Raman spectroscopy analysis of molecular configuration forms of the macular xanthophylls

Article abstract of DOI:10.1002/jrs.5818

Macula lutea, the yellow spot of the retina of the human eye, comprises three xanthophyll pigments: lutein, zeaxanthin, and meso-zeaxanthin, playing numerous important biological functions. Macular xanthophylls are exposed to relatively strong illuminatio

Full text of DOI:10.1002/jrs.5818

Received: 7 February 2019
DOI: 10.1002/jrs.5818
Revised: 11 December 2019
Accepted: 16 December 2019
R E S E A R C H A R T I C L E
Raman spectroscopy analysis of molecular configuration
forms of the macular xanthophylls
A. Sek1,2 | R. Welc1 | M.M. Mendes-Pinto1 | E. Reszczynska3
W. Grudzinski1 | R. Luchowski1 | W.I. Gruszecki1
|
1Department of Biophysics, Institute of
Abstract
Physics, Maria Curie-Sklodowska
University, Lublin, Poland
Macula lutea, the yellow spot of the retina of the human eye, comprises three
xanthophyll pigments: lutein, zeaxanthin, and meso-zeaxanthin, playing
numerous important biological functions. Macular xanthophylls are exposed
to relatively strong illumination in the eye, owing to the fact that the yellow
spot is localized on the optical axis, in the frontal layer with respect to photore-
ceptors. In the present work, we address the problem of photostability and pos-
sible photoisomerization of macular xanthophylls with the application of
resonance Raman spectroscopy. The results show photostability of the two
major isomers of the macular xanthophylls, all-trans and 9-cis, and efficient
photoconversion of the 13-cis isomer to the 9-cis and mostly to the all-trans
form. We report the Raman spectra of the main molecular configuration forms
of the macular xanthophylls, opening an avenue for the examination of their
possible presence and photoconversion in natural systems.
2Department of Interfacial Phenomena,
Faculty of Chemistry, Maria Curie-
Sklodowska University, Lublin, Poland
3Department of Plant Physiology and
Biophysics, Institute of Biology and
Biochemistry, Maria Curie-Sklodowska
University, Lublin, Poland
Correspondence
W. I. Gruszecki, Department of
Biophysics, Institute of Physics, Maria
Curie-Sklodowska University, Pl. M.
Curie-Sklodowskiej 1, 20-031 Lublin
Poland.
Email: wieslaw.gruszecki@umcs.pl
Funding information
Foundation for Polish Science, Grant/
Award Number: TEAM/2016-3/21
K E Y W O R D S
carotenoids, cis-xanthophylls, macula, retina, xanthophylls
1 | INTRODUCTION
xanthophylls, have been identified in the macular pig-
ment pool, namely, lutein (Lut), zeaxanthin (Zea) and
Carotenoid pigments are ubiquitous in the biosphere and
play numerous important physiological roles, including
protection against oxidative damage and filtering harm-
ful, short-wavelength radiation.[1–3] These functions are
particularly important from the standpoint of photo-
protection and integrity of the eye operating under condi-
tions characterized by exposure to light and the presence
of dissolved molecular oxygen in highly vascularized
tissues.[4] An anatomical structure in the retina of the
human eye particularly rich in carotenoids is referred
to as the macula lutea or the yellow spot, due to its
intense colouration.[5] Three polar carotenoids, called
meso-zeaxanthin (m-Zea) (see Figure 1).[4,6] Resonance
Raman spectroscopy of macular xanthophylls can be
effectively applied to examine their properties in the nat-
ural system[7] or even for imaging the retina,[8] owing to
the relatively strong Raman scattering signal of polyene
dyes.[9] On the other hand, a laboratory experience
with carotenoids shows that molecules from these
particular groups can undergo light-induced molecular
configuration changes.[10,11] In the present work, we
address the problem of possible light-induced molecular
reconfiguration of macular xanthophylls, with the appli-
cation of resonance Raman spectroscopy.
J Raman Spectrosc. 2020;1–7.
wileyonlinelibrary.com/journal/jrs
© 2020 John Wiley & Sons, Ltd.
1

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SEK ET AL.
FIGURE 1 Chemical structures of the
macular xanthophylls in the molecular
configuration all-trans. The structures of the
xanthophylls in the molecular configurations cis
are shown in Figures S1–S4
2 | MATERIALS AND METHODS
2.1 | Xanthophylls and solvents
illuminated for 1 hr using a halogen lamp illuminator
(150 W) equipped with a bandpass blue filter (B-390,
Shimadzu, Japan). In order to remove iodine through
reduction reaction, an aqueous solution of 5% sodium
thiosulfate was added. After phase separation, the upper
phase was collected and sodium thiosulfate was removed
by washing with distilled water. The obtained isomer
mixture was dried and dissolved in an HPLC mobile
phase and subjected to chromatographic separation. In
the case of Lut, two separate protocols were applied, pref-
erentially yielding isomerization to 9-cis and 90-cis forms
and to 13-cis and 130-cis forms. In order to obtain a higher
concentration of 9-cis and 90-cis forms, a solution of all-
trans lutein in hexane was supplemented with an I2 solu-
tion in the same solvent (the final concentration of the
catalyst 2% w/w) and incubated for 20 min at 30ꢀC.
Iodine was removed from pigment solutions as in the
case of isomerization of Zea and m-Zea. In order to
obtain higher concentrations of 13-cis and 130-cis forms of
lutein, a solution of all-trans lutein in ethanol was incu-
bated for 2.5 hr at 80ꢀC.
Crystalline xanthophylls (all-trans)-lutein [(3R,30R, 60R)-
β, ε-carotene-3,30-diol)] and (all-trans)-Zeaxanthin
[(3R,30R)-β,β-carotene-3,30-diol)] were obtained from
Extrasynthese. (All-trans) meso-Zeaxanthin [(3R,30S)-β, β-
carotene-3,30-diol)] was obtained from U.S. Pharmaco-
peia. In order to remove possible degradation products,
xanthophylls were repurified directly before use by
means of high-performance liquid chromatography
(HPLC) technique according to the previous report[10]
(more details of purification are presented below). All the
solvents for chromatographic xanthophyll purification
were purchased from POCH (Poland) and were of the
chromatographic quality: methanol (99.8%), methyl tert-
butyl ether (99.8%), acetonitrile (99.9%), and dic-
hloromethane (99.8%). Ultrapure water was obtained
from a Milli-Q system (Millipore, France). The specific
resistivity of water was 18 MΩ cm. Tetrahydrofuran
(THF) for spectroscopic measurements (99.8%) was pur-
chased from POCH (Poland).
Chemical formulas of the main molecular configura-
tion forms of Lut, Zea and m-Zea are shown in
Figures S1–S4.
2.2 | Xanthophyll isomerization
2.3 | Xanthophyll separation and
Isomerization of Zea and m-Zea was performed according
to the procedure described previously.[10] All-trans m-Zea
and all-trans Zea were dissolved in dichloromethane.
Iodine catalyst in hexane was added at a concentration of
2% (w/w). The mixture of iodine and pigment was
purification
Isomers of Zea, m-Zea, and Lut were separated and
purified chromatographically with the application of a
Shimadzu LC-20AD system equipped with an SPD-

SEK ET AL.
3
M20A diode array detector and with a C-30 coated,
phase-reversed column (YMC GmbH, Germany), inter-
nal diameter 4.6 mm, length 250 mm, and particle size
5 μm. In the case of Zea and m-Zea, a mixture of
methanol and methyl tert-butyl ether (95: 5, v/v) was
used as a mobile phase and elution rate was 1 ml/
min. In the case of lutein, two chromatographic phases
(acetonitrile: dichloromethane: methanol, 54: 28: 18, v:
v:v) and (acetonitrile: methanol: water, 72: 8: 3, v:v:v)
were mixed in the proportion 7:3 and applied
isocratically with the elution rate 1.5 ml/min. Chro-
matographic fractions of xanthophylls were identified
according to the literature data.[10,12] Typical HPLC
elution profiles along with the absorption spectra of
the main xanthophyll fractions are presented in Fig-
ures S5–S7.
in the UV-Vis region, were recorded with Cary 60 UV-Vis
Spectrophotometer from Agilent.
3 | RESULTS AND DISCUSSION
Figures 2–4 present the resonance Raman spectra of Lut,
Zea, and m-Zea in the molecular configuration 13-cis,
recorded at 196 K. The spectra recorded at different tem-
peratures, also from the all-trans and 9-cis isomers of the
macular xanthophylls are presented in Figures S8–S16.
For each the pigment sample, spectra were recorded sev-
eral times, with increasing light intensity, in the laser
power range between 0.05% and 10%. Four main bands
can be resolved in the Raman spectra recorded, typical
for carotenoids.[9] The principal ν1 band in the spectral
region between 1,500 and 1,600 cm−1, representing the
C═C stretching vibrations in the conjugated double
bond systems, the ν2 band in the spectral region between
1,100 and 1,250 cm-1, representing the C─C stretching
vibrations in the conjugated double bond systems
coupled either to the C─H in-plane bending or to the
C─CH3 stretching modes, the ν3 band centering at
~1,000 cm−1, representing the CH3 in-plane rocking
vibrations and the ν4 band at ~950 cm−1, representing
the out-of-plane wagging modes of the ═C─H groups.
As can be seen, the exposure to strong light influences
the shape of resonance Raman spectra of individual
2.4 | Raman spectroscopy
Raman spectroscopy measurements were carried out
using an inVia confocal Raman microscope (Renishaw,
UK) with an argon laser (Stellar-REN, Modu-Laser™,
USA) operating at 488 nm (or at 514.5 nm, when indi-
cated), equipped with 20x long working distance objec-
tive (Olympus SLM Plan, NA = 0.25). All spectra were
recorded in the spectral region between 445–1885 cm−1
at 1-s exposure time, 120 accumulations, with EMCCD
Newton 970 camera (Andor Technology, UK) cooled to
223 K. Spectral resolution was 1 cm−1 (2,400 lines/mm
grating). Measurements were performed with varying
output power of the laser, in the range between 1.1 μW
(0.05% of the nominal power) and 240.8 μW (10%), as
measured in the sample compartment. Samples were
scanned at selected temperatures, 298, 196, and 118 K,
stabilized with the application of the Linkam THMS600
temperature-controlled stage from Linkam Scientific
(UK) equipped with LNP96 and T95-PE controller units.
An exact temperature at which spectra were recorded
was monitored by a temperature probe placed within the
sample compartment. Directly before measurements,
xanthophyll samples were dissolved in THF, placed in a
0.1-mm quartz cuvette and incubated for 10 min at the
appropriate temperature. Xanthophyll concentration in
the samples subjected to Raman spectroscopy measure-
ments was in the range between 0.85 and 1.15 × 10−5 M.
Spectroscopic measurements were also conducted on
diluted samples (information provided in appropriate fig-
ure legends). All spectra were preprocessed by cosmic ray
removing and baseline correction using WiRE 4.2 soft-
ware from Renishaw, UK. The procedure of subtraction
of spectra was performed with the application of the
same software. Absorption spectra of carotenoid samples,
FIGURE 2 Resonance Raman spectra of lutein in the
molecular configuration 13-cis and 130-cis recorded with increasing
the light intensity of the laser, in the power range between 0.05%
and 10% (marked). Spectra were recorded at 196 K, with a 488-nm
laser, from the xanthophyll sample in tetrahydrofuran

4
SEK ET AL.
xanthophylls in molecular configuration 13-cis, thus
pointing possible phototransitions between the molecu-
lar configuration forms (Figures 2–4). The splitting of
the ν1 band, visible very clearly in the spectra, is a mani-
festation of this light-induced process. In contrast, pho-
tostability in the same light intensity range can be
observed for the all-trans (Figures S9, S12, and S15) and
9-cis (Figures S10, S13, and S16) molecular configura-
tions. A detailed analysis of the Raman spectra of the
pigments in molecular configuration 13-cis, recorded
with the application of higher laser powers, shows that
among the photoconversion products dominate the iso-
form 9-cis (or 90-cis) with some contribution from the
form all-trans (see Figures S17–S22). It is intriguing why
just one molecular configuration (namely 13-cis), in the
case of all the xanthophylls examined, undergoes the
light-induced isomerization. The solvent was chromato-
graphically pure, and the same solvent was used in the
case of the other xanthophyll isomers, which were found
to be photostable. This minimizes the possibility of a
photoisomerization sensitized by solvent impurities. On
the other hand, there is a certain probability that possi-
ble impurities may possibly interact differently with dif-
ferent carotenoid isomers. A very likely mechanism of
the photo-isomerization observed would be a reaction
driven by the triplet excited state originating from the
singlet-singlet fission in carotenoid molecular aggregated
structures.[13] In order to check the possibility of forma-
tion of molecular ensembles of xanthophylls in a con-
centrated solution subjected to Raman spectroscopic
analysis, absorption spectra were recorded in the UV-Vis
region of the samples both concentrated and diluted.
The spectra recorded for Zea in the molecular configura-
tion 13-cis and all-trans are presented in Figures 5 and
6, respectively. As can be seen from the difference spec-
trum presented in the lower panel of Figure 5, the con-
centrated solution of 13-cis Zea contains a certain
fraction of molecules involved in the excitonic interac-
tions that give rise to the hypsochromic spectral shifts of
both the main S0!S2 absorption band originally located
FIGURE 3 Resonance Raman spectra of zeaxanthin in the
molecular configuration 13-cis recorded with increasing the light
intensity of the laser, in the power range between 0.05% and 10%
(marked). Spectra were recorded at 196 K, with a 488-nm laser,
from the xanthophyll sample in tetrahydrofuran
−
between 400 and 500 nm (11Ag !11Bu+) and the so-
called “cis band” with the maximum at 344 nm
(11Ag !11Ag+). This fraction of molecules can poten-
−
tially sensitize light-driven isomerization observed in the
experiments. As can be seen from the absorption spectra
presented in Figure 6, the concentrated solution of Zea
in the molecular configuration all-trans does not show
spectral shifts diagnostic for formation of molecular
aggregates in this system. The fact that xanthophyll mol-
ecules in such a system are photostable, in contrast to
Zea 13-cis involved in the formation of molecular struc-
tures and undergoing the light-induced isomerization,
leads to the conclusion that this process can be
FIGURE 4 Resonance Raman spectra of meso-zeaxanthin in
the molecular configuration 13-cis recorded with increasing the
light intensity of the laser, in the power range between 0.05% and
10% (marked). Spectra were recorded at 196 K, with a 488-nm laser,
from the xanthophyll sample in tetrahydrofuran. The m-Zea sample
contained a mixture of the 13-cis and 130-cis isoforms that are
indistinguishable both chromatographically and on the basis of
ultraviolet–visible absorption spectroscopy

SEK ET AL.
5
FIGURE 5 Absorption spectra of zeaxanthin in the molecular
configuration 13-cis in tetrahydrofuran solution used for resonance
Raman analysis (absorbance level at the maximum 1.4 in a 1-cm
cuvette) and diluted to the absorbance levels 0.8, 0.3, and 0.2,
indicated. The pure solvent was recorded as a reference. The
spectra were normalized at the maximum. The lower panel
presents the difference spectrum calculated from the spectra
presented in the upper panel: concentrated (absorbance 1.4) minus
diluted (absorbance 0.3)
FIGURE 6 Absorption spectra of zeaxanthin in the molecular
configuration all-trans in tetrahydrofuran solution used for
resonance Raman analysis (absorbance level at the maximum 1.4 in
a 1-cm cuvette) and diluted to the absorbance level 0.3, indicated.
The pure solvent was recorded as a reference. The spectra were
normalized at the maximum. The lower panel presents the
difference spectrum calculated from the spectra presented in the
upper panel (concentrated minus diluted)
potentially sensitized by the triplet states generated via
the singlet-singlet fission. To test whether some other
mechanisms could lead to light-induced isomerization,
observed specifically for the 13-cis xanthophylls, Raman
spectra were recorded from diluted samples in which the
absorption spectra showed no signs of pigment aggrega-
tion (see Figure 5). Moreover, Raman spectra from such
samples were additionally recorded with application of
the laser line 514.5 nm that is out of resonance with the
aggregated forms of the 13-cis isomers of Zea owing to
the hypsochromic spectral shift (see the difference spec-
trum in Figure 5). The results of the experiments (Fig-
ures S23 and S24) show that 13-cis Zea in a monomeric
form also undergoes the light-induced isomerization.
The photoisomerization of the 13-cis isomers can be
explained in terms of different excited state dynamics
that enables effective photoisomerization to other molec-
ular configurations. It is possible that the intramolecular
charge transfer state, observed for the 13-cis isomer of
another polar carotenoid fucoxanthin, distinctively dif-
ferent than in the case of the all-trans form,[14] could be
involved in a photo-conversion of the 13-cis isomers to
other molecular configurations. On the other hand, the
operation of both mechanisms, the singlet fission and
intramolecular charge transfer, and/or other mecha-
nisms may not be excluded. An interesting and impor-
tant issue is the light-induced isomerization of
xanthophylls in the environment of THF that is observed
even at 118 K (see Figure S14). Taking into consider-
ation the fact that the freezing point of THF is at 164.8
K, one can presume that the photoisomerization
observed at 118 K involves a local melting of the pig-
ment-environment, induced by a thermal deexcitation. It
is noteworthy that light-driven isomerization of the 13-
cis forms of the macular xanthophylls was observed to
be substantially more effective at higher temperatures
(196 K and 298 K; see Figures 2–4 andS8). In addition to
the obvious kinetic reasons of such an effect one can
additionally consider a structure-stabilizing effect of
THF that is frozen at 118 K. The subtraction of the spec-
tra of the 9-cis and all-trans forms from the original
Raman spectra recorded from the chromatographically
pure samples of the 13-cis isomers yields the “pure”
spectra that can be attributed to the molecular

6
SEK ET AL.
configuration 13-cis (Figures S17–S22). As can be seen
from the comparison of the spectra derived for the 13-cis
forms of m-Zea, Zea and Lut, the procedure applied
yields very similar final spectra, independently of actual
laser power and differences in scaling factors selected to
subtract the spectra representing the contribution of
the 9-cis and all-trans forms. As mentioned above, the
light-induced conversions of the 13-cis forms of all the
macular xanthophylls are more efficient at the room
temperature (Figure S8) as compared with lower temper-
atures (196 K, Figure S11 and 118 K, Figure S14). This
can be judged based on a comparison of the spectral
splitting in the ν1 region at the same laser powers. On
the other hand, the other molecular configurations of all
the macular xanthophylls (all-trans and 9-cis) were
found to be photostable even at the room temperature,
in the same power range of the laser (see Figures S9 and
S10). The Raman spectra of the main molecular configu-
ration forms of Lut, Zea, and m-Zea, recorded at 196 K,
are presented in Figures 7–9 (and recorded at other tem-
peratures in Figures S25–S30). As can be expected,
FIGURE 8 Comparison of the resonance Raman spectra of
zeaxanthin in the molecular configurations all-trans, 9-cis, and 13-
cis, indicated. Recorded at 196 K, with a 488-nm laser, from the
samples in tetrahydrofuran
pigments in the configuration cis present ν1 bands
shifted towards higher wavenumbers with respect to
molecular configuration all-trans, owing to slightly
shortened conjugation length.[15,16] Importantly, the spe-
cific features of the Raman spectra of macular xantho-
phylls in the ν2 region correspond very well to the same
isoforms of β-carotene.[15,16] It can be concluded that
intensity of the peripheral band in the ν2 spectral region,
centering at ~1133 cm−1, relative to the intensity of the
principal ν2 band, centering at ~1157 cm−1, is diagnostic
and critical for an assignment of appearance of trans-cis
molecular configuration forms of carotenoids, including
the macular xanthophylls Lut, Zea, and m-Zea. Knowl-
edge of Raman spectra of molecular configuration forms
of the xanthophylls opens an avenue to study their pho-
tostability and possible photoconversion in complex nat-
ural systems, including the retina of the eye. The
resonance Raman spectra recorded at room temperature
with the 488-nm laser line from the macula lutea of the
human retina, prepared post mortem[7,17] and in vivo[17]
FIGURE 7 Comparison of the resonance Raman spectra of
lutein in the molecular configurations all-trans, 9-cis, 90-cis and the
mixture of 13-cis and 130-cis, indicated. Recorded at 196 K, with a
488-nm laser, from the samples in tetrahydrofuran

SEK ET AL.
7
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FIGURE 9 Comparison of the resonance Raman spectra of
meso-zeaxanthin in the molecular configurations all-trans, the
mixture of 9-cis and 90-cis, and the mixture of 13-cis and 130-cis,
indicated. Recorded at 196 K, with a 488-nm laser, from the
samples in tetrahydrofuran
are very close to the all-trans lutein, recorded in the pre-
sent work.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
ACKNOWLEDGEMENTS
This work was performed within the project “Xantho-
phylls in the retina of the eye” financed by the Founda-
tion for Polish Science (TEAM/2016-3/21).
How to cite this article: Sek A, Welc R, Mendes-
Pinto MM, et al. Raman spectroscopy analysis of
molecular configuration forms of the macular
xanthophylls. J Raman Spectrosc. 2020;1–7. https://
doi.org/10.1002/jrs.5818
ORCID
W.I. Gruszecki https://orcid.org/0000-0002-8245-3913
REFERENCES
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