutein.
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Study design. Each procedure started at 07:30 AM after the subjects had fasted overnight for 12 h. As previously described (1, 5), each subject was intubated with a single-lumen nasogastric tube (16 Fr, 122 cm, Sherwood Medical Argyle, Tullamore, Ireland) and a single-lumen nasoduodenal tube (duodenographie bilboa-dotter set, 12 Fr, 145 cm, William Cook Europe, Bjaeverkov, Denmark). The stomach tube was located 45-50 cm from the nose in the stomach at the corpus-antrum junction. The duodenal tube was located at the junction of the second and the third portions of the duodenum. The position of the tubes was checked radiographically before meal intake. After the tubes were fitted, the volunteers adopted a sitting position that they maintained until the end of the study to limit variations in gastric emptying rates (19). An antecubital vein was catheterized with an intravenous cannula equipped with disposable obturators (Terumo Europe, Leuven, Belgium).
Fasting stomach juice and duodenal fluid were removed by manual aspiration just before ingestion of the liquid test meal. The 600-ml liquid test meal was fed intragastrically by using a 60 ml-syringe over a 20-min period. A 1-ml sample of the initial liquid test meal was kept for measuring emulsion droplet size. Large samples (100-200 ml) of the stomach contents were aspirated at 20, 40, 60, 90, 120, 150, and 180 min after meal intake by gentle aspiration with a 60-ml syringe to obtain representative samples. A 20-ml aliquot was taken from each sample for analytical determinations. The remaining sample was promptly reinjected into the subject's stomach via the nasogastric tube. Duodenal contents (5-10 ml) were aspirated at the same postprandial times as above by using the nasoduodenal tube. Portions (2 ml) of the stomach content sample and portions (0.5 ml) of the duodenal content sample were placed in glass tubes containing double volumes of chloroform/methanol [2:1 (vol/vol)] to stop lipolysis and subsequently analyze lipids. Portions (0.5 ml) of the sample were placed in tubes and stored at 4°C until the size of emulsion lipid droplets was determined (see Size of emulsified lipid droplets). The remaining fractions of the samples were placed in another tube and stored atSize of emulsified lipid droplets. The median size of emulsion lipid droplets was determined in the initial formulas and in the stomach and duodenal aspirates (just after collection) by using a particle size analyzer (Coulter LS 130, Coultronics, Margency, France). Mean sizes of emulsion lipid droplets were calculated by doing the mean of median size obtained after at least five measurements. In preliminary experiments, vegetable particles had been shown to affect the measurements, so they were discarded beforehand by centrifuging at 1,000 g for 2 min at 10°C.
Separation of fat and aqueous phases from the vegetable particles present in the GI content samples. To assess the transfer of carotenoids from the vegetable matrices to the fat and aqueous phases present in the GI contents, we separated these two phases from the vegetable matrices. For the stomach content samples, the protocol was as follows: 7 ml of stomach contents were placed in polyallomer tubes and centrifuged (2,000 g, 10 min, at 4°C). An aliquot (a few milligrams) of the floating oil phase, when present, was collected to measure carotenoid solubilized into the fat phase of the stomach. An aliquot of the infranatant was filtered through a sintered glass filter tube (100 mm high, 20 mm in diameter, 40- to 100-µm pore size) (Prolabo, Fontenay-sous-Bois, France) to discard large (>40 µm) particles of vegetable matrix. Liposomes and other aqueous soluble structures (phospholipid vesicles, proteins), which can potentially solubilize carotenoids in the aqueous phase of the stomach, were separated from small (<40 µm) fat globules and small vegetable particles (<40 µm) by ultracentrifuge (200,000 g, 335 min, 10°C, in a Kontron TST 41.14 swinging bucket rotor) followed by filtration of the infranatant through a 0.2-µm cellulose acetate filter (Schleicher & Schuell, Prolabo, Fontenay-sous-Bois, France).
For the duodenal samples, the protocol was as follows: 4 ml duodenal content were added with 7 ml cold (4°C) distilled water and placed in polyallomer tubes. The tubes were ultracentrifuged (same conditions as above) to float large lipid droplets and pellet vegetable particles. We noted that, in most of the duodenal samples, no floating oil layer was observed after the centrifugation. An aliquot of the infranatant was collected and filtered through a 0.2-µm cellulose acetate filter to discard small fat globules and vegetable particles and measure carotenoids in micelles. Control optical microscopy showed there were no oil droplets or vegetable particles in the aqueous phase obtained after this filtration.Lipid analysis. Lipids from stomach and duodenal samples were extracted in chloroform/methanol [2:1 (vol/vol)] (13). The lower chloroform phases were evaporated to dryness under nitrogen. Total lipids were determined gravimetrically. The extent of intragastric and intraduodenal triacylglycerol lipolysis was assessed at 40 min after meal intake in the stomach and at 1 h after meal intake in the duodenum. For that purpose triacylglycerols were separated from other lipid classes (diacylglycerols, monoacylglycerols, free fatty acids, free cholesterol, and esterified cholesterol) by two-stage, one-dimensional thin-layer chromatography as described previously (1). They were quantified by densitometry by using a video-densitometry system and the Biolab software package (Visiosoft LND-CNRS patent, Marseille, France).
Chylomicron triacylglycerols were assayed by using an enzymatic colorimetric method with a commercial kit (Biotrol Diagnostic, Chennevières-lès-Louvres, France). The concentrations were measured spectrophotometrically at 490 nm by using an MR 700 microplate reader (Dsynatech Laboratories, Guernsey, UK).Carotenoid analyses. A procedure to accurately extract carotenoids from the three different vegetable matrices and the stomach and duodenal samples was drawn up after preliminary experiments. Several combinations of solvents were tried, with the best combination being the one that gave the whitest vegetable matrix after extraction. The procedure, which was performed under yellow light, was as follows: 1-ml test meal or 2-ml stomach samples or 1-ml duodenal samples were added with 7 ml methanol containing 0.57% MgCO3 (Sigma, Saint Louis, MO) and 0.2 µg/ml internal standard (echinenone, Roche Vitamines France, Neuilly-sur-Seine, France). After homogenization for 30 s with a vortex blender, 7 ml chloroform (containing 0.005% butylated hydroxytoluene as an antioxidant) were added. The sample was homogenized again for 30 s with the vortex blender. After 15 min rest, 7 ml distilled water were added. After being centrifuged (2,000 g, 10 min, room temperature), the lower phase containing most of the carotenoids was collected. Carotenoids remaining in the upper phase were extracted as follows: 5 ml tetrahydrofuran was added, the mixture was then vortexed for 30 s, and dichloromethane (5 ml) was added. It was then vortexed for 30 s, distilled water (3 ml) was added, and it was vortexed again for 30 s. After being centrifuged (2,000 g, 10 min, room temperature), the lower phase was collected and pooled with the previously collected phase. After evaporation to dryness under nitrogen, the dried extract was dissolved in either 200 µl acetonitrile/dichloromethane [50:50 (vol/vol)] for the stomach and test meal samples or 200 µl methanol/dichloromethane [65:35 (vol/vol)] for the duodenal samples. Chylomicron carotenoids were extracted by using a previously published method, i.e., twice with ethanol and hexane (29).
Carotenoids were quantified by reverse-phase HPLC on a Waters system (Waters, Saint-Quentin-en-Yvelines, France). This system was composed of a Waters 660 pump, a Waters 717 plus cooled auto-sampler, and a Waters 996 ultraviolet (UV)-visible diode-array detector. Carotenoids were separated by using two columns fitted in series (41): a 150 × 4.6-nm RP C18, 3-µm Nucleosyl (Interchim, Montluçon, France) coupled with a 250 × 4.6-nm RP C18, 5-µm Vydac TP54 (Hesperia, CA). The mobile phase was an isocratic acetonitrile-dichloromethane-methanol (containing 50 mM ammonium acetate)-water mixture (70:10:15:5, by vol). Carotenoids were detected at 450 nm and identified by retention time and spectral analysis (from 300 to 550 nm) compared with pure (>95%) standards of the following carotenoids: lutein, echinenone, all-trans lycopene,Calculations and Statistical Analysis
The proportions of carotenoids remaining in the vegetable matrices in the stomach and duodenal contents were calculated by subtracting the carotenoids recovered in the lipid phase and in the aqueous phase from the total carotenoids measured in the samples. The extent of intragastric and intraduodenal triacylglycerol lipolysis was calculated as the percentage of triacylglycerols disappearing from the total acylglycerols present (triacylglycerols + diacylglycerols + monoacylglycerols). The area under the curves (AUCs) of the postprandial chylomicron responses (delta from fasting values) were calculated by the trapezoidal rule.Subject characteritics and results are expressed as means ± SE.
Data were tested for normality (Kolmogorov-Smirnov) before using
parametric tests. Changes in measured parameters were analyzed by
ANOVA. Two-factor ANOVA, with time and meal as factors, was used to
study variations in stomach and duodenal pH during digestion. Variations in cis -carotene and lycopene ratio during
digestion were assessed by using two-factor ANOVA, with time and organ
as factors. Proportions of carotenoids in micellar phase were compared by using two-factor ANOVA, with time and carotenoid type as factors. Variations in postprandial chylomicron carotenoid concentrations were
assessed by ANOVA with repeated measures, with time as a factor. When
significant (P < 0.05) differences were detected, means
were compared between each other by using the post hoc Tukey/Kramer's test. Pearson correlation coefficients were obtained from linear regression analyses. Statistics were performed using StatView software
version 5.0 (SAS Institute, Cary, NC).
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RESULTS |
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Stomach and Duodenum pH During Digestion
Figure 1 shows variations in pH in stomach and duodenum contents after the intake of tomato, carrot, and spinach meals. As shown in Fig. 1A, the stomach pH, which was ~1.8 in the fasting state, sharply increased to 5.4-6.2 after meal intake, then continuously decreased to reach 1.8-2.9 after 3 h digestion. A two-factor ANOVA showed there was no meal effect but a time effect on postprandial stomach pH. The duodenal pH (Fig. 1B), which was ~5 in the fasting state, increased to ~6.1-6.6 after meal intake, and remained constant during digestion. Two-factor ANOVA showed a meal effect and no time effect on postprandial duodenal pH. More precisely, the mean postprandial duodenal pH was significantly lower after the tomato meal (5.8 ± 0.1) than after the other two meals (6.2 ± 0.1 and 6.4 ± 0.1 for the spinach and the carrot meal, respectively).
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Size of Emulsion Lipid Droplets in Test Meals and in Stomach and Duodenal Contents During Digestion
In the test meals and in the stomach, lipid droplets exhibited monophasic distributions with bell-shaped curves (data not shown in the figures). In the duodenum, small droplets appeared (size 0.1-1 µm) resulting in biphasic distributions. The mean diameter of lipid droplets found in the carrot, tomato, and spinach meals was significantly different: 10.4 ± 0.4, 2.7 ± 0.2, and 8.1 ± 0.8 µm, respectively. The mean diameter of lipid droplets found in the stomach after the carrot meal (mean of 3 measurements made at each collection time) was significantly higher than that measured after the tomato and the spinach meals: 15.1 ± 0.7 vs. 6.1 ± 0.9 and 7.9 ± 0.5 µm, respectively. The mean diameters of lipid droplets found in the duodenum after the carrot, the tomato, and the spinach test meals were 11.6 ± 1.6, 5.2 ± 0.7, and 4.8 ± 0.6 µm, respectively.Triacylglycerol Lipolysis
The relative amount of triacylglycerols present in the gastric aspirates at 40 min decreased by 12.6 ± 1.8, 14.3 ± 2.5, and 21.8 ± 3.9% for the carrot, tomato, and spinach meal, respectively. In the 1-h duodenal contents, triacylglycerol disappearance was more marked: 48.9 ± 4.7, 56.7 ± 4.2, and 47.0 ± 9.2% for the carrot, tomato, and spinach meal, respectively.Cartenoids in Fasting Stomach and Duodenal Contents
After fasting, no carotenoids were detectable in the samples of stomach contents. Thus assuming a limited detection threshold of 2 ng carotenoid per sample and given that the measurements were made on 2-ml stomach samples, we can assert that the concentration of carotenoids in the fasting stomach was lower than 2 nM. Conversely, carotenoids were detected in the fasting duodenum samples. Specifically, there were detectable amounts of lutein and all-transConcentration of Carotenoids in Stomach and Duodenum During Digestion
Figure 2 shows that the concentration of carotenoids in the stomach gradually decreased during digestion. Conversely, it ranged between 5 and 18 µM in the duodenum and did not significantly vary during digestion. Stomach lutein concentration was significantly lower than
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Proportion of cis Isomers of -Carotene and Lycopene
in Stomach and Duodenum During Digestion
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Distribution of Carotenoids in Different Phases Present in Stomach During Digestion
The distribution of all-trans
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Proportion of Carotenoids in Aqueous (Micellar) Phase Present in Duodenum
Only a very small proportion (between 1.8 and 6.9%) of all-trans carotenoids was recovered in the aqueous phase of the duodenum (Fig. 5). There was an effect of carotenoid species on the proportion of carotenoid found in the aqueous phase. More precisely, the 20- to 150-min mean proportions of lutein and all-trans
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Figure 6 shows that the 20- to 150-min
mean proportions of 13-cis -carotene recovered in the
aqueous phase of the duodenum were significantly (P < 0.001) higher than that of all-trans
-carotene: 14.8 ± 1.6 vs. 4.7 ± 0.3%. The results of lycopene cis
isomers are not presented here owing to the very high variability of
the data obtained.
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Postprandial Chylomicron Lipid and Carotenoid Responses to Test Meals
The postprandial chylomicron triacylglycerol responses, as estimated by 0-8 h AUC, were not significantly different among the three test meals: 0.58 ± 0.10, 0.60 ± 0.09, and 0.54 ± 0.10 mM/h for the tomato, carrot, and spinach meal, respectively (data not shown in the figures). For carotenoids, the first observation was that, although there was detectable all-trans lycopene in the chylomicron fraction, there was no significant variation in the postprandial all-trans lycopene response to the tomato test meal (as assessed by ANOVA with repeated measures, with time as a factor; Fig. 7). In contrast, there was an effect of time on the all-trans
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DISCUSSION |
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The aim of this study was to obtain basic data on the fate of
carotenoids in the human upper GI tract. Specifically, we wanted to
determine 1) whether there is a
trans-to-cis isomerization of -carotene and
lycopene in the acidic environment of the stomach, 2)
whether the stomach plays a role in the bioavailability of carotenoids,
3) how efficient the transfer of carotenoids from their
vegetable matrix to micelles is, and 4) whether
cis and trans isomers of carotenoids have
different metabolisms in the gut lumen. To answer these questions, we
followed the processing of carotenoids in the human upper GI tract by
collecting chyme samples during the digestion of liquid test meals.
This approach has already been used to study the metabolism of fat
soluble vitamins in the human upper GI tract (5).
The first observation concerns the concentration of carotenoids in the
stomach and duodenum during digestion. Curves exhibited by -carotene
and lycopene in the stomach were expected, with a gradual decrease in
carotenoid concentration explained by the gastric emptying of the
meals. The curve of lutein was significantly lower. It is difficult to
explain such a difference, because the same amount of the main
carotenoids was provided in each meal. The most likely hypothesis is
that, although large samples of stomach content were collected to have
a representative sample, the distribution of the vegetable matrix,
i.e., chopped spinach leaves, was inhomogeneous in the stomach leading
to an underestimation of lutein concentration. The relatively constant
concentration of carotenoids in the duodenum can be explained by an
equilibrium between the entry of carotenoids coming from the stomach
and the exit of carotenoids either absorbed or transfered in the lower parts of the intestine.
Given that isomerization of trans carotenoids to
cis carotenoids is promoted by contact with acids
(36), our first goal was to assess whether there was any
isomerization of carotenoids in the acidic environment of the stomach.
The lack of any significant increase in the cis-trans
-carotene and cis-trans lycopene ratios in the stomach
during digestion led to the conclusion that there was no significant
cis-trans isomerization of these carotenoids in the human
stomach. This result seems at variance with a recent result obtained in
vitro (34), which showed an effect of acidic pH on
lycopene isomerization. However, in that study, the isomerization was
observed at the very low pH of 1.6, a value found only in the fasting
stomach and not during the digestion of the complex lycopene-rich meal
used in this study (see Fig. 1).
The fact that the cis-trans -carotene and lycopene ratios
were significantly higher in the duodenum than in the stomach was noteworthy. A first hypothesis is that there was a lower
bioaccessibility (defined as the ease with which the carotenoids are
solubilized within the mixed micelles from the vegetable matrix) of the
cis isomers compared with the trans isomers,
resulting in a lower absorption efficiency for the cis
isomers. However, this is unlikely because there was a higher
proportion of 13-cis
-carotene than of
all-trans
-carotene in the aqueous phase of the duodenum
(Fig. 6), suggesting a better bioaccessibility of the cis
isomers, and it has been shown that, when artificially incorporated in
micelles, both isomers are absorbed with the same efficiency. A second
hypothesis is that cis isomers were secreted in the bile,
resulting in an increased cis-trans ratio in the duodenum.
However, although we detected all-trans
-carotene and
lutein in the fasting duodenal samples, in agreement with previous
results (24), we did not detect significant amounts of
carotenoid cis isomers in the fasting samples of the
duodenum. A third hypothesis is that there was a
cis-to-trans isomerization of carotenoids in the
duodenum, but we have no evidence of this.
Because the carotenoids were initially present only in the vegetable
matrices, their presence in the fat phase of the stomach content must
have resulted from their transfer from the vegetable matrices to this
phase. Although such a transfer has been described in vitro
(35), this is the first time that it has been measured in
humans. The data collected show that the different carotenoids were
transferred with different efficiencies. This may be explained in
several ways. The in vitro results (35) suggest that there was an effect of pH on the transfer efficiency. However, this hypothesis can be rejected, because there was no significant meal effect on stomach pH. A second hypothesis is that there was a matrix
effect, i.e., that the different vegetable matrices (carrot, spinach,
and tomato) have different abilities to release carotenoids (due to
different fiber composition or different intracellular locations of
carotenoids). This second hypothesis can also be rejected because the
kinetics of transfer of -carotene from spinach (data not shown in
the figures) mimicked that of
-carotene from carrots and was very
different from that of lutein from spinach. A third hypothesis is that
there was an effect of triacylglycerol lipolysis on the transfer.
However, although there was a significant matrix effect on gastric
triacylglycerol lipolysis, there was no relationship between the
proportion of lipids remaining as triacylglycerols, which reflects
triacylglycerol lipolysis efficiency, and transfer efficiency. A fourth
hypothesis is that the transfer of carotenoids depends on their
relative solubility in the fat phase. This hypothesis seems plausible
because there was a strongly positive relationship (r = 0.99, P = 0.01) between carotenoid solubility in
tri-C18 triacylglycerols (3) and the proportion of
carotenoids in the fat phase at 180 min digestion. The last hypothesis
is that the size of the lipid droplets, and thus the area for exchange,
affected the transfer efficiency. The positive relationship
(r = 0.98, P = 0.08) between the mean
diameter of emulsions recovered in the stomach and the transfer
efficiency at 180 min suggested that some parameter related to the
quality of the interface might also be involved in the transfer.
Whatever the mechanism involved, these data suggest that the stomach
plays an important role in the bioaccessibility of carotenoids by
initiating their release from their vegetable matrices.
Because it is assumed that carotenoids are absorbed only when they are
solubilized in micelles, the very low percentage of carotenoids
recovered in the aqueous (micellar) phase of the duodenum suggests that
only a very low proportion of carotenoids, supplied in their natural
vegetable matrix, is available for absorption. This result is in
remarkable agreement with a recent study performed in CaCo-2
(10). Indeed it was shown that the extent of absorption of
carotenoids was comprised of between 11% for all-trans
-carotene and 2.5% for all-trans lycopene. It is also in
agreement with a study that estimated absorption efficiency from
chylomicron response (32). With this method, it was
estimated that 1.4 mg of 40 mg
-carotene (3.5%) and 1 mg of 40 mg
lycopene (2.5%) were absorbed in humans, values close to the
solubility values measured here, i.e., 4.8 and 2.0%. However, note
that these solubility values would have been probably different if
purified carotenoids were used. Indeed, it is well known that
carotenoids compete for absorption (42, 44) and the
vegetables ingested contained several carotenoids (see MATERIALS
AND METHODS). However, the aim of this study was to provide basic
data on the digestion of vegetable-borne carotenoids, which are mostly
recovered with other carotenoids species in vegetables.
The fact that the percentage of all-trans lycopene recovered
in the micellar phase of the duodenum was significantly lower than that
of all-trans -carotene and lutein was noteworthy. It is
in remarkable agreement with a recent result that showed that the
extent of absorption of
-carotene,
-carotene, and lutein by
CaCo-2 cells was higher than that of lycopene (10). It
suggests that the availability for absorption of lycopene is less
efficient than that of the other carotenoids. The question arises
whether this difference depends on a vegetable matrix effect or on some other effect. Although it was not possible to accurately measure the
proportion of
-carotene in micelles after the tomato meal (due to
the very low amount of
-carotene provided by this meal, i.e., ~1
mg), the fact that the proportion of
-carotene from spinach was
similar to that of
-carotene from carrot (data not shown in the
figures) suggests that the efficiency of transfer to micelles does not
depend on vegetable matrix characteristics. Because carotenoids are
assumed to be entrapped in the fat phase of dietary emulsions and
because it has been suggested that triacylglycerol lipolysis enhances
the release of the carotenes (3, 43), we suggested that
the efficiency of triacylglycerol lipolysis affects the carotenoid
transfer to micelles. This hypothesis seemed valid, because there was a
strong negative relationship (r =
0.998, P = 0.036) between the concentration of
triacylglycerols in the duodenum, which reflects lipolysis, and the
percentage of carotenoids found in the micellar phase at 1 h of
digestion. The lowest solubility of lycopene in the micellar phase may
result from the lower pH observed in the duodenum during the digestion
of the tomato meal (Fig. 1). It has recently been shown that the
efficiency of carotenoid transfer from emulsion lipid droplets to
micelles diminishes as the pH decreases (43). It may also
result from its higher hydrophobicity. It is remarkable that the least
hydrophobic carotenoid (7), lutein, had the highest
solubility in micelles, whereas the most hydrophobic carotenoid,
lycopene, had the lowest solubility. This hypothesis is in agreement
with previous results (14, 45) that found a higher
relative bioavailability of lutein compared with
-carotene.
The higher proportion of 13-cis -carotene in the micellar
phase may be due to either a higher micelle transfer efficiency, a
lower absorption efficiency of micellar 13-cis
-carotene,
or both. This hypothesis is supported by the fact that the absorption efficiency of 13-cis
-carotene is significantly lower
than that of the all-trans isomer (10) and by
the fact (27) that the cis isomer is more
efficiently transferred to micelles than the all-trans
isomer. The reason for this is unknown but could be due to either a
different partitioning of the two isomers between the core and the
surface of emulsion lipid droplets (3) or different
solubilities of the two isomers in micelles. Finally, the fact that the
13-cis-to-all-trans
-carotene ratio measured in the micellar phase (13.3) was very close to the
13-cis-to-all-trans
-carotene ratio measured
in the chylomicrons (11.5) suggests that 13-cis
-carotene
does not undergo the cis-to-trans isomerization previously demonstrated for 9-cis
-carotene
(48) in the enterocyte.
Before discussion on the postprandial chylomicron carotenoid responses,
it should be stated that these responses could not be compared in
intensity with those obtained in other studies with similar doses of
lipids and carotenoids, because an important proportion of lipids and
carotenoids was taken in the gut during digestion for the different
analysis. The fact that chylomicron all-trans lycopene did
not significantly vary in the postprandial period, whereas
all-trans -carotene and lutein did, is in remarkable agreement with the data obtained in the duodenum, with a lower solubility of lycopene in micelles compared with the two other carotenoids. The fact that cis isomers of lycopene were
hardly detected in chylomicrons can be explained by a low absorption efficiency, an isomerization to the trans isomer in the
enterocyte, or a specific transport via the portal pathway. The first
hypothesis can reasonably be rejected, because it has been suggested
that cis isomers of lycopene artificially incorporated in
micelles are more bioavailable than all-trans isomers of
lycopene (2). The fact that cis isomers of
lycopene are detected in human plasma and tissues (18, 38,
40), accounting for >50% total plasma lycopene
(38), plus the fact that our data suggest that they are
poorly transported by the chylomicrons and thus poorly absorbed strongly suggests that a trans-to-cis
isomerization of this carotenoid, possibly due to antioxidant reactions
of lycopene (30), occurs in the body at a postenterocyte level.
In conclusion, this study provides some new data on the fate of
carotenoids in the human upper GI tract. The main findings are that
1) there is no significant cis-trans
isomerization of -carotene and lycopene in the human stomach,
2) the stomach plays a significant role in the
bioavailability of carotenoids by initiating their transfer from the
vegetable matrix to the fat phase of the meal, 3) the
proportion of carotenoids recovered in the micellar phase of the
duodenum is very low (<7%), which probably explains the poor
bioavailability of these phytomicroconstituents, 4)
13-cis
-carotene is more fully solubilized in micelles
than the all-trans isomer, explaining its higher
bioavailability, and 5) cis isomers of lycopene
are sparingly secreted in the chylomicrons, suggesting a postenterocyte
origin for these isomers in the body.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. S. Southon (Institute of Food Research, Norwich, UK) for the excellent coordination of this European project, Dr. R. Faulk (Institute of Food Research) for advice on carotenoid extraction from vegetables, C. G. Rodenas (Nutrition Department, Nestlé Research Center, Lausanne, Switzerland) for a gift of whey proteins, Dr. H. van Amelsvoort (Unilever Health Institute, Unilever Research, Vlaardingen, Netherlands) for a gift of stripped sunflowerseed oil, L. Morin and P. Rousset for help in blood collection, and Marion Brandolini for the analysis of the diet diaries.
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FOOTNOTES |
---|
This research was supported by the European Union FAIR project CT97-3100.
Address for reprint requests and other correspondence: P. Borel, INSERM U476, Faculté de Médecine, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 5, France (E-mail: Patrick.Borel{at}medecine.univ-mrs.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 10, 2003;10.1152/ajpgi.00410.2002
Received 24 September 2002; accepted in final form 27 December 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Armand, M,
Borel P,
Pasquier B,
Dubois C,
Senft M,
Andre M,
Peyrot J,
Salducci J,
and
Lairon D.
Physicochemical characteristics of emulsions during fat digestion in human stomach and duodenum.
Am J Physiol Gastrointest Liver Physiol
271:
G172-G183,
1996
2.
Boileau, AC,
Merchen NR,
Wasson K,
Atkinson CA,
and
Erdman JW, Jr.
cis-Lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymph-cannulated ferrets.
J Nutr
129:
1176-1181,
1999
3.
Borel, P,
Grolier P,
Armand M,
Partier A,
Lafont H,
Lairon D,
and
Azais-Braesco V.
Carotenoids in biological emulsions: solubility, surface-to-core distribution, and release from lipid droplets.
J Lipid Res
37:
250-261,
1996[Abstract].
4.
Borel, P,
Grolier P,
Mekki N,
Boirie Y,
Rochette Y,
Le Roy B,
Alexandre-Gouabau MC,
Lairon D,
and
Azais-Braesco V.
Low and high responders to pharmacological doses of beta-carotene: proportion in the population, mechanisms involved and consequences on beta-carotene metabolism.
J Lipid Res
39:
2250-2260,
1998
5.
Borel, P,
Pasquier B,
Armand M,
Tyssandier V,
Grolier P,
Alexandre-Gouabau MC,
Andre M,
Senft M,
Peyrot J,
Jaussan V,
Lairon D,
and
Azais-Braesco V.
Processing of vitamin A and E in the human gastrointestinal tract.
Am J Physiol Gastrointest Liver Physiol
280:
G95-G103,
2001
6.
Chug-Ahuja, JK,
Holden JM,
Forman MR,
Mangels AR,
Beecher GR,
and
Lanza E.
The development and application of a carotenoid database for fruits, vegetables, and selected multicomponent foods.
J Am Diet Assoc
93:
318-323,
1993[ISI][Medline].
7.
Cooper, DA,
Webb DR,
and
Peters JC.
Evaluation of the potential for olestra to affect the availability of dietary phytochemicals.
J Nutr
127, Suppl 8:
S1699-S1709,
1997[Medline].
8.
De Pee, S,
West CE,
Permaesih D,
Martuti S,
Muhilal,
and
Hautvast JG.
Orange fruit is more effective than are dark-green, leafy vegetables in increasing serum concentrations of retinol and beta-carotene in schoolchildren in Indonesia.
Am J Clin Nutr
68:
1058-1067,
1998[Abstract].
9.
Dubois, C,
Armand M,
Azais-Braesco V,
Portugal H,
Pauli AM,
Bernard PM,
Latge C,
Lafont H,
Borel P,
and
Lairon D.
Effects of moderate amounts of emulsified dietary fat on postprandial lipemia and lipoproteins in normolipidemic adults.
Am J Clin Nutr
60:
374-382,
1994[Abstract].
10.
During, A,
Hussain MM,
Morel DW,
and
Harrison EH.
Carotenoid uptake and secretion by CaCo-2 cells: beta-carotene isomer selectivity and carotenoid interactions.
J Lipid Res
43:
1086-1095,
2002
11.
Erdman, JW,
Poor CL,
and
Dietz JM.
Factors affecting the bioavailability of vitamin A, carotenoids, and vitamin E.
Food Technol
42:
214-221,
1988[ISI].
12.
Faulks, RM,
Hart DJ,
Wilson PD,
Scott KJ,
and
Southon S.
Absorption of all-trans and 9-cis beta-carotene in human ileostomy volunteers.
Clin Sci (Colch)
93:
585-591,
1997[Medline].
13.
Folch, J,
Lees M,
and
Sloane Stanley GH.
A simple method for the isolation and purification of total lipids from animal tissues.
J Biol Chem
226:
497-509,
1957
14.
Gartner, C,
Stahl W,
and
Sies H.
Preferential increase in chylomicron levels of the xanthophylls lutein and zeaxanthin compared to -carotene in the human.
Int J Vitam Nutr Res
66:
119-125,
1996[ISI][Medline].
15.
Gaziano, JM,
Johnson EJ,
Russell RM,
Manson JE,
Stampfer MJ,
Ridker PM,
Frei B,
Hennekens CH,
and
Krinsky NI.
Discrimination in absorption or transport of beta-carotene isomers after oral supplementation with either all-trans- or 9-cis-beta-carotene.
Am J Clin Nutr
61:
1248-1252,
1995[Abstract].
16.
Hieber, AD,
King TJ,
Morioka S,
Fukushima LH,
Franke AA,
and
Bertram JS.
Comparative effects of all-trans beta-carotene vs. 9-cis beta-carotene on carcinogen-induced neoplastic transformation and connexin 43 expression in murine 10T1/2 cells and on the differentiation of human keratinocytes.
Nutr Cancer
37:
234-244,
2000[ISI][Medline].
17.
Hollander, D,
and
Ruble PE.
-carotene intestinal absorption: bile, fatty acid, pH, and flow rate effects on transport.
Am J Physiol Endocrinol Metab Gastrointest Physiol
235:
E686-E691,
1978
18.
Holloway, DE,
Yang M,
Paganga G,
Rice-Evans CA,
and
Bramley PM.
Isomerization of dietary lycopene during assimilation and transport in plasma.
Free Radic Res
32:
93-102,
2000[ISI][Medline].
19.
Horowitz, M,
Jones K,
Edelbroek MA,
Smout AJ,
and
Read NW.
The effect of posture on gastric emptying and intragastric distribution of oil and aqueous meal components and appetite.
Gastroenterology
105:
382-390,
1993[ISI][Medline].
20.
Krinsky, NI.
Actions of carotenoids in biological systems.
Annu Rev Nutr
13:
561-587,
1993[ISI][Medline].
21.
Krinsky, NI.
Mechanism of action of biological antioxidants.
Proc Soc Exp Biol Med
200:
248-254,
1992[Abstract].
22.
Lampe, JW.
Health effects of vegetables and fruit: assessing mechanisms of action in human experimental studies.
Am J Clin Nutr
70 Suppl 3:
475S-490S,
1999
23.
Lavy, A,
Amotz AB,
and
Aviram M.
Preferential inhibition of LDL oxidation by the all-trans isomer of beta-carotene in comparison with 9-cis beta-carotene.
Eur J Clin Chem Clin Biochem
31:
83-90,
1993[ISI][Medline].
24.
Leo, MA,
Ahmed S,
Aleynik SI,
Siegel JH,
Kasmin F,
and
Lieber CS.
Carotenoids and tocopherols in various hepatobiliary conditions.
J Hepatol
23:
550-556,
1995[ISI][Medline].
25.
Lessin, WJ,
Catigani GL,
and
Schwartz SJ.
Quantification of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables.
Agr Food Chem
45:
3728-3732,
1997.
26.
Levin, G,
and
Mokady S.
Antioxidant activity of 9-cis compared to all-trans beta-carotene in vitro.
Free Radic Biol Med
17:
77-82,
1994[ISI][Medline].
27.
Levin, G,
and
Mokady S.
Incorporation of all-trans- or 9-cis-beta-carotene into mixed micelles in vitro.
Lipids
30:
177-179,
1995[ISI][Medline].
28.
Levin, G,
Yeshurun M,
and
Mokady S.
In vivo antiperoxidative effect of 9-cis beta-carotene compared with that of the all-trans isomer.
Nutr Cancer
27:
293-297,
1997[ISI][Medline].
29.
Lyan, B,
Azais-Braesco V,
Cardinault N,
Tyssandier V,
Borel P,
Alexandre-Gouabau MC,
and
Grolier P.
Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method.
J Chromatogr B Biomed Appl
751:
297-303,
2001[ISI].
30.
Muller, H,
Bub A,
Watzl B,
and
Rechkemmer G.
Plasma concentrations of carotenoids in healthy volunteers after intervention with carotenoid-rich foods.
Eur J Nutr
38:
35-44,
1999[ISI][Medline].
31.
Novotny, JA,
Dueker SR,
Zech LA,
and
Clifford AJ.
Compartmental analysis of the dynamics of beta-carotene metabolism in an adult volunteer.
J Lipid Res
36:
1825-1838,
1995[Abstract].
32.
O'Neill, ME,
and
Thurnham DI.
Intestinal absorption of -carotene, lycopene and lutein in men and women following a standard meal: response curves in the triacylglycerol-rich lipoprotein fraction.
Br J Nutr
79:
149-159,
1998[ISI][Medline].
33.
Parker, RS,
Swanson JE,
You CS,
Edwards AJ,
and
Huang T.
Bioavailability of carotenoids in human subjects.
Proc Nutr Soc
58:
155-162,
1999[ISI][Medline].
34.
Re, R,
Fraser PD,
Long M,
Bramley PM,
and
Rice-Evans C.
Isomerization of lycopene in the gastric milieu.
Biochem Biophys Res Commun
281:
576-581,
2001[ISI][Medline].
35.
Rich, GT,
Fillery-Travis A,
and
Parker ML.
Low pH enhances the transfer of carotene from carrot juice to olive oil.
Lipids
33:
985-992,
1998[ISI][Medline].
36.
Rodriguez-Amaya, DB.
Changes in carotenoids during processing and storage of foods.
Arch Latinoam Nutr
49, Suppl 3:
38S-47S,
1999[ISI][Medline].
37.
Shi, J,
and
Le Maguer M.
Lycopene in tomatoes: chemical and physical properties affected by food processing.
Crit Rev Biotechnol
20:
293-334,
2000[ISI][Medline].
38.
Stahl, W,
Schwarz W,
Sundquist AR,
and
Sies H.
cis-trans Isomers of lycopene and beta-carotene in human serum and tissues.
Arch Biochem Biophys
294:
173-177,
1992[ISI][Medline].
39.
Stahl, W,
Schwarz W,
Vonlaar J,
and
Sies H.
All-trans beta-carotene preferentially accumulates in human chylomicrons and very low density lipoproteins compared with the 9-cis geometrical isomer.
J Nutr
125:
2128-2133,
1995[ISI][Medline].
40.
Stahl, W,
and
Sies H.
Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans.
J Nutr
121:
2161-2166,
1992.
41.
Steghens, JP,
Lyan B,
Le Moel G,
Galabert C,
Fayol V,
Faure H,
Grolier P,
Cheribi N,
Dubois F,
and
Nabet F.
Measurement of carotenoids by high pressure liquid chromatography: from difficulties to solutions.
Ann Biol Clin (Paris)
58:
327-335,
2000[Medline].
42.
Tyssandier, V,
Cardinault N,
Caris-Veyrat C,
Amiot MJ,
Grolier P,
Bouteloup C,
Azais-Braesco V,
and
Borel P.
Vegetable-borne lutein, lycopene, and beta-carotene compete for incorporation into chylomicrons, with no adverse effect on the medium-term (3-wk) plasma status of carotenoids in humans.
Am J Clin Nutr
75:
526-534,
2002
43.
Tyssandier, V,
Lyan B,
and
Borel P.
Main factors governing the transfer of carotenoids from emulsion lipid droplets to micelles.
Biochim Biophys Acta
1533:
285-292,
2001[ISI][Medline].
44.
Van den Berg, H.
Carotenoid interactions.
Nutr Rev
57:
1-10,
1999[ISI][Medline].
45.
Van Het Hof, KH,
Brouwer IA,
West CE,
Haddeman E,
Steegers-Theunissen RP,
van Dusseldorp M,
Weststrate JA,
Eskes TK,
and
Hautvast JG.
Bioavailability of lutein from vegetables is 5 times higher than that of beta-carotene.
Am J Clin Nutr
70:
261-268,
1999
46.
Van Het Hof, KH,
West CE,
Weststrate JA,
and
Hautvast JG.
Dietary factors that affect the bioavailability of carotenoids.
J Nutr
130:
503-536,
2000
47.
Van Vliet, T,
Schreurs WH,
and
van den Berg H.
Intestinal beta-carotene absorption and cleavage in men: response of beta-carotene and retinyl esters in the triglyceride-rich lipoprotein fraction after a single oral dose of beta-carotene.
Am J Clin Nutr
62:
110-116,
1995[Abstract].
48.
You, CS,
Parker RS,
Goodman KJ,
Swanson JE,
and
Corso TN.
Evidence of cis-trans isomerization of 9-cis-beta-carotene during absorption in humans.
Am J Clin Nutr
64:
177-183,
1996[Abstract].