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Isomerization of Cholecalciferol through Energy Transfer as a Protective Mechanism Against Flavin Sensitized Photooxidation Regina S. Scurachio, Willy Glen Santos, Eduardo S. P. Nascimento, Leif H. Skibsted, and Daniel Rodrigues Cardoso J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505958c • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 27, 2015
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Journal of Agricultural and Food Chemistry
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Isomerization of Cholecalciferol through Energy Transfer as a Protective
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Mechanism Against Flavin Sensitized Photooxidation
3 4
Regina S. Scurachioa, Willy G. Santosa, Eduardo S. P. do Nascimentoa, Leif H. Skibsted*,b, Daniel R. Cardoso*,a a
Instituto de Química de São Carlos, Universidade de São Paulo, Avenida Trabalhador
São Carlense 400, CP 780, 13560-470, São Carlos, Brazil. 5
6
b
7
Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Food Chemistry, Department of Food Science, University of Copenhagen,
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
* To whom the correspondence should be addressed: D.R.C e-mail:
26
[email protected] (Tel.: +55 16 33 73 99 76) or L.H.S. e-mail:
[email protected] 27
(Tel.: +45 35 33 32 21)
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Abstract
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Cholecalciferol, vitamin D3, was found to isomerize to 5,6-trans-vitamin-D3 with a
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quantum yield of 0.15 ± 0.01 in air-saturated tert-butanol/water 7:3 (v/v) at 25
32
increasing to 0.32 ± 0.02 in absence of oxygen, through quenching of triplet excited
33
flavin mononucleotide, FMN, rather than to become oxidized. The quenching was
34
found by laser-flash photolysis to have a rate constant of 1.4×108 L mol-1 s-1 in tert-
35
butanol:water 7:3 v/v at 25
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reactive vit.D3 diradical. Vit.D3 forms a 1:1 precomplex with FMN by hydrophobic
37
stacking with ∆Ho = -36 ± 7 kJ·mol-1 and ∆So = -4 ± 3 J·mol-1·K-1 as shown by single
38
photon counting fluorescence spectroscopy and steady state fluorescence spectroscopy.
39
Both ground state precomplex formation and excited state energy transfer seem
40
important for vit.D3 protection against flavin sensitized photooxidation of nutrients in
41
food and biological system.
C,
C assigned to energy transfer from 3FMN* to form a
42 43 44 45 46 47 48 49 50 51 52 53 54
Keywords: vitamin D, riboflavin, photochemistry, 5,6-trans-vitamin D
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Introduction
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Vitamins have varying reactivity depending on their biological functions. For
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some vitamins, the reactivity is modulated in pro-vitamins with better stability, and the
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vitamin function depends on an activation step. The lipophilic vitamin D provides such
67
examples as 7-dehydrocholesterol is found as a pro-vitamin D in food of animal origin
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and ergosterol is found as pro-vitamin D in food from the plant kingdom1,2. The
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activation step involves for both pro-vitamins a photochemical reaction to form
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cholecalciferol, vit.D3, or ergocalciferol , vit.D2, respectively, as occurring in human
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skin upon exposure to UV-B sunlight1-3. Among the water-soluble vitamins, vitamin B2
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is also photoactive since riboflavin, the form of vitamin B2 normally in foods, and the
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biological forms of vitamin B2, flavin mononucleotide (FMN) and flavin adenine
74
dinucleotide (FAD), are potent photosensitizer inducing oxidative damage in food and
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skin including degradation of the isoalloxazine ring common for these flavins4,5.
76 77
Vitamin B2 and vitamin D2 or D3 (Figure 1) or their pro-vitamin forms will
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interact upon light exposure when the two types of vitamins are present together. Milk
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and dairy products provides such examples where the lipophilic vitamin D forms are
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found in the lipid phase of the emulsions, while vitamin B2 is water-soluble2. Human
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skin provides another example, since both the different vitamin B2 forms including
82
flavoproteins located at plasma membrane and pro-vitamin D2 or D3 are present in the
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skin and become exposed to light
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of important nutrients in dairy products upon light exposure in reactions normally
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assigned to oxidation5. A more detailed understanding of the interaction between light
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activated forms of the flavins and the vitamin D homologues is, however, not available
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at present. Such knowledge at the mechanistic level would be important both in relation
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to improvement of stability of food exposure to light and also in relation to skin health5-
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7
6,7
. Riboflavin is known to initiate photodegradation
.
90 91
Vitamin B2 and vitamin D apparently have opposite effects on skin health upon
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light exposure, since riboflavin induces oxidative damage and radical formation with
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increased cancer risk, while the different forms of vitamin D apparently protects against
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light damage to the skin8-10. Most importantly, exposure of biological system containing
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both vitamin B2 and vitamin D to visible light and UV-A light will activate only the
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flavin vitamin forms due to the spectral characteristic of the two types of vitamins, and
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the activated flavin may react with vitamin D. We have accordingly undertaken
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photophysical studies using visible and UV-A light for activation of flavins and studied
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the interaction with vitamin D, the results of which investigations should be valuable for
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both protection of nutritive value of foods and for nutritional advises for better skin
101
health.
102 103
Materials and methods
104 105
Chemicals
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Cholecalciferol (vitamin D3), flavin mononucleotide (FMN), LiClO4 salt, and
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deuterated-methanol (CD3OD) were purchased from Sigma-Aldrich (Steinheim,
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Germany). Acetonitrile HPLC grade was purchased from Mallinckrodt (Phillipsburg,
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NJ). Tert-butanol (99.5%) and Tween®-20 (pure) were purchased from Acros Organics
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(Geel, Belgium). Water was purified (18 MΩ·cm) by means of a Milli-Q purification
111
system from Millipore (Billerica, MA).
112 113
Steady-State Fluorescence Spectroscopy
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Fluorescence measurements were carried out using a Hitachi F-7000 Fluorescence
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Spectrometer (Hitachi High-Tech, Tokyo, Japan). The samples were excited in 1.0 cm x
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1.0 cm fluorescence cuvettes from Hellma (Mulheim, Germany) at 440 nm. Emission
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spectra were recorded using a 2.0 nm band-pass for the excitation monochromator and a
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4.0 nm band-pass for the emission monochromator. The recorded spectra were corrected
119
for instrument response. The fluorescence was measured at 25.0 ± 0.2 oC in tert-
120
butanol/water solution (7:3 v/v).
121 122
Time Resolved Fluorescence Spectroscopy
123
Time-resolved fluorescence were measured by time-correlated single-photon counting
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using an picosecond spectrometer equipped with Glan-Laser polarizers (Newport,
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Irvine, CA), a Peltier-cooled PMTMCP from Hamamatsu model R3809U-50
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(Hamamatsu, Japan) as the photon detector, and Tennelec-Oxford (Oxford, Abingdon,
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UK) counting electronics. The light pulse was provided by frequency doubling the 200
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fs laser pulse of a Mira 900 Ti-Sapphire laser pumped by a Verdi 5 W coherent laser
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(Santa Clara, CA), and the pulse frequency was reduced to 800 kHz using a Conoptics
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pulse picker. The fluorescence decays were taken at the magic angle (λexc = 400 nm)
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and analyzed by a re-convolution procedure with instrument response function (irf) with
132
exponential decay models, and the goodness of the fit was evaluated by the statistical
133
parameters χ2. The fluorescence was measured at 25.0 ± 0.2 oC in tert-butanol/water
134
solution (7:3 v/v).
135 136
Laser Flash Photolysis
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Laser flash photolysis experiments were carried out with LFP-112 ns laser flash
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photolysis spectrometer from Luzchem (Ottawa, Canada) using the third harmonic (355
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nm) of a pulsed Q-switched Nd:YAG laser (BrilliantB, LesUlis, France) attenuated to
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10 mJ·cm−2 as the excitation source with 8 ns of pulse duration. Appropriate UV cuto
141
filters were used to minimize the sample degradation by the monitoring light. The signal
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from the monochromator/photomultiplier detection system was captured by a Tektronix
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TDS 2012 digitizer (Beaverton, OR). The laser system and the digitizer were connected
144
to a personal computer via General Purpose Instrumentation Bus (GPIB) and serial
145
interfaces controlling all the experimental parameters and providing suitable processing
146
and data storage capabilities. Each kinetic trace was averaged 16 times, and observed
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rate constants were determined by parameter fitting with MatLab R2013 to exponential
148
decay functions (Mathworks Inc., Natick, MA). All measurements were made with
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fresh tert-butanol/water 7:3 (v/v) solutions or Tween®-20 micelle (0.1 mol·l-1)
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thermostatted at 25.0 ± 0.5 °C and purged with high-purity N2 for at least 30 min before
151
the experiment.
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Steady State Photolysis
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A tert-butanol/water 7:3 (v/v) solution containing 1.0×10−4 mol·L−1 of FMN and
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1.0×10−3 mol·L−1 of vitamin D3 was exposed to 440 nm monochromatic light generated
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by a mercury lamp (HBO 200 w /4 short arc, Osram, Augsburg, Germany)
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accommodated with a focusing quartz lens, a water-filled heat filter and an Oriel
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narrow-band interference filter (center wavelength, 440 nm; Oriel Corp., Irvine, CA).
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The intensity of light, q0n,p , was measured by ferrioxalate actinometry11 and had the
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average value of 2.53×10−8 Einstein·min−1. The absorbance measurements were carried
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out employing a Hitachi U-3501 (Hitachi-Hitech, Japan) spectrophotometer.
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Cyclic Voltammetry
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Cyclic voltammetry of vit.D3 (1.0×10-3 mol L-1) dissolved in nitrogen-purged
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acetonitrile containing LiClO4 (0.5 M) were carried out using a PAR 264A Potentiostat
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(Princeton Applied Research, Princeton, NJ) equipped with a glass carbon as the
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working electrode, a platinum wire as the auxiliary electrode, and employing an
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Ag/AgCl reference electrode. The scan rate was 100 mV s-1, and the electrochemical
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cell was thermostated at 25.0 ± 0.2 oC.
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LC-DAD-SPE
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The irradiated sample of FMN (1×10-4 mol L-1) and vit. D (1×10-4 mol L-1) was
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analyzed by LC-DAD (Shimadzu Prominence 20A system, Shimadzu Co., Kyoto,
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Japan). Chromatographic separation was carried out on an Agilent Extend C18 reverse
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phase column (2.1mm x 150 mm x 50 µm) .The injection volume was 100 µL. The
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chromatographic separation was performed at a flow rate of 0.25 mL/min in isocratic
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mode consisting of 15% of solvent B in solvent A (A = ACN/H2O (80:20 v/v) and B =
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MeOH). The peaks of interest were detected using the UV response at 265 nm. After the
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chromatographic separation, peaks of interest were trapped on Hysphere C18 cartridges
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from Spark-Holland using a Prospek-II automatized SPE system (Spark-Holland,
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Emmen, Holland) hyphenated to the LC-DAD instrument; the target peak from 10
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chromatographic runs were sequentially trapped on the SPE cartridge to ensure enough
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quantity for the NMR analysis. Solvents used were non-deuterated. A make-up flow of
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eluent A was added to the post column eluate in a ratio of 10:1 in order to improve the
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retention. After the trapping process, the cartridges were dried with high purity nitrogen
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to remove residual solvents. Pure deuterated methanol was used to flush the peak from
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the SPE cartridge directly into the sample vial for posterior NMR analysis.
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Nuclear Magnetic Resonance Spectroscopy
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1
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spectrometer equipped with a 1H {13C, 15N} Triple-resonance Inverse Cryoprobe (TCI).
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The one-dimension 1H NMR experiment was based on the 1D version of the NOESY
194
sequence, using double presaturation suppression (methanol and water) during the
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relaxation delay (4 sec). The spectrum was acquired using 16 k data points in a
H NMR experiments were performed using a Bruker Avance-III 600 MHz NMR
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9578.54 Hz spectral width, giving an acquisition time of 3.42 sec. Processing was
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performed using exponential multiplication, applying a line broadening factor of 0.5 Hz.
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High-resolution Accurate Mass Spectrometry
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Direct infusion high-resolution accurate ESI-MS spectra of reaction products were
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performed on an LTQ-Orbitrap Thermo Fisher Scientific mass spectrometry system
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(Bremen, Germany) operating in the positive ion detection mode. General conditions:
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heater temperature: 50 oC, sheath gas flow rate: 5 a.u, spray voltage: 3.7 kV, capillary
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temperature: 275 oC, flow rate: 5µL/min.
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Results and discussion
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The biological active forms of vitamin B2, i.e. flavin mononucleotide (FMN) and
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flavin adenine dinucleotide (FAD) may be present in human skin together with vitamin
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D, cholecalciferol (vit. D3) or ergocalciferol (vit. D2), and certain vitamin D
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precursors3,5. FMN and FAD absorb visible light in contrast to vit. D as is seen from
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Figure 1 for FMN and vit.D3. Vitamin D precursors as well as vit. D absorb only UV-
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B light, which, however, initiates their conversion. Exposure to sunlight with its UV-B
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component is important for the vit. D status of humans due to these photoconversions
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and to other sterol photoreactions6. FAD and FMN form in contrast excited states by
216
absorption of visible light with the risk of inducing oxidative damage to the skin12. The
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clear spectral separation between FMN/FAD and vit. D3, as seen from Figure 2, gave
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inspiration to a novel approach for a photophysical investigation of a possible protective
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effect of vit. D3 against vit. B2 induced pathological conditions in human skin upon
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exposure to sunlight. No complications from spectral interference from vit. D3 was to be
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expected, as seen from Figure 2, upon excitation of FMN with 355 nm or with 440 nm
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monochromatic light, the wavelengths used for the time-resolved photophysical
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investigation of the triplet states of riboflavin and of the excited singlet state of
224
riboflavin, respectively, and their interaction with vit. D3.
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Exposure of FMN to 440 nm monochromatic light resulted in intense
227
fluorescence which for tert-butanol/water (7:3 v/v) as solvent centered at 540 nm with a
228
lifetime of 4.8 ns for 25 oC 13, see Figure 3 and 4. The fluorescence intensity decreased
229
in the presence of increasing concentrations of vit. D3 without affecting the fluorescence
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lifetime as probed by time resolved single photon counting fluorescence spectroscopy
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with 355 nm light excitation for experiments with a FMN concentration of 1.0×10-5
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mol L-1 and a vit. D3 concentrations of 1.0×10-5 mol L-1 or 1.0×10-4 mol L-1. The
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quenching of FMN fluorescence intensity by vit. D3 was analyzed accordingly to the
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Stern-Volmer equation for vit. D3 ranging from 8.0×10-7 mol L-1 to 1.0×10-6 mol L-1:
235
236
= 1 + × × [ . ]
(1)
237 238
where I0 is the fluorescence intensity at 525 nm in the absence of vit. D3, I is the
239
fluorescence emission intensity for increasing concentration of vit. D3, τ is the natural
240
lifetime of FMN singlet-excited state determined to have the value of 4.8 ns, see Figure
241
4, confirming previous findings13, and kq is the specific rate constant for singlet-excited
242
state quenching, see insert in Figure 3. The experimentally obtained Stern-Volmer
243
constants for different temperatures (15 oC, 25 oC, 35 oC and 45 oC) are collected in
244
Table 1. The high values observed for the second-order rate constant, which were above
245
the diffusion limit, indicate static quenching involving formation of a FMN-vit.D3
246
ground state precursor complex. The increase in temperature from 15 to 45 °C reduces
247
the observed quenching rate constant indicating formation of the ground state precursor
248
complex to be an exothermic process. Formation of a ground state complex was further
249
investigated by analyzing FMN fluorescence emission decay curves in the presence of
250
increasing concentration of vit. D3 (Figure 4). The normalized time decay curves for the
251
FMN singlet-excited emission did not change for increasing concentration of vit. D3
252
confirming the formation of the ground state precursor complex and static quenching.
253 254 255
The formation of this precursor complex may be represented by the following reaction:
256 257 258 259 260
Ka
n vit.D3 + FMN ⇌ [vit.D3]n-FMN
(2)
The number of quenchers binding () to the fluorophore FMN was calculated according to:
261 262
log[ − ] = log + × log[ . ]
(3)
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where is the equilibrium constant for the reaction of eq.(2). The experimental values
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determined for and for 4 different temperatures were obtained by fitting eq.(3) to
266
the data plotted in Figure 5A and are collected in Table 2. The thermodynamic
267
parameters for the ground state complex formation were found to have the values of
268
∆Ho = -36 ± 7 kJ mol-1 and ∆So = -3.9 ± 3.0 J mol-1 K-1 according to the Van’t Hoff
269
equation for a 1:1 stoichiometry, see Figure 5A and 5B. The binding of FMN to vit. D3
270
is suggested to be due to hydrophobic interaction, which often is an exothermic process
271
as is the present case. The small change in entropy for binding of FMN to vit. D3 seems
272
to indicate little solvent involvement. The isoalloxazine ring of FMN is hydrophobic
273
and has the possibility of binding to hydrophobic parts of the vitamin D3 through π-π
274
stacking14. The observed FMN fluorescence quenching by vit. D3 indicates interaction
275
between these two vitamins when in their ground states due to hydrophobic interaction.
276 277
As a consequence of an efficient inter-system crossing in singlet excited FMN
278
(and riboflavin and FAD) to their lowest-energy triplet states, these vit. B2 forms
279
become strongly reactive upon light exposure as in skin or in dairy products15. Vit. D3 is
280
a possible reactant for 3FMN in the skin, and reaction between 3FMN and vit. D3 was
281
studied using transient absorption spectroscopy following laser flash photolysis. The
282
triplet-tiplet absorption of 3FMN around 720 nm13,16 was used to monitor the decay of
283
the reactive 3FMN in the presence of increasing concentrations of Vit. D3, see Figure 6.
284
3
285
solvent according to:
FMN was found to decay mono-exponentially for tert-butanol/water (7:3 v/v) as
286 287
=
!
× "#$%&'() ×
(4)
288 289
and kobs was determined for increasing vit. D3 concentration. As may be seen from the
290
insert in Figure 6, kobs, showed a linear dependence on vit. D3 concentration:
291 292
*+, = + - [ .D3]
(5)
293 294
where k0 is the rate constant for the triplet-state self-decay and k2 is the second-order
295
rate constant for the triplet-state deactivation by vit. D3. The value of k2 = 1.4×108
296
L·mol-1·s-1 at 25 ⁰C (tert-butanol/water (7:3 v/v)) was calculated by linear regression
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(Figure 6) together with k0 = 5.0×104 s-1 corresponding to a natural triplet-FMN
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lifetime of 13 µs at 25 ⁰C in agreement with previous findings16. However, in aqueous
299
Tween®-20 micelles, as a probe for specific micro-domain reactivity of 3FMN towards
300
vit. D3 that is preferentially located inside the hydrophobic core of the micellar
301
aggregates, 3FMN was found to shown a tri-exponential decay curve (experimental
302
goodness of fit χ2 = 1.05) according to:
303 304
= .,
!
× "#$%&0,'() × + -,
!
× "#$%&1,'() × + ,
!
× "#$%&2,'() ×
(6)
305 306 307
From fitting eq.(6) to the experimental data in Figure 7A, the kobs for each
308
micro-domain were determined for increasing concentrations of vit. D3 together with the
309
estimated percentage distribution of 3FMN in the 3 observed micro-domains (average of
310
89.4 % in the continuous phase, 9.6 % at the interface, and 1 % at the disperse phase in
311
the presence of 0.83 mM of vit. D3) as extracted from the pre-exponential term of
312
eq.(6). Figure 7B, shows the linear dependence of kobs for each micro-domain with
313
increasing concentrations of vit. D3 yielding the second-order rate constant for the
314
triplet-state deactivation by vit. D3 in the three different environment: k2 = 6.9×109
315
L·mol-1·s-1 for the reacting at the continuous phase, k2 = 7.1×108 L·mol-1·s-1 for reaction
316
at the micelle interface and k2 = 7.2×107 L·mol-1·s-1 for the reaction inside the
317
hydrophobic core of the micellar aggregates. Figure 7A right panel illustrate the three
318
different micro-domains for deactivating 3FMN by vit. D3 in aqueous Tween®-20
319
micelles. The kinetic results in aqueous Tween®-20 micelles provides a strong evidence
320
that 3FMN may be deactivated by vit. D3 at the interface of the cell membrane or inside
321
the plasma membrane when 3FMN is present as a flavoprotein like NAD(P)H oxidase
322
and NAD(P)H quinone oxidoreductase.
323 324
The photochemical reaction quantum yield (3) for vit.D3 disappearance provides
325
a more practical measurement of the efficiency with which the reaction between the two
326
vitamins occurs upon visible UV-A light exposure occurs and may be determined from
327
eq.(7) for 440 nm light exposure:
328 329
3=
4*567856,9:; ?@* *A,;()'9(:>
=
B [BC D]>;9E % [BC D]F99;>F;=:> % .IJK G,H
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in which the ratio between the number of molecules of vit.D3 reacted and the number of
332
photons absorbed by vit.B2 was calculated from the results of HPLC analysis of vit.D3
333
exposed to and protected against light in a solution together with FMN and from
334
chemical actinometry. q0n,p is the light intensity for the 440 nm monochromatic light
335
used for exposure and Aλ is the absorbance at 440 nm of the tert-butanol/water 7:3 (v/v)
336
solution with vit.D3 and FMN. 3 for disappearance of vit.D3 in the photoreaction
337
sensitized by FMN was found to have the value of 0.15 ± 0.01 in air-saturated
338
butanol/water 7:3 (v/v) for a concentration of 1.0×10-3 mol.L-1 for vit.D3 and 1.0×10-4
339
mol.L-1 for FMN. For anaerobic conditions, a value of 0.32 ± 0.01 was found, both
340
values are means of three independent determinations at 25 oC.
341 342
The photoprocess of vit.D3 considered relates to the triplet state of FMN as a
343
photosensitizer, 3FMN*, and not of the singlet state, 1FMN*, since the lifetime of
344
3
345
seen from Figure 6 and 7 and Figure 5, respectively. The photochemical reaction
346
occurs accordingly as a sequence:
FMN* decreases in the presence of vit.D3 in contrast to the lifetime of 1FMN*, as is
347
FMN + hv → 1FMN*
348
(8)
349 1
ISC
FMN* → 3FMN*
350
(9)
351 352
3
FMN* + vit.D3 → FMN + photoproduct
(10)
353 354
in which initial excitation of FMN to form the excited singlet state is followed by an
355
efficient intersystem crossing (ISC) conversion to the reactive triplet state. 3FMN* is
356
reacting with vit.D3 to form a photoproduct, which was identified by high-resolution
357
accurate ESI-MS and LC-SPE-NMR to be 5,6-trans-vit.D3. The FMN/vit.D3 solution
358
was exposed to 440 nm monochromatic light for two hours. Prior to and following
359
irradiation, the solution was analyzed by LC-SPE and the chromatograms obtained are
360
shown in Figure 8. In chromatogram A shown in Figure 8, the peak 1 eluting at 20 min
361
refers to standard vit.D3. In chromatogram B shown in Figure 8, peak 2 eluting at 18
362
min refers to the photoproduct formed. Peak 2 was isolated by LC-SPE and further
363
analyzed by 1H NMR and ESI-MS. The electrospray high resolution accurate mass
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spectrum, recorded in the positive ion mode, showed a peak related to the photoproduct
365
with a pseudo-molecular ion at 407.3287 m/z with a error of 0.6 ppm and the expected
366
isotopic pattern calculated for C27H44ONa and assigned to a sodium adduct of an isomer
367
of vit.D3, see Figure 9.
368
With the purpose to obtain more information about the identity of the
369
photoproduct, the isolated photoproduct was analyzed by 1H NMR. The 1H NMR
370
spectrum obtained is shown in Figure 9A. The spectrum display signals in the region of
371
0 – 7 ppm. Based on the chemical shifts as reported in Table 3, in comparison with
372
literature values for vit.D3 isomers, it was concluded that the photoproduct was the
373
isomer 5,6-trans-vit.D317. The UV-vis absorption spectrum of the isolated photoproduct
374
(Figure 9B) with maximum absorption at 274 nm match the 5,6-trans-vit.D3
375
isomer17,18.
376 377
Riboflavin and its biological active forms, FMN and FAD, are known as strong
378
oxidants in their triplet excited state with E⊝ = + 1.7 V5. This very reactive triplet state
379
is formed by an efficient intersystem crossing from the initially populated singlet
380
excited state as the result of light absorption. For vit.D3, FMN was found to form a 1:1
381
precursor complex in the ground state resulting in static quenching as was previously
382
observed for cholesterol and ergosterol and assigned to hydrophobic L-L stacking13. The
383
singlet-excited state of FMN was found too short-lived for any bimolecular reactions to
384
occur with the lifetime of 4.8 ns. The reaction of light-activated FMN was accordingly
385
assigned to the triplet state and found to have a second-order rate constant of 1.4×108
386
L.mol-1.s-1 for tert-butanol/water (7:3 v/v) as solvent or of 6.9×109 L·mol-1·s-1 at the
387
continuous phase, 7.1×108 L·mol-1·s-1 at the micelle interface and 7.2×107 L·mol-1·s-1 at
388
the hydrophobic core of the micellar aggregates 13. This is faster than 3FMN* reaction
389
with most lipids including cholesterol but comparable with 3FMN* reaction with
390
ergosterol with a rate constant of 6.2×108 L.mol-1.s-1 13. The most important observation
391
for 3FMN* reaction with vit.D3 is, however, that 3FMN* is not oxidizing vit.D3. Other
392
lipophilic substrates and uric acid have been found to be oxidized by flavin triplet
393
states13,19. An estimate of the redox potential for vit.D3 was obtained by cyclic
394
voltammetry, see Figure 10, and with the value of +1.5 V, oxidation of vit.D3 by
395
3
396
dominates completely and no oxidation products of vit.D3 were detected. Isomerization
FMN* was shown to be possible with +1.7 V for triplet FMN. However, isomerization
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397
of vit.D3, to yield 5,6-trans-vit.D3 requires rotation around the conjugated double
398
bounds in vit.D3 and would be facilitated in a diradical triplet-state of vit.D3. The
399
formation of the diradical form of vit.D3 followed by rotation and recombination to a
400
vit.D3/5,6-trans-vit.D3 mixture apparently competes efficiently with electron transfer
401
from vit.D3 to 3FMN*:
402 403
3
FMN* + vit.D3 → FMN + 3vit.D3-diradical
(11)
3
FMN* + vit.D3 → 2FMN•- + 2vit.D3•+
(12)
404 405 406 407
An energy transfer mechanism involving the 3vit.D3 diradical is outlined in
408
Scheme 1A for photosensitized isomerization of vit.D3. The triplet energy of vit.D3 has
409
been estimated to be around 200 kJ.mol-1 while the energy of the triplet 5,6-trans-vit.D3
410
is lower with a value around 170 kJ.mol-1, both comparable to 3FMN* 20. The observed
411
quenching constant of k2 = 1.4×108 L.mol-1s-1 may thus be assigned to the reaction of
412
eq.(11) with the electron transfer reaction of eq.(12) being significantly slower.
413 414
An energy transfer mechanism rather than electron transfer could be explained
415
by a stepwise charge transfer exchange of electrons where vit.D3 donates an electron to
416
the lowest energy SOMO orbital of
417
[2FMN•- … 2vit.D3•+] followed by electron transfer from the SOMO orbital of the
418
2
419
FMN this exchange will deactivate 3FMN* to form ground state FMN, while for vit.D3
420
a triplet diradical is formed in order to conserve spin. The 3vit.D3 may rotate as shown
421
in Scheme 1A around the 5,6- and 7,8- bonds to form an equilibrium mixture prior to
422
deactivation. Notably, this mechanism entails that the trans-product formed may reform
423
the vit.D3 also in a photochemical process. The 5,6-trans-vit.D3 still have biological
424
effects in relation to calcium biomineralization7.
3
FMN* with formation of a radical ion pair
FMN•- to the LUMO orbital of 2vit.D3•+ of the radical ion pair, see Scheme 1B. For
425 426
The competition between deactivation of 3FMN* by 3O2 and vit.D3 depends on
427
second-order rate constants 9.8×108 L mol-1s-1 for oxygen and 1.4×108 L mol-1s-1 for
428
vit.D3 in the reaction of eq.(11) and the actual concentrations of quenchers to yield the
429
relative deactivation by vit.D3 to yield 5,6-trans-vit.D3:
430
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Relative FM ∗ deactivation by vit. D =
&]F=.^2 [_C .D2] &]F=.^2 [_C .D2 ]`&a1 [b1 ]
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(13)
432 433
The relative deactivation by vit.D3 corresponds accordingly, using [O2] = 3×10-4
434
mol.L-1 the value for water also for the mixed solvent, and [vit.D3] = 1×10-3 mol.L-1, to a
435
fraction of 0.32 which should be compared to the ratio between the quantum yield in the
436
presence and in the absence of oxygen which is seen to have an approximate value of
437
0.5. This is an acceptable agreement, and in the absence of electron transfer, this will
438
explain the surprising protective effect of oxygen against isomerization. Ground state
439
oxygen will form singlet oxygen, 1O2, but the reaction of 1O2 with vit.D3 apparently is
440
too slow to result in oxidation under the present conditions, and 1O2 may deactivate
441
thermally or react with FMN21.
442 443
Vitamin D3 was found to be an efficient quencher of triplet excited state of
444
FMN, one of the biological active forms of riboflavin, vitamin B2. Triplet flavins like
445
FMN are a strong oxidant, which is known to be harmful for skin and eyes under light
446
exposure and to deplete nutrients in milk during retail display4,21. A number of, also
447
otherwise biological important nutrients, are known to deactivate these photosensitizers
448
derived from vitamin B2 in food and tissue exposed to light5. Our findings are important
449
in the respect that we have shown that vit.D3 isomerize rather than is getting oxidized as
450
the result of this quenching reaction. The isomerization lead to a photostationary state
451
and vit.D3 is still active as a vitamin in this mixture and may reform vit.D3 from the
452
isomerized 5,6-trans-vit.D3 photochemically. These observations may adds a new
453
perspective to the role of vit.D3 accumulated in the skin as a protector against light
454
induced oxidative damage and radical formation from flavins acting as photosensitizers.
455 456
Acknowledgements
457
This research is part of the bilateral Brazilian/Danish Food Science Research Program
458
“BEAM - Bread and Meat for the Future” supported by FAPESP (Grant 2011/51555-7
459
and EMU 2009/54040) and by the Danish Research Council for Strategic Research
460
(Grant 11-116064). D.R.C. thanks the Brazilian National Research Council - CNPq for
461
the financial support (305385/2009-7 and 142145/2012-2). Prof. Marcelo H. Gehlen
462
(IQSC-USP) is acknowledged for the time-resolved single photon counting
463
measurements (FAPESP 2011/18215-8).
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464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512
References 1. Tsiaras, W. G.; Weinstock, M. A. Factors Influencing Vitamin D Status. Acta Derm. Venereol. 2011, 91, 115-124. 2. Gorman, A. A.; Hamblett, I.; Rodgers, M. A. J. Ergosterol (Provitamin D2) Triplet State: An Efficient Sensitiser of Singlet Oxygen, O2, Formation. Photochem. Photobiol. 1987, 45, 215-221. 3. Norman, A. W. Sunlight, Season, Skin Pigmentation, Vitamin D, and 25hydroxyvitamin D: Integral Components of the Vitamin D Endocrine System. Am. J. Clin. Nutr. 1998, 67, 1108–1110. 4. Li, T.; King, J. M.; Min, D. B. Quenching Mechanisms and Kinetics of Carotenoids in Riboflavin Photosensitized Singlet Oxygen Oxidation of Vitamin D2. J. Food Biochem. 2000, 24, 477-492. 5. Cardoso, D. R.; Libardi, S. H.; Skibsted, L. H. Riboflavin as a Photosensitizer. Effects on human health and food quality. Food Funct. 2012, 3, 487−502. 6. Wacker, M.; Holick M. F. Sunlight and Vitamin D a Global Perspective for Health. Dermato. Endocrinol. 2013, 5, 51-108. 7. Wondrak, G. T.; Jacobson, M. K.; Jacobson, E. L. Endogenous UVAphotosensitizers: mediators of skin photodamage and novel targets for skin photoprotection. Photochem. Photobiol. 2006, 5, 215-237. 8. Moan, J.; Porojnicu, A. C.; Dahlback, A.; Setlow, R. B. Addressing the Health Benefits and Risk, Involving Vitamin D or Skin Cancer, of Increased Sun Exposure. Proc. Natl. Acad. Sci. 2008, 105, 668-673. 9. Garland, C. F.; Gorham, E. D.; Mohr, S. B.; Garland, F. C. Vitamin D for Cancer Prevention: Global Perspective. Ann. Epidemiol. 2009, 19, 468-483. 10. Bikle, D. D. Protective Actions of Vitamin D in UVB Induced Skin Cancer. Photochem. Photobiol. Sci. 2012, 11, 1808-1816. 11. Harchard, C. G.; Parker, C. A. A New Sensitive Chemical Actinometer II. Potassium Ferrioxalate as a Standard Chemical Actinometer. Proc. R. Soc. London A. 1956, 235, 518−536. 12. Haywood, R.; Andrady, C.; Kassouf, N.; Sheppard, N. Intensity-dependent Direct Solar Radiation- and UVA-induced Radical Damage to Human Skin and DNA, Lipids, and Proteins. Photochem. Photobiol. 2011, 87, 117-130. 13. Cardoso, D. R.; Scurachio, R. S.; Santos, W. G.; Homem-de-Mello, P.; Skibsted, L. H. Riboflavin-Photosensitized Oxidation Is Enhanced by Conjugation in Unsaturated Lipids. J. Agric. Food Chem. 2013, 61, 2268-2275.
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513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
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14. Kamat, B. P.; Seetharamappa, J.; Melwanki, M. B. Spectroscopic Studies on the interaction of riboflavin with bovine serum albumin. Indian J. Biochem. Biophys. 2004, 41, 173-178. 15. Heelis, P. F. The Photophysical and Photochemical Properties of Flavins (Isoalloxazines). Chem. Soc. Rev. 1982, 11, 15-39. 16. Cardoso, D. R.; Franco, D. W.; Olsen, K.; Andersen, M. L.; Skibsted, L. H. Reactivity of Bovine Whey Proteins, Peptides and Amino Acids Toward Triplet Riboflavin as Studied by Laser Flash Photolysis. J. Agric. Food Chem. 2004, 52, 6602−6606. 17. Okamura, W. H.; Hammond, M. L.; Rego, A.; Norman, A. W.; Wing, R. M. Studies on Vitamin D (Calciferol) and its Analogues 12. Structural and Synthetic Studies of 5,6-Dihydrovitamin D3 and the Stereoisomers of 10,19-Dihydrovitamin D3 Including Dihydrotachysterol. J. Org. Chem. 1977, 42, 2284-2291. 18. Yamada, S.; Nakayama, K.; Takayama, H. Studies of Vitamin D Oxidation. 3. Dyesensitized Photooxidation of Vitamin D and Chemical Behavior of Vitamin D 6,19Epidioxides. J. Org. Chem. 1983, 48, 3477-3483. 19. Clausen, M. R.; Huvaere, K.; Skibsted, L. H.; Stagsted, J. Characterization of Peroxides Formed by Riboflavin and Light Exposure of Milk. Detection of Urate Hydroperoxide as a Novel Oxidation Product. J. Agric. Food Chem. 2010, 58, 481487. 20. Gielen, J. W. J.; Koolstra, R. B.; Jacobs, H. J. C.; Havinga, E. Triplet-sensitized Interconversion and Photooxidation of Vitamin D and trans-Vitamin D. J. Royal Netherl. Chem. Soc. 1980, 99, 306-311. 21. Jung. M. Y.; Oh, Y. S.; Kim, D. K.; Min, D. B. Photoinduced Generation of 2,3butanedione from Riboflavin. J. Agric. Food Chem. 2007, 55, 170-174.
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Figure Captions
Figure 1. Chemical structures for FMN, vitamin D3, and vitamin D2. Figure 2. UV−vis absorption spectra for solutions of flavin mononucleotide and vitamin D3 (both 1.0×10-4 mol L-1) in tert-butanol/water 7:3 (v/v) at 25 oC. Figure 3. Fluorescence emission spectra at 25 oC of flavin mononucleotide 1.0 × 10−4 mol·L−1 (in tert-butanol/water 7:3 (v/v)) for increasing concentration of vit.D3 from 8.0×10−7 to 8.0×10−6 mol L−1. Inset: Stern−Volmer plot, see equation 1. λexc = 440 nm and λemis =520 nm. Figure 4. Fluorescence lifetime measurements at 25 oC by time-resolved single photon counting for flavin mononucleotide 1.0×10−5 mol·L−1, in the absence or presence of 1.0×10−5 mol·L−1 or 1.0×10−4 mol·L−1 vitamin D3 in tert-butanol/water 7:3 (v/v). Irf = instrument response function.
Figure 5. A) Quenching of singlet excited state flavin mononucleotide fluorescence by vit.D3 at 25 0C in tert-butanol/water 7:3 (v/v), plotted according to equation 2 for static quenching. B) Van’t Hoff plot of the data extracted from Figure 5A for varying temperature (15, 25, 35 and 45 0C).
Figure 6. Kinetic traces for triplet-excited flavin mononucleotide decay monitored at 720 nm following 8 ns laser pulses of 10 mJ cm2 at 355 nm for increasing concentrations of vitamin D3 in N2-saturated in tert-butanol/water 7:3 (v/v) at 25 °C. Inset: kobs from exponential fitting of absorption decay (720 nm) versus vit.D3 concentration.
Figure 7. A) Kinetic traces for triplet-excited flavin mononucleotide decay monitored at 720 nm following 8 ns laser pulses of 10 mJ cm2 at 355 nm for increasing concentrations of vitamin D3 in N2-saturated aqueous Tween®-20 micelles at 25 °C (right panel) and graphical illustration of the micro-domains for triplet-excited flavin mononucleotide in aqueous Tween®-20 micellar solution. B) kobs from tri-exponential fitting of absorption decay (720 nm) versus vit.D3 concentration for the reaction at
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different micro-domains (continuous phase, micelle interface and hydrophobic core of the micellar aggregates).
Figure
8.
LC-DAD-SPE
chromatograms
monitored
at
265
nm
of
flavin
mononulceotide/vit.D3 solution in tert-butanol/water 7:3 (v/v) prior to light exposure (A) and following 120 min of exposure to 440 nm monochromatic light.
Figure 9. Spectroscopic characterization of photoproduct from exposure of flavin mononucleotide/Vit.D3 solution in tert-butanol/water 7:3 (v/v) for 120 min with 440 nm monochromatic light. A) 1H-NMR spectrum of photoproduct from isolated peak 2 of chromatogram B of Figure 6. B) ESI-MS high-resolution accurate mass spectrum of photoproduct from peak 2 of chromatogram B in Figure 6. Inset in B: UV-vis absorption spectrum of photoproduct. Figura 10. Cyclic Voltammetry of vitamin D3 (1×10-3 mol·L-1) in acetonitrile solution containing 50 mM of LiClO4 at 25 °C.
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Table 1. Second-order rate constant of singlet-excited state riboflavin quenching, 1kq, by vitamin D3 at different temperatures. Temperature (ºC)
1
kq ( L mol-1 s-1)
15
3.6×1012
25
3.5×1012
35
3.4×1012
45
3.3×1012
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Table 2. Association constant (Ka) and number of binding quenchers (n) for formation of the vit.D3−FMN ground state precomplex at different temperatures in tertbutanol/water 7:3 (v/v).
T (⁰C)
Ka (L.mol-1)
n
15
6.9 ± 0.1×104
1.10 ± 0.02
25
3.7 ± 0.2×10
4
0.75 ± 0.01
35
3.4 ± 0.2×104
1.04 ± 0.03
45
1.5 ± 0.2×104
0.96 ± 0.02
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Table 3. Chemical shift for hydrogen in
1
H-NMR spectrum of product of
cholecalciferol reaction with light-activated flavin mononucleotide in tert-butanol/water 7:3 (v/v), see Figure 7A, in comparison with chemical shift in spectrum of 5,6-transvit.D3 from literature17,18,20. Assignment
δ (measured)
δ (literature)
H-6
6.43
6.50
H-7
5.85
5.84
H-19Z
4.97
4.95
H-19E
4.63
4.65
H-3α
3.90
3.86
H-4α
2.85
2.84
H-9β
2.85
2.84
H-1β
2.42
2.44
H-4β
2.31
2.31
H-1α
2.19
2.17
H-2α
1.91
1.94
H-2β
1.55
1.58
CH3-21
0.91
0.92
(CH3)2-26,27
0.85
0.87
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CH3
N
CH3
CH 3
CH3
R
Page 22 of 33
CH 3
CH3 CH3
CH3
N
O
H
NH
N
H
H
H
CH2
CH 2
CH3
O
R= C5H 11PO 7 (FMN)
HO
HO vitamin D3
vitamin D2
Figure 1.
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Journal of Agricultural and Food Chemistry
1.2
1.0
Absorbance
Vitamin D3 FMN 0.8
0.6
0.4
0.2
0.0 300
400
500
600
λ (nm)
Figure 2.
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1.20
10000 I0/I
1.15
8000
1.10
Intensity a.u.
1.05
6000
1.00 0.0
-6
2.0x10
-6
4.0x10
-6
6.0x10
-6
8.0x10
-5
1.0x10
[vitamin D3] (molL-1)
4000
2000
0 450
500
550
600
650
λ (nm)
Figure 3.
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FMN irf FMN + vit D3 1.0. 10
log (Photon Counting)
1000
FMN + vit D3 1.0. 10
-5
-1 mol.L
-4
-1 mol.L
100
10
1 0
10
20
30
40
time (ns)
Figure 4.
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-0.6
Page 26 of 33
A)
-0.8
log[(I0 - I) / I]
-1.0 -1.2 -1.4 -1.6 -1.8 -2.0 -6.2
-6.0
-5.8
-5.6
-5.4
-5.2
-5.0
log [vit D3]
11.2
B)
11.0 10.8
ln K
10.6 10.4 10.2 Van't Hoff equation 0 0 ln(K) = -∆Η /RT + ∆S /R
10.0 9.8 9.6 9.4 3.8x10
-4
3.9x10
-4
4.0x10
-4
4.1x10
-4
4.2x10
-4
1/RT
Figure 5.
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4
2.0x10
∆Abs Normalized at 720 nm
kobs (s-1 )
1.2 1.0
4
1.5x10
4
1.0x10
-5
0.0
-5
1.0x10
-5
2.0x10
3.0x10
-5
4.0x10
[vitamin D3] (mol.L-1)
0.8
-4
FMN 1.0 10 M -4 -5 FMN 1.0 10 M+ Vit D 5.0 10 M -4 -5 FMN 1.0 10 M+ Vit D 1.0 10 M -4 -5 FMN 1.0 10 M+ Vit D 2.0 10 M -4 -5 FMN 1.0 10 M+ Vit D 3.0 10 M -4 -5 FMN 1.0 10 M+ Vit D 4.0 10 M -4 -4 FMN 1.0 10 M+ Vit D 5.0 10 M -4 -4 FMN 1.0 10 M+ Vit D 1.0 10 M
0.6 0.4 0.2 0.0 0
5
10
15
20
25
time (µs)
Figure 6.
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A) 0.030 0.025
∆Abs at 720 nm
FMN
FMN 1.0 10-5 M -5 -4 FMN 1.0 10 M + Vit D 1.7 10 M -5 -4 FMN 1.0 10 M + Vit D 3.3 10 M FMN 1.0 10-5 M + Vit D 5.0 10-4 M -5 FMN 1.0 10 M + Vit D 8.3 10-4 M
0.020
Vitamin D3 Tween-20
0.015
Micellar border 0.010 0.005 0.000 -0.005 0
20
40
60
80
100
time (µs)
-1
kobs (s )
Continuum phase
Interface phase
B) 6x106 3x106
k2 = 6.9X109 M-1s-1
0 6x105 3x105
k2 = 7.1X108 M-1s-1
Disperse phase
0 8x104 4x104
k2 = 7.2 X 107 M-1s-1
0 0
2x10-4
4x10-4
6x10-4
8x10-4
-1
[vitamin D3] (M )
Figure 7.
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2000
A 1
Intensity
1500
1000
500
0 0
3
6
9
12
15
18
21
24
time (min)
1600 1400
B
1200
1
Intensity
1000 800 600 400 2 200 0 0
3
6
9
12
15
18
21
24
time (min)
Figure 8.
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A
vitD pos_130320165910 #14 RT: 0.29 AV: 1 NL: 2.24E6 T: FTMS + p ESI Full ms [150.00-500.00] 407.3287 R=33601 C 27 H 44 O Na 0.5821 ppm 100 95
B
90 85 80 75 70
Relative Abundance
65 60 55 50 45 40
408.3319 R=33404 C 26 13C H 44 O Na 0.3617 ppm
35 30 25 20 15 10 5 0 407.2
407.3
407.4
407.5
407.6
407.7
407.8
407.9 m/z
408.0
408.1
408.2
408.3
408.4
408.5
408.6
Figure 9.
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-5
6.0x10
-5
5.0x10
-5
4.0x10
-5
I(A)
3.0x10
-5
2.0x10
-5
1.0x10
0.0 -5
-1.0x10
-5
-2.0x10
-0.5
0.0
0.5
1.0
1.5
2.0
Potential (V vs Ag/AgCl)
Figura 10.
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Scheme 1. Proposed reaction mechanism for vitamin D3 isomerization throught charge transfer exchange of electrons (energy transfer) from triplet state flavin mononucleotide yielding 5,6-trans-vitamin D3 A)
R N
N
O
NH N O
energy transfer
R N
HO N
O
HO
vitamin D3
NH N O
OH
5,6-trans-vitamin D3
B) SOMO beta
LUMO
SOMO
LUMO
SOMO alpha
HOMO
HOMO
SOMO
3 FMN*
vit.D3
2FMN
2vit.D
3
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LUMO
SOMO beta
HOMO
SOMO alpha
FMN
3vit.D * 3
32
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