Isomerization of Cholecalciferol through Energy Transfer as a

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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

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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:

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[email protected] (Tel.: +55 16 33 73 99 76) or L.H.S. e-mail: [email protected]

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(Tel.: +45 35 33 32 21)

ACS Paragon Plus Environment

1


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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.

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Both ground state precomplex formation and excited state energy transfer seem

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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

55 56 57 58 59 60 61


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Introduction

63 64

Vitamins have varying reactivity depending on their biological functions. For

65

some vitamins, the reactivity is modulated in pro-vitamins with better stability, and the

66

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

68

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

70

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

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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

78

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

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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-

89

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

93

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

98

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

115

Spectrometer (Hitachi High-Tech, Tokyo, Japan). The samples were excited in 1.0 cm x

116

1.0 cm fluorescence cuvettes from Hellma (Mulheim, Germany) at 440 nm. Emission

117

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

124

using an picosecond spectrometer equipped with Glan-Laser polarizers (Newport,

125

Irvine, CA), a Peltier-cooled PMTMCP from Hamamatsu model R3809U-50

126

(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

128

fs laser pulse of a Mira 900 Ti-Sapphire laser pumped by a Verdi 5 W coherent laser

129

(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)

131

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

137

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

139

nm) of a pulsed Q-switched Nd:YAG laser (BrilliantB, LesUlis, France) attenuated to

140

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

142

from the monochromator/photomultiplier detection system was captured by a Tektronix

143

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

147

rate constants were determined by parameter fitting with MatLab R2013 to exponential

148

decay functions (Mathworks Inc., Natick, MA). All measurements were made with

149

fresh tert-butanol/water 7:3 (v/v) solutions or Tween®-20 micelle (0.1 mol·l-1)

150

thermostatted at 25.0 ± 0.5 °C and purged with high-purity N2 for at least 30 min before

151

the experiment.

152 153

Steady State Photolysis

154

A tert-butanol/water 7:3 (v/v) solution containing 1.0×10−4 mol·L−1 of FMN and

155

1.0×10−3 mol·L−1 of vitamin D3 was exposed to 440 nm monochromatic light generated

156

by a mercury lamp (HBO 200 w /4 short arc, Osram, Augsburg, Germany)

157

accommodated with a focusing quartz lens, a water-filled heat filter and an Oriel

158

narrow-band interference filter (center wavelength, 440 nm; Oriel Corp., Irvine, CA).

159

The intensity of light, q0n,p , was measured by ferrioxalate actinometry11 and had the

160

average value of 2.53×10−8 Einstein·min−1. The absorbance measurements were carried

161

out employing a Hitachi U-3501 (Hitachi-Hitech, Japan) spectrophotometer.

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163 164

Cyclic Voltammetry

165

Cyclic voltammetry of vit.D3 (1.0×10-3 mol L-1) dissolved in nitrogen-purged

166

acetonitrile containing LiClO4 (0.5 M) were carried out using a PAR 264A Potentiostat

167

(Princeton Applied Research, Princeton, NJ) equipped with a glass carbon as the

168

working electrode, a platinum wire as the auxiliary electrode, and employing an

169

Ag/AgCl reference electrode. The scan rate was 100 mV s-1, and the electrochemical

170

cell was thermostated at 25.0 ± 0.2 oC.

171 172

LC-DAD-SPE

173

The irradiated sample of FMN (1×10-4 mol L-1) and vit. D (1×10-4 mol L-1) was

174

analyzed by LC-DAD (Shimadzu Prominence 20A system, Shimadzu Co., Kyoto,

175

Japan). Chromatographic separation was carried out on an Agilent Extend C18 reverse

176

phase column (2.1mm x 150 mm x 50 µm) .The injection volume was 100 µL. The

177

chromatographic separation was performed at a flow rate of 0.25 mL/min in isocratic

178

mode consisting of 15% of solvent B in solvent A (A = ACN/H2O (80:20 v/v) and B =

179

MeOH). The peaks of interest were detected using the UV response at 265 nm. After the

180

chromatographic separation, peaks of interest were trapped on Hysphere C18 cartridges

181

from Spark-Holland using a Prospek-II automatized SPE system (Spark-Holland,

182

Emmen, Holland) hyphenated to the LC-DAD instrument; the target peak from 10

183

chromatographic runs were sequentially trapped on the SPE cartridge to ensure enough

184

quantity for the NMR analysis. Solvents used were non-deuterated. A make-up flow of

185

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

187

to remove residual solvents. Pure deuterated methanol was used to flush the peak from

188

the SPE cartridge directly into the sample vial for posterior NMR analysis.

189 190

Nuclear Magnetic Resonance Spectroscopy

191

1

192

spectrometer equipped with a 1H {13C, 15N} Triple-resonance Inverse Cryoprobe (TCI).

193

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

195

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

197

performed using exponential multiplication, applying a line broadening factor of 0.5 Hz.

198 199

High-resolution Accurate Mass Spectrometry

200

Direct infusion high-resolution accurate ESI-MS spectra of reaction products were

201

performed on an LTQ-Orbitrap Thermo Fisher Scientific mass spectrometry system

202

(Bremen, Germany) operating in the positive ion detection mode. General conditions:

203

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

207 208

The biological active forms of vitamin B2, i.e. flavin mononucleotide (FMN) and

209

flavin adenine dinucleotide (FAD) may be present in human skin together with vitamin

210

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

212

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

214

component is important for the vit. D status of humans due to these photoconversions

215

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

218

inspiration to a novel approach for a photophysical investigation of a possible protective

219

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

222

monochromatic light, the wavelengths used for the time-resolved photophysical

223

investigation of the triplet states of riboflavin and of the excited singlet state of

224

riboflavin, respectively, and their interaction with vit. D3.

225 226

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

231

with 355 nm light excitation for experiments with a FMN concentration of 1.0×10-5

232

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)

263


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264

where is the equilibrium constant for the reaction of eq.(2). The experimental values

265

determined for and for 4 different temperatures were obtained by fitting eq.(3) to

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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

298

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


(7)

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330 331

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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364

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


12

Page 13 of 33


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


13


431

Relative FM ∗ deactivation by vit. D =

&]F=.^2 [_C .D2] &]F=.^2 [_C .D2 ]`&a1 [b1 ]

Page 14 of 33

(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).


14

Page 15 of 33


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.


15


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

Page 16 of 33

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.

559


16

Page 17 of 33


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


17


Page 18 of 33

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.


18

Page 19 of 33


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


19


Page 20 of 33

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


20

Page 21 of 33


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


21


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.


22

Page 23 of 33


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.


23


Page 24 of 33

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.


24

Page 25 of 33


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.


25


-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.


26

Page 27 of 33


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.


27


Page 28 of 33

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.


28

Page 29 of 33


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.


29


Page 30 of 33

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.


30

Page 31 of 33


-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.


31


Page 32 of 33

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


LUMO

SOMO beta

HOMO

SOMO alpha

FMN

3vit.D * 3

32

Page 33 of 33


For Table of Contents Only


33

Isomerization of Cholecalciferol through Energy Transfer as a

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