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Nutrition Research 28 (2008) 532 – 538

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Cellular transport of lutein is greater from uncooked rather than cooked

spinach irrespective of whether it is fresh, frozen, or canned
Laurie O'Sullivan, Lisa Ryan, S. Aisling Aherne, Nora M. O'Brien⁎
Department of Food and Nutritional Sciences, University College Cork, Cork, Republic of Ireland
Received 7 January 2008; revised 26 May 2008; accepted 28 May 2008

Abstract

Lutein, a carotenoid found in significant levels in spinach, has attracted a great deal of attention
owing to its reported function as a shield against the photooxidative effects of blue light. Therefore,
the rationale of this study was to examine the effects of various processing and cooking methods on
lutein bioavailability from spinach (Spinacia oleracea) using an in vitro digestion procedure coupled
with the use of a human intestinal Caco-2 cell model. Fresh, frozen, and canned spinach were
analyzed uncooked and after boiling or microwave cooking. Lutein content from the uncooked and
cooked digested food (digestate) and appropriate micelles was determined. Micellarized lutein from
the spinach samples was adjusted to 0.1 μmol/L and added to Caco-2 cells. Cellular uptake and
secretion (cellular transport) of lutein were determined. Our results showed that digestate obtained
from uncooked canned spinach had greater lutein content (P b .05) than uncooked fresh or frozen
spinach. Microwave cooking, but not boiling, significantly lowered the lutein content of canned
spinach digestate and micelles compared with their uncooked counterparts. Interestingly, there were
no differences in the micellarization of lutein between the cooking and processing methods. Cellular
transport of lutein was greater from uncooked spinach micelles compared with boiled or microwave-
cooked spinach. To conclude, although the lutein content of digesta and micelles may have been
modified, its micellarization was not significantly affected by any of the cooking or processing
methods tested. In general, cellular transport of lutein was greatest in uncooked spinach irrespective
of whether the spinach was fresh, frozen, or canned.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Bioavailability; Carotenoid; Caco-2 cells; Digestion; In vitro; Lutein; Micelles; Spinach (Spinacia oleracea)
Abbreviations: HBSS, hanks balanced salt solution; HPLC, high-performance liquid chromatography.

1. Introduction number of years, there has been an increasing body of

research conducted on lutein; however, limiting data exist on
The xanthophyll carotenoid lutein is present in significant
the impact of processing (eg, freezing and canning) and
amounts in the macular tissue and lens of the eye where it is
domestic cooking on both the bioavailability and bioacces-
thought to function as an antioxidant and a shield against the
sibility of this carotenoid.
photooxidative effects of blue light [1-4]. In addition, high
Bioavailability is termed as the fraction of an ingested
dietary intake of lutein has been associated with reduced
nutrient available for use in normal physiologic functions
risks of certain cancers, coronary heart disease, and stroke
and storage in the body [7]. There are a number of steps
[5]. Rich dietary sources of lutein include green leafy
involved in the bioavailability of carotenoids from foods.
vegetables such as spinach (Spinacia oleracea), which is
These include (1) the release of carotenoids from a food
reported to contain 6.3 mg of lutein per 100 g [6]. In the past
matrix, (2) the transfer of carotenoids to micelles, (3) the
uptake of carotenoids by intestinal cells, (4) the incorpora-
⁎ Corresponding author. Tel.: +353 21 4902884; fax: +353 21 4270244. tion of carotenoids into chylomicrons and their secretion by
E-mail address: nob@ucc.ie (N.M. O'Brien). intestinal cells, and (5) the transport of carotenoids to the
0271-5317/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2008.05.011
 L. O'Sullivan et al. / Nutrition Research 28 (2008) 532–538
blood [8]. Carotenoid bioaccessibility is defined as the
amount of the ingested carotenoid(s) that are available for
absorption in the gut after digestion [9,10] (ie, steps 1 and
2), whereas the term carotenoid micellarization refers to
the transfer of carotenoids from the digested food
(digestate) to the micelles (ie, step 2) [10-12]. The
efficiency of carotenoid micellarization may depend on
many factors such as food treatments and the presence of
dietary fat and/or fiber [8,12,13].
Steps 3 and 4 of carotenoid bioavailability (cellular
transport) can be mimicked in the laboratory by the use of a
differentiated Caco-2 cell model [11,14]. Caco-2 cells are
human colon adenocarcinoma cells that, when differentiated,
exhibit both functional and morphological characteristics
similar to enterocytes [15]. When supplemented with certain
fatty acids, such as oleic acid and taurocholate, highly
differentiated Caco-2 cells are capable of forming and
secreting chylomicrons [16,17], which are necessary for
effective carotenoid transport to the blood [17]. Conse-
quently, numerous studies have involved the use of an in
vitro digestion procedure coupled with a Caco-2 cell model
to investigate potential bioavailability of carotenoids from
various foods, meals, and supplements [9-12,14,18-22].
Therefore, the present study used this coupled system to
investigate various aspects of lutein bioavailability from
fresh, frozen, and canned spinach that were uncooked (raw),
boiled, or microwave cooked.
2. Methods and materials
2.1. Materials
Fresh whole leaf, frozen, and canned spinach (S.
oleracea) were purchased from a local supermarket (Super-
Valu, Cork, Ireland). Lutein was purchased from Fluka
(Buchs SG, Switzerland). Fetal bovine serum was purchased
from Biosciences Ltd (Invitrogen Ltd, Paisley, Scotland). All
other chemical reagents were purchased from Sigma Aldrich
Ireland Ltd (Dublin, Ireland) unless otherwise stated. Cell
culture plastics were supplied by Cruinn Diagnostics Ltd
(Greiner Bio-One, Kremsmunster, Austria). Transwell plates
were purchased from Costar (Corning Life Sciences,
Schiphol-Rijk, The Netherlands). All solvents used were of
high-performance liquid chromatography (HPLC) grade.
2.2. Sample preparation
All preparation of foods was performed under amber light
to minimize photooxidation of lutein. Spinach samples were
cooked by boiling or microwave cooking (see Section 2.3).
Uncooked (raw) samples of the 3 spinach products served as
a control. A sample weight of approximately 5 g of fresh and
canned spinach was microwave cooked for 1 minute at the
high setting (Hinari Lifestyle, Alba plc, Herts, UK; 800-W
microwave oven, 100% power). The frozen spinach was
microwave cooked for 1.5 minutes. For the boiling method,
500 mL of distilled water was brought to boil, the spinach
533
was added, and the cooking time began; the heat was
adjusted to maintain a steady rolling boil for 10 minutes.
Canned spinach was heated directly in a beaker for
approximately 5 minutes (until bubbling).
2.3. In vitro digestion
The in vitro digestion procedure was carried out as
previously described by Garrett et al [11]. Briefly, 2 to 2.5 g of
sample was weighed and homogenized twice (Janke and
Kunkel, Ultra-Turrax T25; IKA-Labortechnik, Staufen,
Germany) with 5 mL of saline solution for 10 seconds
(8000 rpm). The resulting sample homogenate was trans-
ferred to an amber jar. An additional 13 mL of saline, which
was used to rinse both the apparatus and the homogenate
vessel, was added to each homogenate. The pH was acidified
to 2.0 with 0.1 mol/L HCl, and 1 mL of pepsin (0.04 g/mL
HCl) was added, creating a final volume of 20 mL. The
samples were overlaid with nitrogen gas and incubated at
37°C for 1 hour in a shaking water bath (Grant, OLS200;
Keison Products, Chelmsford, Essex, UK) at 95 rpm to mimic
the gastric phase of human digestion. The intestinal phase
involved increasing the pH to 5.3 with 0.9 mol/L sodium
bicarbonate. The bile salts glycodeoxycholate (0.8 mmol/L),
taurodeoxycholate (0.45 mmol/L), and taurocholate
(0.75 mmol/L), as well as porcine pancreatin (0.04 g/mL of
0.1 mol/L HCl) were added to the samples. The final pH was
adjusted to 7.4 using NaOH. Samples were overlaid with a
layer of nitrogen gas and incubated for 2.5 hours at 37°C to
mimic the duodenal phase of human digestion. Aliquots of
the digesta were frozen at −80°C until further analysis.
2.4. Micelle fraction isolation
The micelle (aqueous) fraction of the digestate that
contains the accessible carotenoids was isolated using
ultracentrifugation as previously described by O'Connell
et al [23]. Briefly, aliquots of the digestate (5 mL) were
placed into ultracentrifuge tubes (Beckman Life Sciences,
Brea, Calif) and centrifuged at 53 000 rpm for 95 minutes
(NVT90 rotor; Beckman Instruments Inc, Fullerton, Calif).
The micelle fraction was removed using a needle and was
filter sterilized using a surfactant-free cellulose acetate filter
(0.2 μm; Millipore, Bedford, Mass) to remove any micro-
crystalline aggregates. The resulting filtrate was stored at
−80°C until further analysis.
2.5. Cellular transport of micellarized lutein obtained
from spinach
Human colon adenocarcinoma Caco-2 cells were pur-
chased from the European Collection of Animal Cell
Cultures (Salisbury, Wilts, UK). Cells were maintained in
the Dulbecco's modified Eagle's medium supplemented
with 10% (vol/vol) fetal bovine serum and 1% (vol/vol)
nonessential amino acids. Cells were grown at 37°C in a
humidified incubator with 5% CO2 in the absence of
antibiotics. For the experiments, cells were seeded at a
534 L. O'Sullivan et al. / Nutrition Research 28 (2008) 532–538
5
density of 1.25 × 10 cells/mL on transwell plates (6-well
plate, 24-mm diameter, 0.4-μm pore size membrane). Cells
were grown for 21 to 25 days to obtain a differentiated cell
monolayer. Transepithelial electrical resistance measure-
ments were taken twice weekly by a transepithelial electrical
resistance voltohmmeter to ensure the monolayer was intact.
Diffusion of phenol red was also performed to ensure that
cell monolayers were undamaged (results not shown). At the
beginning of each experiment, the apical side of the transwell
plate received 2 mL of carotenoid-enriched media (contain-
ing 0.1 μmol/L of micellarized lutein with sterile media).
The basolateral chamber received 2 mL of serum-free media.
The incubation time for all experiments was 4 hours, after
which time, the micelle-enriched media were removed and
the cell layer was washed with warm sterile Hanks balanced
salt solution (HBSS). Media (2 mL) containing 0.5 mmol/L
taurocholate, 1.6 mmol/L oleic acid, and 45 mmol/L glycerol
were added to the apical chamber and incubated for 16 hours
for the stimulation and secretion of chylomicrons [17]. After
the incubation time, media from each side of the membrane
were removed, the monolayer was washed twice with HBSS,
and cells were scraped into 1 mL of HBSS. Cell samples
were sonicated for 30 seconds on ice. Samples were stored at
−80°C until further analysis.
2.6. Carotenoid extraction and analysis
All samples were allowed to thaw and were briefly
vortexed. A recovery standard consisting of either β-Apo-8′-
carotenol or lycopene was added to all samples, which were
extracted twice with hexane/ethanol/acetone (50:25:25, vol/
vol/vol) [24]. Samples were centrifuged (Sorvall TC6, H400
rotor originally purchased from DuPont Instruments, Herts,
UK) at 3000 rpm for 5 minutes, and the supernatant layers
were removed, pooled, and dried under nitrogen gas. The
residues were reconstituted in 100 μL of mobile phase, and
the carotenoid content of the samples was analyzed by
reversed-phase HPLC. The HPLC method used was based on
the methodology of Hart and Scott [25]. The HPLC system
(Finnigan SpectraSYSTEM; Thermo Scientific, Philadelphia,
PA) consisted of a P2000 pump connected to a AS3000
autoinjector and UV1000-visible detector. The column
system consisted of a Spherisorb ODS-2 C18 5 μm PEEK
guard column (Alltech Associate Applied Science Ltd.,
Lancs, UK; supplied by Ocon Chemicals Ltd, Cork, Ireland)
connected to a Vydac 201TP54 (250 × 4.6 mm) reversed-
phase C18 column (Separations Group; supplied by AGB
Scientific Ltd, Dublin, Ireland). Column temperature was
maintained at 28°C by an internal column oven. The injection
volume was 50 μL; samples were eluted using isocratic
mobile phase composed of acetonitrile/methanol/dichloro-
methane (75:20:5, vol/vol/vol) containing 10 mmol/L
ammonium acetate, 4.5 mmol/L butylated hydroxytoluene,
and 3.6 mmol/L triethylamine at a flow rate of 1.5 mL/min.
Carotenoids were detected at 450 nm. The mobile phase was
filtered through a 0.5-μm organic filter and degassed using
ultrasonic agitation. Results were collected and analyzed
using ChromQuest software (version 4.2, Thermo Fisher
Scientific). Lutein, lycopene, and β-Apo-8′-carotenol recov-
ered from all the analyzed samples were extrapolated from
pure carotenoid standard curves after correction for extraction
efficiency, based on recovery of lycopene or β-Apo-8′-
carotenol, and quantified after correction for initial weight and
dilution factors. In view of the fact that carotenoid isomers and
a C30 column were not available to our laboratory at the time
of this study, we could not analyze the potential effects of
cooking and/or processing on carotenoid isomerization.
2.7. Statistical analyses
Micellarization was expressed as the percentage of
carotenoid transfer from the digestate to the micelles. Lutein
transport was determined by expressing the uptake and
secretion of lutein by the Caco-2 cells as a percentage of the
initial amount added to the cells. Data are expressed as the
mean ± SEM of 3 independent experiments. Data were
analyzed by 1-way analysis of variance (ANOVA) and, where
appropriate, Tukey multiple comparison test (Prism 4.03;
GraphPad Inc, San Diego, Calif). This analysis compared the
means of all 3 columns representing 3 different treatments
[26] and also all 3 spinach types. P b .05 was considered
statistically significant (Prism 4.03, GraphPad Inc).
3. Results
3.1. Lutein content of digesta obtained from
spinach samples
Fresh, frozen, and canned spinach were subjected to
either the in vitro digestion procedure directly (uncooked) or
after a domestic cooking procedure such as boiling or
microwave cooking. There was no significant difference in
the lutein content between the starting materials (whole
foods) and the foods after simulated digestion (data not
shown). The lutein content in the uncooked canned spinach
Table 1
Lutein content (μg/100 g) of digesta from uncooked and cooked fresh and
processed spinach a
Lutein (μg/100 g) b
Uncooked Boiled Microwave
cooked
Fresh spinach 2705.3 ± 166.7 c 3893.6 ± 1113.0 3945.5 ± 416.9
Frozen spinach 2990.9 ± 76.5 c 5059.6 ± 857.6 3385.3 ± 290.4
Canned spinach 7624.1 ± 1038.0 d,e 8213.3 ± 829.1 2499.7 ± 664.0 c
a
All samples were subjected to an in vitro digestion procedure. Lutein
from digested samples (digesta) was extracted and quantified by HPLC.
b
Data are the means ± SEM for 3 independent experiments (n = 3) and
were analyzed by 1-way ANOVA and, where appropriate, Tukey multiple
comparison test.
c
Significantly different (P b .05) from uncooked canned spinach.
d
Significantly different (P b .05) from uncooked fresh or frozen
spinach.
e
Significantly different (P b .05) from microwave-cooked canned
spinach.
 L. O'Sullivan et al. / Nutrition Research 28 (2008) 532–538 535

Table 2 3.3. Lutein micellarization from spinach

Lutein content (μg/100 g) of micelles from uncooked and cooked fresh and
processed spinach a Data on the effects of cooking and processing on lutein
Lutein (μg/100 g) b micellarization are presented in Table 3. Micellarization was
Uncooked Boiled Microwave cooked
expressed as the percentage transfer of lutein from the
digesta to the micelles. In general, the uncooked and cooked
Fresh spinach 1734.0 ± 131.8 2554.0 ± 532.3 1459.3 ± 138.7
fresh spinach samples had the greatest transfer of lutein to the
Frozen spinach 1083.3 ± 50.3 2530.7 ± 381.1 776.6 ± 141.9
Canned spinach 1803.3 ± 207.5 c 2293.5 ± 282.3 775.8 ± 207.8 d micelles. Again, boiling slightly increased lutein micellar-
a ization from all 3 samples (fresh, frozen, and canned);
All samples were subjected to an in vitro digestion procedure. Micelles
were isolated from digesta by ultracentrifugation, extracted, and quantified however, the effect was not significant. Similarly, microwave
by HPLC. cooking had no significant effect on the transfer of lutein to
b
Data are the means ± SEM for 3 independent experiments (n = 3) and the micelles when compared with uncooked spinach.
were analyzed by 1-way ANOVA and, where appropriate, Tukey multiple Therefore, neither cooking nor processing significantly
comparison test.
affected lutein micellarization from spinach.
c
Significantly different (P b .05) from microwave-cooked canned
spinach. 3.4. Cellular transport of micellarized lutein from
d
Significantly different (P b .05) from uncooked canned spinach.
spinach micelles
After the isolation and quantification of lutein in micelles
obtained from spinach samples, 0.1 μmol/L of micellarized
digestate was significantly (P b .05) greater than uncooked
lutein was added to differentiated Caco-2 cells cultured on
fresh or frozen spinach (Table 1). When all 3 spinach types
were boiled, the lutein content of the digesta increased
slightly but not significantly. There was a significant
reduction (P b .05) in the lutein content of the microwave-
cooked canned spinach digestate when compared with its
uncooked counterpart (Table 1). Boiling or microwave
cooking had no significant affect on the lutein content of
the fresh or frozen spinach samples.
3.2. Lutein content of micelles obtained from all
spinach samples
Table 2 shows the lutein content of the micelles obtained
from all the spinach samples. There was no significant
difference between the lutein content of micelles obtained
from uncooked spinach and boiled spinach (Table 2). In
addition, their processed form (ie, fresh, frozen, or canned)
had no affect on lutein levels in the micelle fraction. Once
again, there was a significant reduction (P b .05) in the lutein
content of the microwave-cooked canned spinach micelles
when compared with its uncooked counterpart (Table 2).

Table 3
Lutein micellarization from uncooked and cooked fresh and processed
spinach a
Fig. 1. Cellular transport of lutein by Caco-2 cells supplemented with
Lutein micellarization (%) b micellarized lutein from (A) fresh spinach, (B) frozen spinach, and (C)
Uncooked Boiled Microwave cooked canned spinach. Lutein content of all the relevant micelles was adjusted to
0.1 μmol/L and added to Caco-2 cells for 4 hours. After treatment, the
Fresh spinach 64.0 ± 1.9 73.5 ± 20.3 38.0 ± 5.7
micelle-enriched media were removed, the cell layer was washed, and
Frozen spinach 36.2 ± 1.6 50.4 ± 1.2 23.9 ± 5.8
the Caco-2 cells were grown in media containing chylomicron-stimulating
Canned spinach 23.8 ± 0.7 29.0 ± 5.9 40.9 ± 19.2
compounds for 16 hours. Cellular transport was determined by expressing
a
All samples were subjected to an in vitro digestion procedure. Micelles the uptake and secretion of lutein by the Caco-2 cells as a percentage of the
were isolated from digesta by ultracentrifugation. Lutein from both digesta initial amount added to the cells (0.1 μmol/L). Data are means ± SEM for 3
and micelles was extracted and quantified by HPLC. independent experiments (n = 3) and were analyzed by 1-way ANOVA and,
b
Lutein micellarization is expressed as the percentage transfer of lutein where appropriate, Tukey multiple comparison test. aSignificantly different
from the digestate to the micelles. Data are means ± SEM for 3 independent (P b .05) from microwave-cooked spinach. bSignificantly different (P b .05)
experiments (n = 3). Data were analyzed by 1-way ANOVA and were found from uncooked spinach. cSignificantly different (P b .05) from boiled
not to significantly differ. spinach.
536 L. O'Sullivan et al. / Nutrition Research 28 (2008) 532–538
transwell plates. Lutein content of the cells and lutein
transported to the basolateral compartment of the transwell
plate was quantified separately, and the values were
combined. Cellular transport of lutein was calculated by
expressing lutein cell uptake plus secretion by Caco-2 cells
as a percentage of the lutein concentration (0.1 μmol/L) that
was added to the cells (Fig. 1). The uncooked fresh, frozen,
and canned spinach samples had similar levels of lutein
cellular transport (Fig. 1). The boiled or microwave-cooked
fresh spinach had reduced lutein cellular transport compared
with the uncooked fresh spinach samples (Fig. 1A). In fact,
cooking by microwave significantly reduced (P b .05)
cellular transport compared with the uncooked fresh sample
(Fig. 1A). Similarly, cooking the canned spinach, by either
boiling or microwave cooking, significantly decreased (P b
.05) the transport of lutein by the Caco-2 cells (Fig. 1C).
When Caco-2 cells were supplemented with lutein from
uncooked and cooked frozen spinach micelles, there was no
significant difference in the cellular transport of lutein
between all 3 samples (Fig. 1B). Looking at Fig. 1A to C, the
uncooked spinach samples did show greater cellular lutein
transport compared with their cooked counterparts.
4. Discussion
We report the application of an in vitro digestion model to
compare the bioavailability of lutein from fresh, frozen, and
canned spinach that were uncooked and cooked. This model
consisted of 2 steps: first, in vitro digestion where the
spinach samples were subjected to simulated gastric and
intestinal phases of human digestion [11], and second,
micelles containing the accessible lutein were then added to
differentiated Caco-2 cells. A potential limitation to mimic
human physiologic absorption of carotenoids by the in vitro
digestion procedure is that these models incorporate
additional emulsifiers that may not be ingested along with
carotenoid-rich fruits and vegetables as a part of the human
diet [22]. However, the in vitro digestion procedure does
provide a rapid and cost-effective model for screening the
bioaccessibility of carotenoids and, coupled with the Caco-2
cell model, offers a good indicator of bioavailability [11].
Furthermore, under conditions mimicking the postprandial
state, Caco-2 cells grown on membranes take up and
incorporate carotenoids into chylomicrons and secrete them
via chylomicrons [16]. We have previously shown that
differentiated Caco-2 cells are taking up and secreting
carotenoids, including lutein, via chylomicrons [27].
It has been well documented that the nature of the food
matrix is a determining factor on carotenoid bioavailability
[8,13,21,28-35]. In order for a carotenoid to become accessible
for potential absorption, it must be released from this matrix by
both chemical and physical disruption [36]. In the present
study, we report that digested canned spinach had a greater
content of lutein compared with fresh or frozen spinach. The
enhanced content of lutein in the digestate may have arisen as a
result of the breakdown of the plant structure during the
canning process. Subsequent microwave cooking of the
canned spinach resulted in lower amounts of lutein detected
in the digestate, possibly because of partial destruction and/or
isomerization of the carotenoid [37-39]. For example, Aman et
al [37] reported that blanching fresh spinach (blanched with
vapor at approximately 100°C over a covered water bath for 2
minutes) resulted in a 17% decrease in total lutein content.
Compared with their canning process, the lower temperature
and much shorter heat exposure of blanching resulted in
substantial degradation of lutein. Glaser et al [38] examined the
lutein content of home-grown and commercial spinach. The
authors report a 10% decrease of total lutein content after
microwave cooking of the commercial spinach and conclude
that this was due to destruction of the carotenoid. In addition,
Zhao et al [39] studied the different effects of microwave and
ultrasound on the stability of all-trans-astaxanthin and reported
that microwave cooking induced significant isomerization of
astaxanthin to its cis analogues. As mentioned earlier, lutein
isomers were not commercially available, and a C30 HPLC
column, which is required for the detection of carotenoid
isomers, was also not available during this study.
In general, the lutein content of micelles from spinach was
similar, with the exception of the microwave-cooked canned
spinach that contained lower amounts than its uncooked
counterpart. The amount of lutein in the micelle fraction
from spinach that was frozen or canned did not differ from
the fresh variety. Micellarization of carotenoids is an
essential step during digestion as the micelles deliver
carotenoids to the brush border membrane of enterocytes.
Therefore, efficiency of micellarization is often used as an
estimate of the relative availability of carotenoids
[10,18,19,40]. Interestingly, although some modifications
were shown in the lutein content of the digesta and micelles,
the efficiency of lutein transfer to the micelles was not
affected by the cooking and/or processing methods tested.
Lutein micellarization was approximately 38% in the
microwave-cooked fresh spinach. This finding is in agree-
ment with other studies [14,31]. For instance, Chitchum-
roonchokchai et al [14] report values of 40% to 53%
micellarization of lutein from microwave-cooked fresh
spinach puree. Castenmiller et al [31] report similar values
of 45% to 55% from microwave-cooked fresh spinach. Goñi
et al [41] found that 70% of lutein is accessible from boiled
fresh spinach. Similarly, we showed that micellarization of
lutein was approximately 73.5% from boiled fresh spinach.
Comparing uncooked fresh spinach with their processed
counterparts, there was a nonsignificant reduction in lutein
micellarization with fresh spinach enabling the greatest
transfer of lutein, followed by frozen and then canned. This
trend was also evident with the boiled spinach.
Highly differentiated Caco-2 cells are routinely used to
study the intestinal absorption, metabolism, and secretion
of carotenoids [16,42,43]. In the present study, we applied
micellarized lutein obtained from the various spinach
samples to Caco-2 cells. The concentration of lutein that
was added to the cells, that is, 0.1 μmol/L, was selected
 L. O'Sullivan et al. / Nutrition Research 28 (2008) 532–538
based on the literature [27,43]. During and Harrison [43]
report that 0.1 to 10 μmol/L is considered representative of
the normal range of carotenoid intake from the diet and/or
supplements. In addition, to compare fresh, frozen, and
canned spinach and to evaluate the effects of cooking, we
standardized the amount of lutein in the micelles. Our
uptake and secretion values for micellarized lutein from
uncooked fresh spinach are in agreement with the data
reported for the uptake and secretion of pure lutein in
Caco-2 cells [27]. A similar study conducted by Chit-
chumroonchokchai et al [14] report that there was a 6.3%
transfer of micellarized lutein from microwave-cooked
spinach to the basolateral chamber, which is in agreement
with data from the present study. A human bioavailability
study conducted by Castenmiller et al [31] investigated the
effect of the spinach matrix on carotenoid concentrations in
human serum. The authors conclude that the relative
bioavailability of whole leaf spinach is 45% [30], which is
in agreement with our present findings.
After cooking, the cellular transport of lutein from
spinach was lower than the uncooked samples. The reasons
for this are unclear; however, spinach contains significant
amounts of β-carotene [14,20,37,38], and after cooking,
β -carotene may have also been released from the spinach
matrix and incorporated into the micelle fraction. When
micelles were then applied to the Caco-2 cells; competitive
uptake between the 2 predominant carotenoids may have
occurred [11,14,18,32,44]. Kostic et al [44] demonstrated
that there is an interaction between β-carotene and lutein
when they are administered in a combined dose and that
β -carotene negatively affects lutein uptake.
Another possible explanation for the reduced cellular
transport of lutein from cooked spinach is that there may
have been preferential uptake of trans isomers in the Caco-2
cells. Earlier reports suggest that trans to cis isomerization
of lutein in chloroplasts occurs after a heating or cooking
process [28,45]. During et al [46] reported that there was
preferential uptake of all trans isomers of β-carotene over
9-cis and 13-cis isomers in Caco-2 cells; a finding that is
also in line with a human study [47]. Therefore, it could be
speculated that after cooking (boiling and microwave
cooking), there was a higher cis to trans ratio of lutein
within the micelle fraction. However, in a recent publica-
tion, Aman et al [37] report no increase in cis-lutein
stereoisomers in processed spinach. This finding is in
contrast to previous studies [45]; thus, it becomes apparent
that the influence of thermal treatment of all-trans-
carotenoids sensitized by chlorophyll on degradation and
isomerization is still unknown.
To summarize, our findings provide information on the
effects of cooking and processing on the bioavailability of
lutein sourced from spinach. Despite the significantly higher
lutein content in the digestate from canned spinach, the amount
incorporated into the micelles was similar to that from the fresh
or frozen spinach. Whereas boiling had no significant affect,
microwave cooking significantly decreased the lutein content
537
of the micelles derived from canned spinach. In addition,
cellular transport was highest from uncooked spinach
irrespective of the spinach being fresh, frozen, or canned.
Further work is warranted to assess whether this may be due to
competition with β-carotene uptake, enhanced degradation,
and/or isomerization of lutein after the cooking process.
Spinach, when consumed in various forms, has the
potential to enhance lutein status, which may possibly reduce
and/or prevent the risk of age-related macular degeneration
and cataract formation. This study clearly shows that
cooking, more than processing, may impact the bioavail-
ability of lutein. Gaining knowledge on the effects of
cooking and processing on carotenoid-containing foods is
useful to encourage food manufacturers to maximize
carotenoid bioavailability potential and to help consumers
obtain adequate/optimal amounts of carotenoids [48].
Acknowledgment
This research was funded by the Science Foundation
Ireland under the PI Award 04/IN3/B509. The authors thank
Karen Galvin for helpful technical suggestions on carotenoid
analysis.
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