Kinetics for Singlet Oxygen Formation by Riboflavin Photosensitization and the Reaction between Riboflavin and Singlet Oxygen

ABSTRACT: The formation of singlet oxygen by riboflavin and the kinetics and mechanisms of riboflavin degradation in aqueous solution under light were determined. The singlet oxygen formation rate by riboflavin was 2.31 μmole oxygen/mL headspace/h of serum bottle. The degradations of riboflavin were 66% in D₂O and 40% in H₂O, respectively, under light after 24 h. The results indicate that singlet oxygen is involved in riboflavin destruction under light. The riboflavin destructions were 94.0% and 15.7% with 0 mM or 160 mM ascorbic acid, respectively, under light after 96 h. The reaction rate between riboflavin and singlet oxygen was 1.01 × 10¹⁰/M/s, which is a diffusion-controlled reaction rate. This explains the extremely fast degradation of riboflavin in foods under light. Ascorbic acid and sodium azide reduce the degradation of riboflavin under light with different quenching mechanisms. Ascorbic acid quenched both singlet oxygen and excited triplet riboflavin. Sodium azide quenched only the singlet oxygen in riboflavin solution with a quenching rate of 1.547 × 10⁷/M/s. With the involvement of both the Type-I and Type-II mechanisms in the riboflavin degradation under light, singlet oxygen quencher alone could not protect the riboflavin from degradation completely. Addition of ascorbic acid can protect riboflavin oxidation in foods exposed to light.     

Antioxidant mechanisms of Trolox and ascorbic acid on the oxidation of riboflavin in milk under light

Antioxidant activities and the mechanism of water-soluble Trolox and ascorbic acid on the oxidation of riboflavin in milk were studied. Trolox or ascorbic acid at 0, 100, 250, 500, or 1000 ppm was added to milk with or without added 50 ppm riboflavin and stored under light at 27 °C. Headspace oxygen was analysed by GC and Trolox, ascorbic acid, and riboflavin were determined by HPLC. The headspace oxygen of milk with added 50 ppm riboflavin depleted faster than that of milk without added riboflavin (p < 0.05). Trolox and ascorbic acid decreased during storage under light and riboflavin was completely destroyed within 24 h. As the concentration of Trolox or ascorbic acid increased, the riboflavin loss decreased. Riboflavin, Trolox, and ascorbic acid competed to react with singlet oxygen which was formed in the presence of riboflavin under light. Trolox and ascorbic acid protected riboflavin in milk under light by reacting with singlet oxygen.     

Effects of Riboflavin Photosensitization on the Degradation of Bisphenol A (BPA) in Model and Real-Food Systems

ABSTRACT:  Effects of riboflavin photosensitizations on the stability of bisphenol A (BPA), a well-known endocrine disrupting chemical, were studied in model and real-food systems by high-performance liquid chromatography (HPLC). Concentration of BPA was significantly decreased under light exposure ( P < 0.05) as the concentration of riboflavin increased while those without riboflavin under light or those with riboflavin in the dark did not change significantly ( P > 0.05). Addition of 50, 100, and 200 μM sodium azide significantly increased the stability of BPA in riboflavin photosensitization with concentration dependent manner ( P < 0.05), implying that a singlet oxygen or type II pathway played a role in the photodegradation of BPA. Stability of BPA in riboflavin was significantly increased in the presence of high concentration of tert -butanol, a hydroxyl radical quencher, under light storage for 80 min, indicating hydroxyl radicals were involved and contributed to the degradation of BPA, at least in part. Availability of riboflavin photosensitization on the photodegradation of BPA was tested in 2 canned tea beverages with different phenolic contents. BPA was more stable in the beverage sample with higher total phenolic contents and free radical scavenging ability. The photodegradation of BPA in riboflavin photosensitization can be an efficient way to decrease the concentration of BPA from environmental or food systems.     

Effects of Trolox and ascorbic acid on the riboflavin photosensitised oxidation of aromatic amino acids

The effects of 0, 100, 250, 500, 750 or 1000 ppm Trolox and ascorbic acid on the singlet oxygen oxidation of aromatic amino acids viz. phenylalanine, tryptophan, tyrosine in the presence of 25 ppm riboflavin were determined by measuring depleted headspace oxygen by gas chromatography and aromatic amino acid content by high performance liquid chromatography. The coefficients of variations (CVs) for headspace oxygen analysis and HPLC analysis of aromatic amino acids were 1.4% and 4.7%, respectively. Samples were stored under light (1000 lux) at 30 °C for 10 h. Both Trolox and ascorbic acid acted as antioxidants. As the concentration of Trolox and ascorbic increased from 0 to 1000 ppm, the head space oxygen depletion increased. This was due to the oxidation of Trolox and ascorbic acid along with amino acids in the presence of riboflavin. High performance liquid chromatography analysis of the samples clearly indicated that both Trolox and ascorbic acid minimised the degradation of phenylalanine, tryptophan, tyrosine significantly (p < 0.05), but did not prevent their oxidation completely. Trolox acted as a better antioxidant than ascorbic acid in protecting phenylalanine, tryptophan and tyrosine. Type-I mechanism was mainly responsible for riboflavin photosensitised degradation of aromatic amino acids.     

Singlet Oxygen Detection in Skim Milk by Electron Spin Resonance Spectroscopy

ABSTRACT: 2,2,6,6-Tetramethyl-4-piperidone (TMPD) can react with singlet oxygen to produce the corresponding nitroxide, 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TAN), which can be detected by electron spin resonance (ESR) spectroscopy. ESR detected the formation of TAN in 5 μM riboflavin phosphate buffer, or skim milk containing 20 mM TMPD during illumination. The effects of illumination, riboflavin, and oxygen on the formation of TAN indicated that the presence of each was required for singlet oxygen formation. The photo reduced riboflavin might reduce TAN to the hydroxylamine of TAN, thus decreasing the paramagnetic TAN signal. Maximum concentration of TAN was obtained at an added 10 mM riboflavin in buffer solution or in skim milk after 15 min of illumination in the presence of oxygen.     

Singlet Oxygen Quenching Activities of Various Fruit and Vegetable Juices and Protective Effects of Apple and Pear Juices against Hematolysis and Protein Oxidation Induced by Methylene Blue Photosensitization

ABSTRACT:  Effects of various fruit and vegetable juices on rubrene oxidation induced by a chemical source of singlet oxygen in a microemulsion system have been studied. The singlet oxygen quenching activities of fruit and vegetable juices were greatly different with different juices. The apple and pear juices exhibited the highest antioxidative activity among the tested juices in singlet oxygen–induced rubrene oxidation, showing 56.69% and 59.34% inhibition, respectively. The grape, kumquat, red cabbage, and spinach juices also showed relatively strong antioxidative activity against singlet oxygen–induced rubrene oxidation. Lemon juice showed the least activity, resulting in 0.63% inhibition of rubrene oxidation. The singlet oxygen quenching activities of 1 mL of apple and pear juices were equivalent to 33.97 and 34.64 mg ascorbate, respectively. Singlet oxygen quenching activities of juices had very low correlation with both ABTS radical scavenging activity ( R ²= 0.11) and total phenolic contents ( R ² < 0.1). However, a high correlation ( R ²= 0.66) was found between the ABTS radical scavenging activities and total phenolic contents of juices. The apple and pear juices also significantly inhibited both erythrocyte lysis and protein oxidation induced by fluorescence light illumination in the presence of methylene blue. Electron spin resonance (ESR) spectroscopy data showed that the protective activities of these juices against biological damages induced by photodynamic ways were, to at least some extent, due to their singlet oxygen quenching abilities. This represents first report on the singlet oxygen quenching activities of the apple and pear juices, and their protective activities against photodynamically induced biological damages.     

Stability of Bisphenol A (BPA) in Oil-In Water Emulsions under Riboflavin Photosensitization

Effects of riboflavin photosensitization on the degradation of bisphenol A (BPA) were determined in oil-in-water (O/W) emulsions containing ethylenediaminetetraacetic acid (EDTA) or sodium azide, which are a metal chelator or a singlet oxygen quencher, respectively. Also, the distribution of BPA between the continuous and dispersed phases in O/W emulsions was analyzed by high-performance liquid chromatography (HPLC). The concentration of BPA in O/W emulsions significantly decreased by 38.6% after 2 h under visible light irradiation and in the presence of riboflavin (P < 0.05). Addition of EDTA and sodium azide protected the decomposition of BPA significantly in a concentration dependent manner (P < 0.05), which implies both transition metals and singlet oxygen accelerate the photodegradation of BPA in O/W emulsions. Approximately 21.7% of the BPA was distributed in the 2.5% (w/v) dispersed lipid particles and 78.3% was in the continuous aqueous phase of the emulsions. The amount of BPA in aqueous phase decreased faster than the amount of BPA in the lipid phase during riboflavin photosensitization (P < 0.05). Thus, the BPA in the aqueous phase was the major target of riboflavin photodegradation in O/W emulsions. Practical Application: Concentration of BPA, an endocrine disrupting chemical, was decreased significantly in oil-in-water emulsions under riboflavin and visible light irradiation. BPA in continuous aqueous phase was major target of riboflavin photosensitization. However, BPA was distributed more densely in lipid phase and more protected from riboflavin photosensitized O/W emulsions. This study can help to decrease the level of BPA in foods made of O/W emulsions containing riboflavin, which could be displayed under visible light irradiation.     

Flavonoids protecting food and beverages against light

Flavonoids, which are ubiquitously present in the plant kingdom, preserve food and beverages at the parts per million level with minor perturbation of sensory impressions. Additionally, they are safe and possibly contribute positive health effects. Flavonoids should be further exploited for the protection of food and beverages against light‐induced quality deterioration through: (1) direct absorption of photons as inner filters protecting sensitive food components; (2) deactivation of (triplet‐)excited states of sensitisers like chlorophyll and riboflavin; (3) quenching of singlet oxygen from type II photosensitisation; and (iv) scavenging of radicals formed as reaction intermediates in type I photosensitisation. For absorption of light, combinations of flavonoids, as found in natural co‐pigmentation, facilitate dissipation of photon energy to heat thus averting photodegradation. For protection against singlet oxygen and triplet sensitisers, chemical quenching gradually decreases efficiency hence the pathway to physical quenching should be optimised through product formulation. The feasibility of these protection strategies is further supported by kinetic data that are becoming available, allowing for calculation of threshold levels of flavonoids to prevent beer and dairy products from going off. On the other hand, increasing understanding of the interplay between light and matrix physicochemistry, for example the effect of aprotic microenvironments on phototautomerisation of compounds like quercetin, opens up for engineering better light‐to‐heat converting channels in processed food to eventually prevent quality loss. © 2014 Society of Chemical Industry     

Stability of isoflavone daidzein and genistein in riboflavin, chlorophyll b, or methylene blue photosensitization

ABSTRACT:  Effects of photosensitizers including riboflavin, chlorophyll b , or methylene blue on the stability of daidzein and genistein were studied in model systems by high-performance liquid chromatography (HPLC). Concentration of daidzein and genistein in 80% methanol with riboflavin under light for 7 h was significantly decreased with the apparent 1st-order rate constants of 0.234 and 0.193/h, respectively, ( P < 0.05), while those without riboflavin under light did not change significantly ( P > 0.05). The stability of isoflavone aglycones in the dark did not change significantly irrespective of the presence of riboflavin ( P > 0.05). The concentrations of daidzein and genistein in chlorophyll b or methylene blue model systems under light were not significantly different from those in the dark for 7 h ( P > 0.05). Addition of sodium azide increased the stability of daidzein and genistein in riboflavin photosensitization with concentration dependent manner. However, the protective effects of β-carotene addition on the photodegradation of isoflavones were not high. The stability difference of daidzein and genistein in riboflavin photosensitization may be due to the high reactivity of riboflavin through type I pathway, although singlet oxygen may be involved in part.     

Quenching Mechanism and Kinetics of Ascorbic Acid on the Photosensitizing Effects of Synthetic Food Colorant FD&C Red Nr 3

ABSTRACT:  The pH effect on the oxidative stability of ascorbic acid in the presence of food colorant FD&C Red Nr 3 during storage with or without light was investigated. The quenching mechanism and kinetics of ascorbic acid on the FD&C Red Nr 3 photosensitized oxidation in an aqueous system at 25 °C were also studied by measuring the degradation of ascorbic acid or depletion of headspace oxygen. Red Nr 3 had no influence on the oxidation of ascorbic acid under dark storage, but accelerated its oxidation rate under light storage. The oxidative stability of ascorbic acid decreased as the pH increased from 4 to 7 under light without FD&C Red Nr 3. The quenching rates of ascorbic acid on the singlet oxygen by measuring the degradation of ascorbic acid in the presence of Red Nr 3 under light storage were 1.53 ± 0.15 × 10⁸, 1.86 ± 0.25 × 10⁸, and 1.19 ± 0.12 × 10⁸ M⁻¹S⁻¹ at pH 4, 5.6, and 7, respectively.     

QUENCHING MECHANISMS AND KINETICS OF CAROTENOIDS IN RIBOFLAVIN PHOTOSENSITIZED SINGLET OXYGEN OXIDATION OF VITAMIN D2

The oxidation of vitamin D₂ in a solution of 12% water, 88% acetone and 15 ppm ribqflavin under light or dark was studied by measuring the headspace oxygen. Ribofiavin accelerated the oxidation of vitamin D₂ by singlet oxygen under light, but did not affect vitamin D₂ oxidation under dark. Quenching mechanisms and kinetics within the range of 0–20 ppm β‐cawtene or fucoxanthin and with 0–80 ppm retinyl acetate or retinol on 15 ppm riboflavin photosensitized singlet oxygen oxidation of vitamin D₂ were also studied. The rate of singlet oxygen formation by 15 ppm riboflavin was 1.78 umole oxygen/mL headspace‐hour. The reaction rate constant of vitamin D₂ with singlet oxygen was 2.2 times 10⁷ M^?1A s^?1. The carotenoids minimized the oxidation of vitamin D₂ by quenching singlet oxygen. The total quenching rate constants of retinol, retinyl acetate, jucoxanthin and ft‐carotene were in the order of 1.22 times 10⁸, 5.98 times 10⁸, 1.78 times 10⁹ and 5.00 times 10⁹M^?1. s^?1 respectively, which suggests that as the number of double bonds of carotenoids increases, the quenching rate constant ofcarotenoid increases.     

Comparative determination of riboflavin in milk by HPLC and lumiflavin methods

A sensitive method for the determination of riboflavin in milk is described. It involves HPLC at room temperature with fluorescence detection. The technique has good recovery, for milk 98% and for yogurt 93%. The VC for milk is 8.5 and for yogurt 6.5%. The method proposed is more rapid, reproducible and technically simple then the commonly used lumiflavin method. The HPLC technique provides opportunity for a large number of measurements using a small sample volume.     

Effects of green tea catechin on the lipid oxidation,volatile compound formation,and losses of retinol and α-tocopherol in whole milk during light illumination as compared with ascorbic acid

Effects of green tea catechin (GTC, catechin content 61.4%) on the formations of hydroperoxide, malonaldehyde, and volatile compounds, and losses of retinol and tocopherol in milk during light illumination were studied. GTC showed surprisingly high protective activity of milk photooxidation, in a dose dependent manner. GTC at 25 ppm exerted 82.1 and 75.0% inhibitions of hydroperoxide and malonaldehyde formation during 30 h light illumination, respectively. GTC at 25 ppm exerted 88.7 and 94.3% inhibition of hexanal and heptanal formation in milk during 30 h light illumination, respectively. The 25 ppm GTC greatly suppressed the losses of retinol and α-tocopherol, showing 53.6 and 58.7% protections of their losses in milk after 12 h light illumination, respectively. The protective activity of GTC was at least 4 times higher than ascorbic acid. The inhibitory activities of GTC and ascorbic acid against the photooxidation of milk were in consistent with their singlet oxygen quenching abilities.     

Photosensitized amino acid degradation in the presence of riboflavin and its derivatives

The addition of photosensitizers to water can accelerate disinfection in sunlight-based systems by enhancing oxidation of target compounds through direct reaction with the excited sensitizer or through production of another oxidant, such as singlet oxygen (1O?). The kinetics of the oxidation of selected amino acids in the presence of the sensitizer riboflavin (Vitamin B2), its primary photoproduct lumichrome, and its derivative riboflavin tetraacetate (2',3',4',5'-tetraacetylriboflavin; RTA) were quantified and the mechanisms of reaction were determined during exposure to 365 ± 9 nm light. 1O?-mediated reactions contributed to the rapid photodegradation of the four amino acids, but its contribution was sensitizer-dependent and varied from 5.4-10.2% for tyrosine, 7.1-12.4% for tryptophan, 18.7-69.0% for methionine, and 64.7-100.2% for histidine. Riboflavin was subject to rapid photodegradation (t? < 8 min), while the half-lives of lumichrome and RTA were 100 and 30 times longer, respectively. Lumichrome and RTA also were more efficient 1O? sensitizers (quantum yield (Φ) = 0.63 and 0.66) compared to riboflavin (Φ = 0.48). Of the three flavin-based compounds, RTA shows the most promise as a sensitizer in sunlight-based disinfection systems because it absorbs both visible and UV light, is an efficient 1O? sensitizer, is a strong oxidant in its triplet state, and exhibits greater photostability.     

Gum arabic microcapsules as protectors of the photoinduced degradation of riboflavin in whole milk

Microcapsules (MC) made with gum arabic (GA) as shell material without and with β-carotene (βc) as core material were prepared by the spray-drying technique. The effect of these MC on the photodegradation of riboflavin (Rf) in whole milk by fluorescent daylight lamp irradiation was evaluated at a storage temperature of 4°C. The additions of 1.37 mg/mL of MC without βc (MC-GA) and with 0.54 μg/mL of βc (MC-βc-GA) decreased the apparent first-order rate constant of Rf photodegradation by approximately 26 and 30%, respectively. A systematic kinetic and mechanistic analysis of the results indicates that the global protective effect of the MC is mainly due to the combination of quenching of the electronically excited triplet state of Rf and scavenging of the photogenerated reactive oxygen species, such as singlet molecular oxygen, superoxide radical anion and hydroxyl radical. A minor contribution to the photoprotective effect can be also associated with the inner-filter effect exerted by the MC, which partially blocks the direct excitation of Rf. These results allow us to conclude that photodegradation of Rf in milk can be considerably reduced by the addition of small amounts of MC, avoiding large losses in the nutritional value of milk.     

Chemistry and Reaction of Singlet Oxygen in Foods

Singlet oxygen is a highly reactive, electrophilic, and nonradical molecule. It is different from diradical triplet oxygen in its electron arrangement. Photosensitizers can form singlet oxygen from triplet oxygen in the presence of light. The reaction rate of singlet oxygen with foods is much greater than that of triplet oxygen due to the low activation energy. Singlet oxygen oxidation produces undesirable compounds in foods during processing and storage. However, carotenoids and tocopherols in foods can minimize singlet oxygen oxidation. The in‐depth scientific knowledge on the formation, reactions, quenching mechanisms, and kinetics of singlet oxygen can greatly improve the quality of foods by minimizing the oxidation during processing and storage. The single oxygen oxidation of foods has contributed to the explanation of several important chemical reactions in the reversion flavor in soybean oil, sunlight flavor in milk products, and the rapid losses of vitamin D, riboflavin, and ascorbic acid in milk under light storage.     

Direct Spectroscopic Observation of Singlet Oxygen Quenching and Kinetic Studies of Physical and Chemical Singlet Oxygen Quenching Rate Constants of Synthetic Antioxidants (BHA,BHT, and TBHQ) in Methanol

Abstract: Singlet oxygen quenching by synthetic antioxidants (BHA, BHT, and TBHQ) was directly observed by spectroscopic monitoring of luminescence at 1268 nm. The luminescence data showed unambiguous evidence of singlet oxygen quenching by synthetic phenolic antioxidants with the highest activity for TBHQ, followed by BHA and BHT. The protective activities of these synthetic antioxidants on α-terpinene oxidation with chemically-induced singlet oxygen under dark further confirmed their singlet oxygen quenching abilities. Total singlet oxygen quenching rate constants (k_r + k_q) of BHA, BHT, and TBHQ were determined in a system containing α-terpinene (as a singlet oxygen trap) and methylene blue (as a sensitizer) during light irradiation, and the values were 5.14 × 10⁷, 3.41 × 10⁶, and 1.99 × 10⁸ M⁻¹s⁻¹, respectively. After the k_r value of α-terpinene was first determined, the k_r values of the synthetic antioxidants were calculated by measuring their relative reaction rates with singlet oxygen to that of α-terpinene under the identical conditions. The k_r values of the BHA, BHT, and TBHQ were 3.90 × 10⁵, 1.23 × 10⁵, and 2.93 × 10⁶, M⁻¹s⁻¹. The percent partition of chemical quenching over total singlet oxygen quenching (k_r × 100)/(k_r + k_q) for BHA, BHT, and TBHQ were 0.76%, 3.61%, and 1.47%, respectively. The results showed that the synthetic antioxidants quench singlet oxygen almost exclusively through the mechanism of physical quenching. This represents the first report on the singlet oxygen quenching mechanism of these synthetic antioxidants. Practical Application: The synthetic antioxidants, especially TBHQ, have been found to have a strong singlet oxygen quenching ability. This article also clearly showed that singlet oxygen quenching by synthetic antioxidants was mainly by the physical quenching mechanism. The results suggested that these synthetic antioxidants, especially TBHQ, could be used practically for the protection of the food components such as edible oils and vitamins against singlet oxygen induced oxidations without significant losses of antioxidant activity during storage under light.     

Effects of Riboflavin Photosensitized Oxidation on the Volatile Compounds of Soymilk

ABSTRACT: Soymilks with or without added riboflavin in serum bottles were stored under light or in dark at 20 °C. The headspace oxygen and volatile compounds were determined by gas chromatography. Riboflavin had significant effects on the headspace oxygen depletion and volatile compounds formation in soymilk under light ( P < 0.05). Riboflavin did not have significant effects on the formation of volatile compounds and the depletion of headspace oxygen in dark ( P > 0.05). The volatile compounds increased under light, but not in dark as the added riboflavin increased. Storage temperature at 4 °C or 20 °C did not have significant difference in the effect of riboflavin on the headspace oxygen depletion in soymilk under light. Hexanal, an important beany flavor compound, was identified as the major volatile compound in the riboflavin photosensitized soymilk. Singlet oxygen oxidation was involved in the formation of volatile compounds in soymilk under light. Hexanal could be formed by singlet oxygen oxidation. Ascorbic acid, a quencher for singlet oxygen and the excited triplet sensitizer, significantly inhibited the formation of hexanal and total volatiles in soymilk under light.     

Singlet Oxygen Quenching Effects of Phosphatidylcholine in Emulsion Containing Sunflower Oil

ABSTRACT:  Effects of phosphatidylcholine (PC) on the oxidation of oil by singlet oxygen in a W/O microemulsion and an emulsion food model containing tocopherol-stripped sunflower oil (TSSO) have been studied. The W/O microemulsion consisted of methylene chloride, butanol, and sodium dodecyl sulfate with PC (0, 250, 1000 ppm) and TSSO (0, 3.3, 16.5, 33 mg/mL). Production of singlet oxygen in the microemulsion was done chemically with hydrogen peroxide in the presence of sodium molybdate, and indirectly evaluated by rubrene oxidation at A₅₂₉. The emulsion food model consisted of TSSO, distilled water, and xanthan gum with addition of 250 ppm PC and 4 ppm chlorophyll b, and was placed at 25 °C under fluorescent lights (1700 lux) for 24 h. The oxidation of TSSO was determined by thin-layer chromatography and values of conjugated dienoic acid (CDA) and peroxides (POV). PC significantly decreased the oxidation of rubrene and TSSO in the W/O microemulsion, but its content was decreased to approximately one-half by a 20-min reaction, indicating its degradation. This clearly shows that PC acted as an antioxidant via chemical quenching of singlet oxygen in the W/O microemulsion. A possible synergism between PC and TSSO was observed in singlet oxygen quenching in the microemulsion. PC also significantly decreased the chlorophyll-photosensitized oxidation of TSSO in the emulsion food model, possibly by singlet oxygen quenching. This study clearly suggested that PC be used as an antioxidant to improve the lipid oxidative stability of an emulsion food containing chlorophyll under light.     

Stability and Photochemistry of Vitamin D2 in Model System

Oxidation of vitamin D₂ in a model system of 12% water and 88% acetone in the presence of 15 ppm riboflavin under light and dark was studied by measuring the depleted headspace oxygen. Riboflavin accelerated the oxidation of vitamin D₂ by singlet oxygen under light, but did not affect the vitamin D₂ oxidation under dark. The effect of 0 and 15 ppm riboflavin on the stability of vitamin D₂ during storage at 25 and 60°C was studied by measuring the contents of vitamin D₂ during 48h. Results indicated that photosensitized singlet oxygen oxidation of vitamin D₂ under light was temperature-independent, and triplet oxygen oxidation of vitamin D₂ both under light and in the dark was temperature-dependent.