Digestive stability of xanthophylls exceeds that of carotenes as studied in a dynamic in vitro gastrointestinal system

The Journal of Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Digestive Stability of Xanthophylls Exceeds

That of Carotenes As Studied in a Dynamic in

Vitro Gastrointestinal System

1–3

Ste´phanie Blanquet-Diot,

* Maha Soufi,

Mathieu Rambeau,

Edmond Rock,

and Monique Alric

Universite´ Clermont-Ferrand I, Centre de Recherche en Nutrition Humaine Auvergne, Institut Fe´de´ratif de Recherche Sante´ 79, 63001 Clermont-Ferrand, France and5_{Unite´ de Nutrition Humaine, INRA Clermont-Ferrand/Theix, Centre de Recherche en}

Nutrition Humaine Auvergne, 63122 Saint-Gene`s Champanelle, France

Abstract

Epidemiological studies have suggested that high consumption of tomato products is associated with a lower risk for chronic diseases. To exert their health effect, the phytochemicals of tomatoes have to be bioavailable and therefore it implies their stability through the digestion process. Here, we assessed the digestive stability of the red-pigmented lycopene and other carotenoids brought in nutritional quantity within different food matrixes, using the TNO gastro-intestinal tract model (TIM). This multicompartmental dynamic system accurately reproduces the main parameters of gastric and small intestinal digestion in human. In vitro digestions of a standard meal containing red tomato (RT), yellow tomato (devoid of lycopene), or lycopene beadlets were performed. Zeaxanthin and lutein were stable throughout artificial digestions, whereas b-carotene and all-trans lycopene were degraded (;30 and 20% loss at the end of digestion, respectively) in the jejunal and ileal compartments. The recovery of b-carotene in the digesta of the RT meal was significantly lower than that in the yellow one, showing a food matrix effect. In the same way, until 180 min of digestion, the recovery percentages of all-trans lycopene from RT were significantly lower than those issued from the supplement. Isomeric conformation also influenced the stability of carotenoids, 5-cis lycopene being the most stable isomer followed by all-trans and 9-cis. No trans-cis isomerization of lycopene occurred in the TIM. By using a relevant dynamic in vitro system, this study allowed us to gain further insight into the parameters influencing the digestive stability of carotenoids, and therefore their bioavailability, in humans. J. Nutr. 139: 876–883, 2009.

Introduction

Numerous epidemiological studies have shown an association

between the consumption of tomatoes or tomato-based food

products and a reduction in the risk of several human chronic

diseases (1,2). Although many scientists have attributed the

protective effect of tomatoes to the red-pigmented lycopene,

other investigations have shown a higher in vivo effect of whole

tomato compared with lycopene alone (3,4). It is thought that

synergetic effects of this carotenoid with other phytochemicals

of tomato (e.g. other carotenoids, polyphenols, or vitamins C

and E) may enhance its benefit. A yellow tomato (YT)

variety

lacking lycopene could be a useful experimental model to

distinguish its effect from that of red tomato (RT), as suggested

by Gitenay et al. (5). By comparing the effects of red tomato, YT,

or lycopene from a supplement, Gitenay et al. clearly showed a

higher potential of tomatoes than lycopene alone to protect

against oxidative stress (5) or to module gene expression in a

prostate cancer cell line (6).

Bioavailability is a critical feature in the assessment of the role

of phytochemicals, such as carotenoids, in human health. In

humans, carotenoid bioavailability is often monitored by

fol-lowing changes in plasma concentrations after the ingestion of a

carotenoid-rich meal (7). However, this approach failed to

indicate the actual amounts that are accessible, absorbed, or

metabolized. Some animal models are also available (8) but they

are costly and limited in application. Alternatively, simple in vitro

digestion models have been developed for assessing carotenoid

bioaccessibility (9,10). They are increasingly being used to study,

with a high predictive value (16), preabsorptive processes and

food-related factors affecting carotenoid bioavailability (11–16).

In these in vitro studies, efficiency of micellarization, i.e. the

amount of carotenoids transferred from the digested food to the

Supported by the French Ministe`re de l’Enseignement Supe´rieur et de la Recherche.

2

Author disclosures: S. Blanquet-Diot, M. Soufi, M. Rambeau, E. Rock, and M. Alric, no conflicts of interest.

3

Supplemental Figure 1 is available with the online posting of this paper at jn.nutrition.org.

* To whom correspondence should be addressed. E-mail: stephanie.blanquet@ u-clermont1.fr.

6

Abbreviations used: LB, lycopene beadlet; RT, red tomato; TIM, TNO gastrointestinal tract model; YT, yellow tomato.

0022-3166/08 $8.00ª 2009 American Society for Nutrition.

876 Manuscript received December 16, 2008. Initial review completed January 16, 2009. Revision accepted February 9, 2009.

micellar fraction, is often used as an indicator of bioavailability.

Nevertheless, these systems are mono-compartmental and static

and are not representative of the continuously changing

varia-bles during passage through the stomach and the small intestine.

Moreover, they give little information about the digestive

stability of carotenoids, especially at the level where degradation

occurs throughout digestion, and about their sensitivity to the

different digestive secretions (HCl, enzymes, etc.).

The TNO gastrointestinal tract model (TIM) system is the in

vitro model that presently allows the closest simulation of in

vivo dynamic physiological processes occurring within the

lumen of the stomach and small intestine of humans (17). This

dynamic, computer-controlled system has been designed to

accept parameters and data from in vivo studies on human

volunteers. The main parameters of digestion, such as pH; body

temperature; peristaltic mixing and transport; gastric, biliary,

and pancreatic secretions; and passive absorption of small

mol-ecules and water are reproduced as accurately as possible. This

in vitro system offers accuracy, reproducibility, easy

manipula-tion, and the possibility of collecting samples at any level of the

digestive tract and at any time during digestion with no ethical

constraints. It has been validated by microbial (18),

pharma-ceutical (19,20), and nutritional (21,22) studies. The actual

limitations of the model include the absence of cellular system

and feedback mechanisms.

The aim of the present study was to assess the luminal

stability of lycopene and/or other carotenoids from RT, YT, or a

lycopene supplement subjected to digestion in the TIM system

within a standard meal. The effects of food matrix and digestive

secretions were of particular interest.

Materials and Methods

Chemicals and supplies. Lipase was purchased from Amano Phar-maceuticals. Unless otherwise stated, all other chemicals and supplies were purchased from Sigma-Aldrich or Fischer Scientific.

TABLE 1 Parameters of in vitro digestion in TIM when

simulating gastrointestinal conditions of healthy adult after intake of a solid meal1,2

Gastric compartment Initial volume 300 mL Time (min)/pH 0/6 15/5.7 45/4.5 90/2.9 120/2.3 240/1.8 300/1.6

Secretions 20.25 mL/min of pepsin (2072 kUI/L) 20.25 mL/min of lipase (250.5 kUI/L) 20.25 mL/min of HCl 1.5 mol/L if necessary t1/2 85 min

b coefficient 1.8 Duodenal compartment

Volume 30 mL pH Maintained at 6.4

Secretions 20.5 mL/min of bile salts (4% during the first 30 min of digestion then 2%) 20.25 mL/min of pancreatic juice (106_USP/L)

20.25 mL/min of intestinal electrolyte solution 20.25 mL/min of NaHCO31 mol/L if necessary

Jejunal compartment

Volume 130 mL pH Maintained at 6.9

Secretions 0.25 mL/min of NaHCO31 mol/L if necessary

Ileal compartment

Volume 135 mL pH Maintained at 7.2

Secretions 0.25 mL/min of NaHCO31 mol/L if necessary

t1/2 250 min

b coefficient 2.5

1_{Adapted from Souliman et al. (20).}

2_{Gastric and ileal deliveries are modeled with a power exponential formula: f}

¼ 1–22(t/t1/2)b_, where f represents the fraction of meal delivered, t the time of delivery, t1/2the half-time of delivery, and b a coefficient describing the shape of the curve (23).

TABLE 2 Carotenoid concentrations in test meals1

Carotenoid YT RT LB mg/kg diet b-Cryptoxanthin 0 0 0 b-13-cis carotene 0 0 0 Zeaxanthin 0 0.22 6 0.02 0 Lutein 0.27 6 0.01 0.21 6 0.02 0.11 6 0.04 b-Carotene 0.13 6 0.01 1.23 6 0.12 0 5-cis Lycopene 0 3.63 6 1.28 2.06 6 0.48 9-cis Lycopene 0 0.60 6 0.08 0.18 6 0.03 All-trans lycopene 0 31.06 6 2.71 16.41 6 0.55

1_{Values are means 6 SEM, n¼ 6.}

TABLE 3 Digestive stability of zeaxanthin and lutein in the

overall TIM1

Zeaxanthin Lutein

Time of digestion, min RT YT RT LB % 60 92 6 5 102 6 4 131 6 41 79 6 12 120 131 6 11 101 6 12 149 6 24 96 6 26 180 113 6 5 91 6 8 124 6 10 70 6 17 240 108 6 16 93 6 5 127 6 9 88 6 31 300 124 6 17 94 6 5 138 6 5* 87 6 28

1_{Values are means 6 SEM, n¼ 3. *Different from 0 min (T0), P , 0.05.}

FIGURE 1 Digestive stability of b-carotene from YT and RT meals in the overall TIM. Values are means 6 SEM, n¼ 3. 1Different from 0 min (T0), P , 0.05. *Different from YT, P , 0.05.

In vitro gastrointestinal system.The gastric-small intestinal system TIM consists of 4 successive compartments simulating the stomach and the 3 segments of the small intestine: the duodenum, jejunum, and ileum (Supplemental Fig. 1). This system has been described elsewhere (17). Briefly, each compartment was composed of glass units with a flexible inside wall. Peristaltic mixing and body temperature were achieved by pumping water at 37C into the space between the glass jacket and the flexible wall at regular intervals. Mathematical modeling of gastric and ileal deliveries with a power exponential formula (23) was used for the computer control of chyme transit. Chyme transport through TIM was regulated by the peristaltic valves that connect the successive compart-ments. The volume in each compartment was monitored by a pressure sensor. The pH was computer-monitored and continuously controlled by adding either HCl or NaHCO3. Simulated gastric, biliary, and

pancreatic secretions were introduced into the corresponding compart-ments by computer-controlled pumps. Water and products of digestion were removed from the jejunal and ileal compartments by pumping dialysis liquid through hollow fiber membranes (HG 600, Hospal Cobe).

Preparation of test meals for in vitro digestion.Three different meals were prepared extemporaneously and subjected to digestion into the TIM. The standard meal was designed to contain all the macronutrients present in a typical Western diet but without carotenoid [adapted from Tyssandier et al. (24)]. The meal consisted of 60 g of cooked egg white that was previously rinsed with water to completely remove egg yolk, 50 g of cooked pasta, 2.5 g of soy lecithin (Les 3 cheˆnes), and 20 g of sunflower oil. All the ingredients (except soy lecithin) were purchased from a local supermarket. A total of 100 g of double-concentrated puree of RT or YT (CTCPA) was added to the standard meal to constitute the RT and YT meals, respectively. The lycopene beadlet (LB) meal contained 78 mg of the hydrosoluble powder (LycoVit 10% DC, BASF). The powder particles (150–850 mm in size) consisted of lycopene embedded in a matrix of gelatin and sucrose coated with starch. The volume of the meals was adjusted to 300 mL with mineral water (Volvic) before homogenizing

20 min with an Ultra Turrax system (T25, IKA, Werke) set at 24,000 rpm.

Experimental design.The TIM system was programmed to reproduce the digestion of a solid meal in healthy human adults (Table 1). Because the current work focuses on the luminal stability of carotenoids, passive absorption of digestion products was not reproduced (the hollow fibers and the dialysis pump were disconnected). To prevent photodecompo-sition and oxidation of the carotenoids, all the digestive compartments and the collection vessels were recovered by tinfoil and maintained under nitrogen flow. The total duration of the digestion was 300 min (n ¼ 3 for each condition). Samples (1 mL) were taken from the meals before introduction into the artificial stomach and were regularly collected from each digestive compartment during digestion. The ileal deliveries were kept on ice and pooled at 0–60, 60–120, 120–180, 180–240, and 240– 300 min. The volumes were measured and aliquots were taken for each period. All samples were stored at 220C prior to analysis.

Extraction and HPLC analysis of carotenoids.Sample extraction was conducted under subdued (yellow) light, as previously described (24). Echinenone (Hoffmann-La Roche) was used as internal standard (0.36 mmol/L). Extracts were dried under nitrogen and dissolved in 200 mL methanol/dichloromethane (65:35, v:v) before injection into an HPLC apparatus. Carotenoids were analyzed by reverse-phase HPLC using a Waters system equipped with a cooled autosampler injector and a UV-visible diode array detector system. Separation was carried out using 2 columns in a series: a Nucleosil C18 (150 3 4.6 mm, 3 mm) followed by a Vydac TP54 (250 3 4.6 mm, 5 mm) purchased from Interchim. The mobile phase was an acetonitrile:methanol (containing 50 mmol/L ammonium acetate):dichloromethane:water mixture (70:15:10:5, v:v:v:v). The flow rate was isocratic (2 mL/min). Carotenoids were identified and quantified by comparing retention times, spectral profiles, and peak areas with pure standards (zeaxanthin, lutein, b-cryptoxanthin, b-carotene, b-13-cis caro-tene, and all-trans lycopene). Quantification of cis-lycopene was conducted using calibration curves constructed with all-trans lycopene.

FIGURE 2 Recovery profiles of b-carotene from YT and RT meals in the stomach (A), duodenum (B), jejunum (C), and ileum (D) of TIM. Values are means 6 SEM, n¼ 3. 1Different from the marker transit, P , 0.05. *Different from YT, P , 0.05. ND, Nondetectable.

Calculations and statistical analyses. The digestive stability of carotenoids was evaluated by calculating percentage recovery (carote-noids in digesta/carote(carote-noids in test meal 3 100).

The results were presented in 2 ways: 1) in the overall TIM, by summing the recovery percentages of carotenoids found in all the digestive compartments and in the ileal deliveries; and 2) in each digestive compartment by comparing the recovery profiles of the carot-enoids and that of a nonabsorbable marker, blue dextran, as previously described (17). Values are given as means 6 SEM (from 3 to 6 independent replicates). Significant differences between treatments were tested by ANOVA with repeated-measure analysis followed by a post hoc test based on a least square difference. The comparison between various times and the initial time (test meal) was made using paired t test. All analyses were performed using the SAS 9.1 software (SAS Institute). Differences of P , 0.05 were considered to be significant.

Results

Carotenoid composition of test meals.

The carotenoid

concentrations in test meals (YT, RT, and LB) are given in Table

2. Our HPLC method allowed the detection of the main dietary

carotenoids (25). b-Cryptoxanthin and b-13-cis carotene were

not found in any of the meals. The main carotenoid measured in

RT and LB meals was the all-trans lycopene (31.06 6 2.71 and

16.41 6 0.55 mg/kg diet, respectively). Two cis isomers of this

carotenoid were also detected and identified as 5-cis and 9-cis

lycopene by comparing their spectral characteristics with those

reported by other authors (26,27). Lycopene was not detected in

the YT meal. Small concentrations of lutein (ranging from 0.11

to 0.27 mg/kg diet) were found in all the tested meals.

Zea-xanthin was detected in a small quantity only in the RT meal.

b-Carotene was found in higher concentrations in the RT than in

the YT meal (1.23 vs. 0.13 mg/kg diet).

In vitro digestive stability of carotenoids.

Zeaxanthin and

lutein were stable during in vitro digestion (Table 3). In the RT

experiment, the high variability in their recoveries may be

ex-plained by the difficulty of separating lutein from zeaxanthin by

HPLC. The stability of b-carotene during digestion depended on

the tested meal (Fig. 1). b-Carotene from YT was stable during

in vitro digestion, whereas that from RT was degraded, with

a recovery percentage of 67.8 6 9.4% (n ¼ 3) at 300 min.

Throughout digestion, the recovery percentages of b-carotene

were lower for the RT meal than the YT meal (P , 0.05). The

recovery profiles of the carotenoid were compared with that

of the transit marker in each digestive compartment of TIM

(Fig. 2). In the RT experiment, the curves of b-carotene and that

of the marker did not differ in the stomach and duodenum,

showing that the degradation of the carotenoid occurred in only

the jejunum and ileum. In these compartments, the recovery

percentages of b-carotene from RT were lower than that from

YT (P , 0.05). All-trans lycopene was not stable during in vitro

digestions of RT or LB meals (Fig. 3A). After 300 min of

digestion, 83.0 6 2.4% and 80.6 6 4.3% (n ¼ 3) of initial

all-trans lycopene were recovered for RT and LB experiments,

respectively. From the beginning of the experiment to 180 min of

digestion, the recovery percentages of all-trans lycopene from

RT were lower than that from LB (P , 0.05). As for b-carotene,

all-trans lycopene was not degraded in the stomach and

duodenum of TIM, but it partly disappeared in the jejunum

and ileum (Fig. 4). The recoveries obtained for RT and LB meals

significantly differed in the duodenal and jejunal compartments.

The behaviors of 5-cis and 9-cis isomers of lycopene were

completely different in the simulated gastrointestinal conditions.

The results obtained with 5-cis lycopene were highly variable

and we did not find significant changes over time or differences

between the meals (Fig. 3B). This isomeric form of lycopene was

thus considered stable during in vitro digestion. On the contrary,

the percentages of 9-cis lycopene fell dramatically from 0 to 120

min of digestion (Fig. 3C). At the end of in vitro digestion, 33.3

6

13.9 and 60.6 6 11.0% (n ¼ 3) of initial 9-cis lycopene were

recovered in the LB and RT experiments, respectively. From 120

to 300 min digestion, the percent recovery of this carotenoid

tended to be lower for the LB than for the RT experiment (P ¼

0.19 at 300 min). 9-cis Lycopene from LB and RT was stable

under gastric conditions but was degraded in the small intestine

from the duodenal compartment (Fig. 5).

Isomerization of lycopene.

We performed further calculations

to follow the trans-cis isomerization of lycopene under the

sim-FIGURE 3 Digestive stability of all-trans (A), 5-cis (B), and 9-cis lycopene (C) from RT and LB meals in the overall TIM. Values are means 6 SEM, n ¼ 3. 1Different from 0 min (T0), P , 0.05. *Different from LB, P , 0.05.

ulated gastrointestinal conditions. In the RT meal, all-trans,

5-cis, and 9-cis isomers represented 88.2, 10.0, and 1.8% of the

total amount of lycopene measured, respectively. Similar

per-centages were found for the LB meal with 87.2, 11.8, and 1%,

respectively. In all the digestive compartments, the isomeric

distribution of lycopene did not differ regardless of the sampling

time and the tested meal (data not shown). Nevertheless, in the

gastric compartment during the RT experiment, the mean

percentages of all-trans lycopene tended to decrease (79.1% at

120 min), whereas those of 5-cis increased (18.8% at 120 min).

Discussion

Consumption of tomato and tomato-based food contributes to

the intake of a wide range of carotenoids, particularly the

well-known lycopene. To mediate their health effect, carotenoids

should be bioavailable, i.e. absorbed and delivered to the target

tissues for utilization or storage. The bioavailability of

carote-noids is extremely variable, being influenced by many dietary

and physiological factors (28). In vitro digestive systems have

been shown to be relevant tools to study the effects of physical

properties of carotenoids, such as trans compared with cis

isomers (14,15,29) or free compared with esterified forms (13);

food source and matrix (12,13,29,30); processing (10,31,32);

and interaction with other dietary compounds (10,33–35). The

systems that have been utilized to date are static and

mono-compartmental (9,10,36). Gastric and duodenal digestions are

successively simulated in a flask at 37C under shaking by

changing pH and adding the appropriate digestive secretions.

Until now, no data, to our knowledge, were available on the

stability of carotenoids in the different gastrointestinal

compart-ments and the influence of pH and digestive secretions in

dynamic conditions.

In the present study, we assessed the luminal stability of

lycopene and other carotenoids (zeaxanthin, lutein, and

b-caro-tene) in the TIM system. Contrary to previously mentioned

models, this model reproduces the main dynamic phenomena

occurring during gastrointestinal digestion in humans: transit of

chyme from one digestive compartment to another, evolution of

pH during gastric digestion and from the duodenum to ileum

(Table 1), and sequential arrival of digestive secretions. Lycopene

and the other carotenoids were supplied by LB, RT, or YT to

evaluate the effect of food matrix on their digestive stability. To

closely mimic real conditions, they were brought within a standard

meal, in a quantity similar to that found in human diet (37).

Zeaxanthin and lutein (regardless of the tested meal) were

stable during digestion in TIM (Table 3). No data are available

about in vitro digestive stability of zeaxanthin from tomato.

Nevertheless, high recovery percentages (from 80 to 100%)

were found for zeaxanthin in orange, spinach, or sweet corn

(12,13,29). Our results for lutein stability are consistent with

those of Granado-Lorencio et al. (12) who found a 100%

re-covery in tomato paste after simulated gastric or duodenal

digestion. This carotenoid seems to be highly resistant to

gas-trointestinal conditions whatever its food source, because

recovery percentages ranging from 80 to 100% were measured

in digesta of orange, spinach, or broccoli (11,12,29). b-Carotene

from YT was stable in the TIM system, whereas a 30% loss was

observed when this carotenoid was from RT (Fig. 1).

Granado-Lorencio et al. (12) found a recovery of b-carotene from tomato

paste of 80% after simulated gastric digestion and of almost

100% after the duodenal phase. This seems to indicate a

sen-sitivity of b-carotene to their gastric conditions (pepsin; pH 1.1;

FIGURE 4 Recovery profiles of all-trans lycopene from RT and LB meals in the stomach (A), duodenum (B), jejunum (C), and ileum (D) of TIM. Values are means 6 SEM, n¼ 3. 1Different from the marker transit, P , 0.05. *Different from LB, P , 0.05.

1 h) that was not found in the present study (Fig. 2A). This may

be explained by differences between our 2 experimental

proto-cols (static vs. dynamic conditions). As for the RT meal, previous

studies showed a degradation of b-carotene during simulated

digestion. Recovery percentages ranging from 50 to 85% were

found in digesta of carrot, spinach, broccoli, or mango (11,

12,29,35). In the present study, the stability of b-carotene

depended on the food matrix. The amount of b-carotene in the

tested meals (Table 2) may have influenced its stability during

digestion through the efficiency of micellarization, as previously

shown for lutein or zeaxanthin (38,39). Our results showed that

the levels of b-carotene from RT significantly decreased in

the lowest parts of the small intestine (Fig. 2C,D). It appears

that trans-cis isomerization of b-carotene does not occur

during digestion either in vitro (15,30) and in vivo (24,

40). Nevertheless, we suppose that nondetected metabolites/

degraded molecules of b-carotene were produced during

small intestinal digestion, but no precise data support our

hypothesis.

Lycopene is found in most food sources primarily as the

all-trans isomer. Similar isomeric distributions were found for

lycopene in LB and RT meals, with a predominance of the

all-trans isomeric form (87–88%), in accordance with previously

published data (27,41). Whatever the food matrix (RT or LB), all

trans-lycopene was significantly degraded during in vitro

diges-tion in TIM (Fig. 3A). Granado-Lorencio et al. (12) found a

recovery of lycopene from tomato paste of 45% and almost

80% after gastric and duodenal digestion, respectively. As

previously discussed for b-carotene, such a low recovery in the

gastric phase may be imputed to static conditions, because with

dynamic ones in TIM, there was no degradation in the artificial

stomach (Fig. 4A). Recovery percentages of all-trans lycopene

similar to that found here (;80%) were obtained by Failla et al.

(14) after gastrointestinal digestion of gac fruit cooked with rice.

Until 180 min of digestion, the recovery percentages of all-trans

lycopene from RT were lower (P , 0.05) than that from the

supplement. As expected, the matrix in which lycopene is

located in RT differs from that of the synthetic beadlets and has

affected lycopene stability during digestion. Similar in vitro

results were obtained by Chitchumroonchokchai et al. (29), who

showed that the digestive stability of lutein and zeaxanthin from

an oil-based supplement was greater than that from spinach. In

humans, lycopene from tomato oleoresin capsules is much more

available than that from raw tomatoes (42). Here, it is difficult

to conclude that there is a relative advantage of the supplement

over RT as a dietary source of lycopene, because at the end of

digestion in TIM, similar recovery percentages were found in

both cases. 5-cis Lycopene was stable during in vitro digestion

(Fig. 3B), whereas 9-cis lycopene was widely degraded (Fig. 3C).

Similar results were obtained by Chasse et al. (43) using ab initio

molecular modeling.

In our in vitro study, b-carotene and lycopene (belonging to the

carotene group) seemed to be more sensitive to digestive conditions

than lutein or zeaxanthin (xanthophylls). The hydrophobicity of

carotenoid may affect their luminal stability, as has already been

observed for their transfer from emulsion lipid droplets to micelles

(44). The difference in their localization in the lipid droplets (45)

may explain their digestive stability and therefore their availability,

as it has already been described in humans for b-carotene and

lutein (46,47). Our results also suggested that the lower plasma

status of lycopene compared with b-carotene in humans following

tomato product ingestion (48), especially in the elderly (49), may

result from postabsorptive events rather than from phenomena

occurring in the digestive lumen.

FIGURE 5 Recovery profiles of 9-cis lycopene from RT and LB meals in the stomach (A), duodenum (B), jejunum (C), and ileum (D) of TIM. Values are means 6 SEM, n¼ 3. 1Different from the marker transit, P , 0.05. *Different from LB, P , 0.05. ND, Nondetectable.

Although lycopene is present in its all-trans isomeric form

in fruits and vegetable, cis-isomers constitute the predominant

form in human serum and tissues (41). A trans-cis isomerization

has been suspected to occur during digestion (26). In our in vitro

conditions, we observed a disappearance of all-trans lycopene

in the lowest part of the small intestine (Fig. 4C,D) that could

not be directly linked with the formation of 5-cis or 9-cis

lyco-pene that was stable and degraded under digestive conditions,

respectively (Fig. 3). We then further analyzed the isomeric

distribution of lycopene throughout digestion and showed that

trans-cis isomerization occurred in none of the digestive

com-partments of TIM. Conflicting results have been obtained about

trans-cis isomerization of lycopene during digestion. In vitro

studies (26,50) have shown that this carotenoid was isomerized

as a result of low pH, whereas another study (14) concluded that

isomeric profiles of lycopene had no marked change during

simulated gastrointestinal digestion. In vivo investigations

showed that modest gastric isomerization occurred in the

stom-ach of ferrets (51) but not in that of humans (24). Our results

support the hypothesis that isomerization in the digestive lumen

does not contribute to the enrichment of cis isomers in the

human blood. This enrichment may be rather due to a

pre-ferential uptake of cis isomers by enterocytes or a more efficient

incorporation into chylomicrons for delivery to tissues (14,41).

As suggested above for b-carotene, we could hypothesize that

nondetected oxidative metabolites as well as enzyme-catalyzed

cleavage products of lycopene (52) were produced during small

intestinal digestion in TIM.

In conclusion, we found that the digestive stability of

carot-enoids depends on their nature, xanthophylls being more stable

than carotenes; their isomeric conformation; the food matrix in

which they are included; and the digestive compartment. These

data clearly confirm that numerous factors influence the

bio-availability of carotenoids in humans and therefore condition

their health effects.

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