Isomerization of Cholecalciferol through Energy Transfer as a Protective Mechanism Against Flavin-Sensitized Photooxidation.

Cholecalciferol, vitamin D3, was found to isomerize to 5,6-trans-vitamin-D3 with a quantum yield of 0.15 ± 0.01 in air-saturated 7/3 tert-butyl alcohol/water (v/v) at 25 °C, increasing to 0.32 ± 0.02 in the absence of oxygen, through quenching of triplet excited state flavin mononucleotide, FMN, rather than becoming oxidized. The quenching was found by laser flash photolysis to have a rate constant of 1.4 × 10(8) L mol(-1) s(-1) in 7/3 tert-butyl alcohol/water (v/v) at 25 °C, assigned to energy transfer from (3)FMN* to form a reactive vit.D3 diradical. vit.D3 forms a 1/1 precomplex with FMN by hydrophobic stacking with ΔH° = -36 ± 7 kJ mol(-1) and ΔS° = -4 ± 3 J mol(-1) K(-1), as shown by single photon counting fluorescence spectroscopy and steady-state fluorescence spectroscopy. Both ground-state precomplex formation and excited-state energy transfer seem important for vit.D3 protection against flavin-sensitized photooxidation of nutrients in food and biological systems.

Photooxidation of other B-vitamins as sensitized by riboflavin.

Pyridoxal phosphate (PLP) was found to deactivate triplet-excited riboflavin (Rib) in aqueous solution with a deactivation constant of 3.0 ± 0.1 × 10(8) L mol(-1) s(-1) at 25 °C. Likewise, PLP was found to quench the fluorescence emission of (1)Rib* with (1)kq = 1.0 ± 0.1 × 10(11) L mol(-1) s(-1) as determined by steady state fluorescence. The rather high quenching constant suggests the formation of a ground state complex, which was further confirmed by time-resolved fluorescence measurements to yield a (1)Rib* deactivation constant of 3.4 ± 0.4 × 10(10) L mol(-1) s(-1). Triplet quenching is assigned as one-electron transfer rather than hydrogen-atom transfer from PLP to (3)Rib*, as the reaction quantum yield, Φ = 0.82, is hardly influenced by solvent change from water to D2O, Φ = 0.78. Neither biotin nor niacin deactivates the singlet- or triplet-excited riboflavin as it is expected from their higher oxidation potentials E > 2 V vs NHE.

Riboflavin-photosensitized oxidation is enhanced by conjugation in unsaturated lipids.

Methyl esters of polyunsaturated fatty acids were found to quench triplet-excited riboflavin ((3)Rib) in efficient bimolecular reactions with rate constants, as determined by laser flash photolysis, linearly depending upon the number of bis-allylic methylene (from 1 to 5). Deactivation of (3)Rib is predicted by combining the experimental second-order rate constants k2 determined for acetonitrile/water (8:2, v/v) at 25 °C with density functional theory (DFT) calculations of bond dissociation energy to have an upper limiting value of 1.22 × 10(7) L mol(-1) s(-1) for hydrogen abstraction from bis-allylic methylene groups in unsaturated lipid by (3)Rib. Still, ergosterol was found to deactivate (3)Rib with k2 = 6.2 × 10(8) L mol(-1) s(-1), which is more efficient than cholesterol, with 6.9 × 10(7) L mol(-1) s(-1). Likewise conjugated (9E,11E) methyl linoleate (CLA) reacts with 3.3 × 10(7) L mol(-1) s(-1), 30 times more efficient than previously found for methyl α-linolenate. Conjugation as in CLA and ergosterol is concluded to enhance (3)Rib deactivation, and dietary plant sterols and CLA may accordingly be important macronutrients for eye and skin health, protecting against light exposure through efficient deactivation of (3)Rib.

Photodegradation of folate sensitized by riboflavin.

Folate is shown to react with singlet-excited state of riboflavin in a diffusion controlled reaction and with triplet-excited state of riboflavin in a somewhat slower reaction with (3)k(q) = 4.8 × 10(8) L mol(-1) s(-1) in aqueous phosphate buffer at pH 7.4, ionic strength of 0.2 mol L(-1), and 25°C. Singlet quenching is assigned as photo-induced reductive electron transfer from ground state folate to singlet-excited riboflavin, while triplet quenching is assigned as one-electron transfer rather than hydrogen atom transfer from folate to triplet-excited riboflavin, as the reaction quantum yield, φ = 0.32, is hardly influenced by solvent change from water to deuterium oxide, φ = 0.37. Cyclic voltammetry showed an irreversible two-electron anodic process for folate, E = 1.14 V versus NHE at a scan-rate of 50 mV s(-1), which appears to be kinetically controlled by the heterogeneous electron transfer from the substrates to the electrode. Main products of folate photooxidation sensitized by riboflavin were pterin-6-carboxylic acid and p-aminobenzoyl-L-glutamic acid as shown by liquid chromatographic ion-trap mass spectrometry (LC-IT-MS).