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The action spectrum for folic acid photodegradation in aqueous solutions
Journal of Photochemistry and Photobiology B: Biology 126 (2013) 11–16
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
The action spectrum for folic acid photodegradation in aqueous solutions Asta Juzeniene a,⇑, Tran Thi Thu Tam a, Vladimir Iani a, Johan Moan a,b a b
Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310 Oslo, Norway Department of Physics, University of Oslo, N-0316 Oslo, Norway
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 8 May 2013 Accepted 29 May 2013 Available online 7 June 2013 Keywords: Folic acid Action spectrum UV radiation
a b s t r a c t Folate is essential for cell division and growth. Deficiency is linked to birth defects, magaloblastic anaemia, cardiovascular disease, etc. Folic acid is a synthetic form of folate and is used to fortify food and in supplements. In aqueous solutions, in blood and even in human skin, folic acid may be degraded by ultraviolet radiation. Consequently, photodegradation of folic acid in human blood may lead to folate deficiency. However, the degree and the health consequences of such photodegradation are unknown. It is not clear which spectral region plays the most important role in the photodegradation of folic acid. In this study the photodegradation of folic acid in aqueous solution under different wavelengths of ultraviolet radiation (260–400 nm) was investigated by fluorescence spectroscopy. The photodegradation rate of folic acid was dependent on wavelength. Action spectrum for 1 lM folic acid photodegradation was determined. Its action spectrum is not identical to its absorption or fluorescence excitation spectra. The action spectrum demonstrated that UVB and UVA degrade folic acid. Protecting skin against UVB and UVA radiation by sunscreens may help to protect folic acid in human blood under intense solar radiation. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Folate, a water-soluble B vitamin, is involved in the synthesis, methylation and repair of DNA [1,2]. Folate deficiency may lead to magaloblastic anaemia, adverse pregnancy outcomes (neural tube defects), fertility problems and neuropsychiatric disorders [1,2]. Mammals cannot produce folate and must obtain it from their diet. Folic acid (FA), a synthetic folate form, is used for food fortification and nutritional supplements. The World Health Organization recommends that all women of reproductive age daily take FA supplements to prevent neural tubes defects in babies [3]. FA undergoes photodegradation in aqueous solutions, in blood irradiated in test tubes and in blood in healthy humans exposed to ultraviolet radiation (UV) from artificial sources and from sunlight [4–8]. Some studies have claimed that solar or artificial UV radiation can also deplete natural folates in human blood and skin [6,9–11], while other studies have claimed that UV radiation has no effect on folate levels [6,12–14]. Discrepancies in the different studies may be due to differences in used UV sources, fluence, skin condition (healthy, vitiligo, psoriasis, etc.), and methods for the measurements of folates in blood [15]. Recently, it was demonstrated by Fukuwatari et al. [6] that FA is more photolabile in blood than natural forms of folate. It seems that the presence of folic acid may stimulate photodegradation of the natural forms of folates. This means that photodegradation of FA in human blood may lead ⇑ Corresponding author. Tel.: +47 22934260; fax: +47 22781207. E-mail address: [email protected] (A. Juzeniene). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.05.011
to folate deficiency. However, the degree, the mechanism, and the health consequences of this photodegradation are unknown. FA has peaks in the UVB and UVA regions (Fig. 2). Due to this, broadband UV sources were used to study FA photodegradation [5,7,16]. It is not clear which wavelength plays the most important role in the initiation of the photodegradation of FA. The action spectrum for FA will show which wavelengths are most efficient in degrading FA molecules. It may help to understand the mechanisms of the photodegradation of FA, not only in aqueous solutions, but also in blood, and to predict its influence on human health. In the present work this action spectrum for FA has been determined by fluorescence spectroscopy. During UV exposure FA is converted into p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-formylpterin (FPT), which is then further oxidized to 6-carboxypterin (CPT) (Fig. 1). Based on our earlier work [5] we have chosen to measure only linear increase of initial fluorescence emission intensity. The formation of FPT is responsible for the increase of fluorescence intensities under UV exposure. The increase of fluorescence intensity by 40% was chosen due to statistical considerations. 2. Materials and methods 2.1. Chemicals Folic acid (FA, catalog number F7876, Lot. Number 081M1546V) and p-aminobenzoyl-L-glutamic acid (PABA-Glu) were purchased from Sigma–Aldrich Norway AS (Oslo, Norway), and both compounds were used without further purification. Dulbecco’s
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phosphate buffered saline (PBS) was obtained from PAA laboratories GmbH (Pasching, Austria). 6-formylpterin (FPT) was obtained from Schircks Laboratories (Jona, Switzerland). Before each experiment stock solutions of 100 mM FA, PABA-Glu or FPT were prepared in 1 M NaOH. Afterwards, they were diluted in PBS (pH = 7.4) to the required concentrations. pH 7.4 was chosen to simulate physiological media, because normal blood pH is also around 7.4. 2.2. Absorption and fluorescence measurements Absorption spectra were registered with a Perkin-Elmer Lambda 40 UV/VIS spectrophotometer (Norwalk, CT, USA). Fluorescence emission and excitation spectra were registered with a Perkin-Elmer LS50B luminescence spectrometer (Norwalk, CT, USA). Fluorescence emission spectra were measured in the range of 300–540 nm. Excitation wavelength was set at 270 nm. For fluorescence excitation spectroscopy, the emission wavelength was set at 445 nm. The complete excitation spectra were recorded in the range of 220–420 nm. 2.3. Photolysis A set-up with a high pressure Xenon arc lamp 900 W (Oriel, Stamford, CT) and a monochromator MODEL (Jarrell Ash, Boston, MA, USA) were used during the experiments. The monochromator was set with relatively wide slits resulting in Gaussian shaped peaks with 12 nm bandwidth. A power meter SOLO 2 with Gentec-EO PH photodetector PH100-SiUV (Gentec-EO, Québec, Canada) was used to measure the fluence rates (U) at the monochromator exit slit at different wavelengths (Fig. 3). FA samples (0.7 ml) in opened quartz cuvettes were exposed to UV radiation in the spectral range from 260 nm to
Fig. 2. Absorption spectra of 1 lM FA, p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-formylpterin (FPT) in PBS.
400 nm. The same quartz cuvette with 10 mm length and 4 mm inside width (Fig. 4) was used for absorption and fluorescence measurements and for UV exposure. The fluorescence excitation wavelength was set at 270 nm and the photodegradation of FA was measured by determining the increase of fluorescence of photoproducts at 445 nm. This increase of fluorescence (data not shown) is due to the photoproducts of FA [5,17]. FA has a low fluorescence quantum yield (<0.005) [18]. However, when the molecule is exposed to UV radiation, the bond between the 6-methylpterin and the p-aminobenzoic acid (Fig. 1) is broken and the fluorescence of the solution increases [5,18,19]. The obtained fluorescence emission values at 445 nm were normalized to 1 at the initial time point for all concentrations. The temperatures of the samples were approximately 22–25 °C. All experiments were performed in constant dim light in order to avoid external, uncontrolled light exposure.
Fig. 1. The structural formula of folic acid (FA), 6-formylpterin (FPT), p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-carboxypterin (CPT). Schematic photodegradation of FA.
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Fig. 3. Measured fluence rates of the lamp in the region from 260 to 400 nm.
Fig. 5. (A) Absorption and fluorescence emission spectra of 0.26, and 1 lM FA in PBS. (B) Normalized absorption, fluorescence emission and excitation spectra of 0.26 and 1 lM FA in PBS. The excitation wavelength was set at 270 nm for the fluorescence emission spectra and the emission wavelength was set at 445 nm for the fluorescence excitation spectra. Fig. 4. Experimental setup of the cuvette in the holder, where ‘1 = 0.2 cm, ‘2 = 0.4 cm and ‘3 = 1.0 cm. For UV exposure 1 cm path length of the cuvette was used.
2.4. Construction of action spectra for FA photogeneration An action spectrum is a plot of a response per number of incident photons, against wavelength. The total number of incident photons hitting the sample (ni) to give the same effect at all wavelengths is given by:
ni ¼
Utka hc
(data not shown). Not more than 5% of FA was photooxidized during given UV exposures [5,20]. FA has absorption peaks at around 280 nm and 350 nm (Figs. 2 and 5). For this reason, these two wavelengths were chosen for the demonstration of FA photodegradation and its photoproduct formation (Fig. 6). From these results the rates of photoproducts formation were determined as well as the fluences needed for a fluorescence intensity increase by 40% at 445 nm of 0.26 and 1 lM FA (Figs. 6 and 7). Photoproducts formation rates were similar (P > 0.05) at 0.26 lM and 1 lM at 280 and 350 nm (Fig. 6).
;
where U is the fluence rate (mW/cm2), t is the radiation exposure time (s) needed for a fluorescence intensity increase by 40% at 445 nm, k is the radiation wavelength (nm), a is the exposed area on the cuvette (0.7 cm2), h is the Planck constant (6.626 1034 J s), and c is the speed of light (3 108 m/s). 2.5. Data analysis Data are represented as mean values and standard errors of the mean of at least three independent experiments are given. The data were also analyzed using the linear fitting routines of SigmaPlot 11.0 from Systat Software Inc. (Richmond, CA, USA). 3. Results Aqueous solutions of 0.26 and 1 lM FA were exposed to UV radiation at every 10 nm in the range between 260 nm and 400 nm with total fluences up to 2 J/cm2. The fluorescence emission spectra and its kinetics at 445 nm were recorded (data not shown). This led to increase in fluorescence intensity by more than 40% as measured at 445 nm. No apparent changes were observed in the absorption spectra of 0.26 and 1 lM FA after UV exposure
Fig. 6. Kinetics of the increase of the fluorescent products resulting from exposure of 0.26 lM (A) and 1 lM (B) FA to radiation at 280 nm and 350 nm. The fluorescence excitation wavelength was set at 270 nm.
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Fig. 7. Fluence doses needed for a fluorescence intensity increase by 40% at 445 nm of 0.26 lM and 1 lM FA. FA was exposed to radiation at 280 and 350 nm.
FA absorbs around 3.8-fold stronger at 280 nm than at 350 nm (Fig. 5B). Correspondingly, the fluence needed for a fluorescence intensity increase by 40% was approximately 4-folds higher at 350 nm than at 280 nm at a concentrations of 0.26 lM and 1 lM FA (Fig. 7). The action spectrum for FA photodegradation showed that UVB quanta were much more efficient to degrade FA than UVA quanta at 1 lM (Fig. 8). The action spectrum is not identical to its absorption or fluorescence excitation spectra (Fig. 8). The absorption spectrum of FA has peak at around 280 nm, while its action spectrum has peak at around 270 nm, at the same wavelength as its fluorescence excitation peak (Fig. 8). While in the UVA region the action spectrum is lower in comparison to the absorption spectrum of 1 lM FA (Fig. 8).
4. Discussion Due to the biological importance of FA, many scientists have studied its photodegradation mechanism in aqueous solutions [4,5,7,16,20–22]. The photolysis of FA has been studied mostly under broadband UVA, UVC (which is absorbed by the ozone layer and does not reach the Earth’s surface) or visible light exposure [4,5,7,23,24]. No data have been published about the photolysis of FA under UVB exposure, and, consequently, the action spectrum for FA degradation has been unknown until now. Photooxidation of FA is initiated by excited states of FA itself [7], and it is usually divided into three phases [5,7,23]. In the first phase, formation of the photoproducts PABA-Glu and FPT occurs and obeys a zero order rate law. In the second phase the presence of FPT sensitizes the degradation of FA, and its degradation process
Fig. 8. The action spectra for 1 lM photodegradation, normalized to maxima fluorescence excitation and absorption spectra of FA.
is accelerating [7,25]. In the third phase the degradation of FPT to CPT is the dominating process, and this reaction follows a first order kinetics. In the present study only the first phase (initial) of the photodegradation of commercially available FA was studied. PABA-Glu, FPT and CPT are not photostable [26–28]. Their formation is dominant in the beginning of the photolysis of FA, while later their degradation becomes dominant. Additionally, FPT and CPT, but not PABA-Glu, act as photosensitizers [5,7,17,20,23]. Furthermore, they act differently at different wavelengths, due to differences in their absorption characteristics (Fig. 2): PABA-Glu absorbs mostly in the UVB region, while FPT and CPT absorb both in the UVB and in the UVA regions [5]. All these factors influence the photobleaching rates at different wavelengths [26–28]. PABA-Glu undergoes photolysis under UVB exposure, but above 320 nm, PABA-Glu does not absorb, then it is photostable. CPT is not photostable under UVB and UVA exposure, but its quantum yield of photodegradation is much lower than those of FA and FPT [27,28]. Besides singlet oxygen production by energy transfer of the triplet excited states of pterins to oxygen, electron transfer processes have been shown to play a dominant role in the formation of other reactive oxygen species and in the p the autocatalytic photooxidation of FA [7,25,29,30]. It has been found that FA is photolabile only in the presence of oxygen [7,28]. Moreover, many endogenous or exogenous photosensitizers might cause the degradation of FA, under UV or even visible light exposure [7,25]. The action spectrum of 1 lM photodegradation is not identical to its absorption and fluorescence excitation spectra (Fig. 8). At the same time fluorescence excitation spectrum is not similar to the absorption spectrum of FA (Figs. 5 and 8). FA has fluorescence excitation peaks at around 270 nm and 350 nm, while absorbance peaks at around 280 nm and 350 nm (Figs. 5 and 8). Thomas et al. [18] reported that fluorescence excitation spectrum of FA obtained by excitation at 450 nm was different from the absorption spectrum. In their case, fluorescence excitation peaks were around 270 nm and 370 nm. The ratio between the fluorescence excitation bands at 370 nm and 270 nm was around 0.3. They did not mention which concentration of FA was measured, but in order to avoid inner filter effect, they kept the absorption of FA solution below 0.1. This means that the concentration of FA was below 4 lM. In our case the ratio between the fluorescence excitation bands at 350 nm and 270 nm was around 1.3 (Fig. 5). We could not find any other study, which would describe the fluorescence excitation spectra of FA. So big discrepancy between our and Thomas et al. fluorescence excitation spectra may be due to impurities of FA itself (different producers) or solution in which it was prepared, aggregation of FA, or differences in used solvents. We were using commercially available FA without further purification, which may contain some impurities. Thomas et al. [18] calculated that the fluorescence quantum yields for the high energy bands are lower by a factor of at least 3 than those corresponding to the low energy bands [18]. This means that only a fraction of the energy of the upper excited state(s) (S2,Sn) is dissipated through internal conversion to the lowest singlet excited state (S1) [18]. Consequently, a photophysical (intersystem crossing to the triplet manifold) or/and a photochemical process should occur from an upper singlet excited state [18,19]. The relatively long chain substituent of FA might act as an ‘‘internal fluorescence quencher,’’ enhancing the radiationless deactivation of the singlet excited state [18,31]. Due to this reason FA does not produce singlet oxygen [17]. The quantum yield of singlet oxygen production is very low for FA (60.02). At the very early stage of the photoconversion (below 10%) the light absorption by the photoproducts can be neglected and the rate is proportional to the quantum yield for FA phototransformation. The quantum yield associated with the disappearance of 600 lM
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FA under UVA exposure was measured by Thomas et al. [28] and a value of 0.025 was observed. On the other hand, the same value was also obtained for the quantum yield of FPT formation. Therefore, consumption of FA to lead FPT is the only process that takes place at very early stage of FA photodegradation [28]. A higher photoreaction quantum yield in the UVB would be consistent with the fluorescence excitation spectra, indicating that some upper excited state does not undergo quantitative internal conversion to the lowest lying (fluorescent) state. FA is used for supplementation and food fortification. It lacks coenzyme activity and must be reduced to dihydrofolate and then to tetrahydrofolate after it enters body [32]. The same enzyme, dihydrofolate reductase, catalyzes both these reactions. The activity of dihydrofolate reductase is much lower in humans than in animals, and it varies greatly between individuals [32,33]. In humans, increased FA intake (over 200 lg) leads to elevated blood concentrations of unmetabolized FA [33–35]. Unmetabolized serum FA was detected in 38% of the older population (aged P 60 years) in the United States (mandatory folic acid fortification) with a mean concentration of 1.2 nM (range 0–273 nM), accounting for 2.25% of total plasma folate [36,37]. In the older Irish population (aged P 60 years) despite the lack of a mandatory fortification program unmetabolized FA was detected in 94.1% of the cohort with a mean concentration of 0.39 nM (range 0–1.59 nM), accounting for 1.3% of total plasma folate [38]. Spending time in the sun or in sunbeds shortly after taking FA supplementation may lead to FA degradation. In healthy volunteers plasma folate levels decreased by 26% only in the group of volunteers who took FA before a single exposure of 2 h to sunlight [6]. The dose of UVA in sunlight was about 12 J/cm2. The natural folate derivative, 5-methyltetrahydrofolate, may also degrade under UV exposure [39–41], but 5-methyltetrahydrofolate is more stable than FA, because it 5-methyltetrahydrofolate almost does not absorb in the UVA region [42]. Earlier studies have demonstrated that FA photodegradation is concentration dependent [5,20]. Due to too low sensitivity of absorption and fluorescence spectroscopy we were not able to study degradation of physiologically relevant concentrations (<2 nM FA, [36–38]) and have chosen to study degradation of 0.26 and 1 lM FA. Solutions of 0.26 lM and 1 lM FA has the same absorbance at 280 and 350 nm, respectively (Fig. 5). It has been reported that a UVA dose of 2 J/cm2 from a broadband lamp degrades 10 and 85 lM FA completely in aqueous solutions in a cuvette (1 cm optical path), while much higher UVA doses are needed to destroy lower concentration of FA [5,20]. While 1 lM FA degrades completely only after UVA dose of 30 J/ cm2 [5]. It will take 5 and 85 min, respectively, to obtain UVA doses of 2 or 30 J/cm2, from the sun at noon under the Equator (where fluence rate is around 6 mW/cm2). In blood much higher UVA doses will be needed to degrade FA. Under physiological conditions (<2 nM FA) the first phase of the degradation of FA will be dominant. However, most studies have been performed using much higher FA concentrations (600 lM FA [28], 450 lM FA [28], 85 lM FA [20], 20–1000 lM FA [7]) where the first phase of FA degradation is rapidly changed into the second and third phases. To obtain conditions more physiologically relevant during the initial stage of FA photodegradation, much lower concentrations of FA must be studied. Additionally, the mechanisms involved in the photoooxidation of FA under UV exposure strongly depend on the pH [4,19]. Excitation of the acidic form of FA in the presence of oxygen leads to cleavage and oxidation of the molecules, yielding FPT and PABAGlu [19]. In the photooxidation of the basic form of FA, an additional reaction pathway exists, leading to an unknown compound with a molecular weight higher than that of FA [19].
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The present results demonstrate that in aqueous solutions UVB radiation is more efficient to degrade FA of the same concentration than UVA radiation. Only a small fraction of UVB radiation reaches blood vessels, and it is unlikely that direct photodegradation of FA takes place in human skin during exposure to UVB radiation. However, the presence of photosensitizers, such as riboflavin, significantly increases the photolysis rates of FA under UVA radiation [24]. In this case the photolysis of FA is mostly mediated via photosensitization rather than via direct photolysis. Exposure of humans taking FA supplementation to solar radiation both processes may be involved in the photolysis of FA, because blood and skin contains many endogenous photosensitizers [43]. Williams and Jacobson [11] have demonstrated that folate depleted human keratinocytes exposed to UV radiation (0.2 J/cm2 UVB and 4 J/cm2 UVA) were unable to repair DNA damage, while non-depleted control keratinocytes exposed to the same UV dose showed a nearly complete removal of DNA strand breaks to reach a level of non-exposed control cells after 24 h. Thus, chronic folate deficiency (due to low intake of natural folates and FA or due to photodegradation of folates and FA) may lead to skin photocarcinogenesis. Additionally, high concentrations of the photoproducts of FA may induce DNA damage under UV radiation and lead to the initiation of skin cancer [20,29]. At the same time, FA is an effective singlet oxygen and free radical (peroxyl, azide, sulfate, hydroxyl, thiyl) scavenger [17,44]. Thus, it may protect human skin and other organs from DNA damage mediated by reactive oxygen and nitrogen species. However, a meta-analysis of six randomized controlled trials showed that folic acid supplementation may boost prostate cancer risk [45]. Further studies are needed to understand a possible mechanism of FA photodegradation in human body. 5. Conclusions The photodegradation rates of FA are wavelength, but not concentration, dependent. The action spectrum for 1 lM FA in aqueous solutions shows that quanta in the UVB region are more efficient than quanta in the UVA region to degrade FA. The action spectrum for 1 lM FA photodegradation has maximum in the UVB region around 267 nm, in the same position as in the fluorescence excitation spectrum, but it is shifted to around 280 nm in the absorption spectrum. The peak in the UVA region of the action spectrum, fluorescence emission and absorption spectra is in the same position at around 350 nm. The action spectrum of 1 lM FA resembles its absorption spectrum, but it is blue shifted. Our experiments indicate that FA is the main chromophore in the initial degradation of FA. 6. Abbreviations
FA FPT CPT PABA-Glu UVA UVB
folic acid 6-formylpterin 6-carboxypterin p-aminobenzoyl-L-glutamic acid ultraviolet A (400–315 nm) ultraviolet B (280–315 nm)
Acknowledgments The present work was supported by the Research Foundation of the Norwegian Radium Hospital and the South-Eastern Norway Regional Health Authority. The authors thank Armend Håti for his help in performing fluorescence spectroscopy.
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References [1] M. Lucock, I. Daskalakis, New perspectives on folate status: a differential role for the vitamin in cardiovascular disease, birth defects and other conditions, Br. J. Biomed. Sci. 57 (2000) 254–260. [2] N.A. Wani, A. Hamid, J. Kaur, Folate status in various pathophysiological conditions, IUBMB Life 60 (2008) 834–842. [3] L.M. De-Regil, A.C. Fernandez-Gaxiola, T. Dowswell, J.P. Pena-Rosas, Effects and safety of periconceptional folate supplementation for preventing birth defects, Cochrane Database Syst. Rev. 6 (2010). CD007950. [4] M.J. Akhtar, M.A. Khan, I. Ahmad, Photodegradation of folic acid in aqueous solution, J. Pharm. Biomed. Anal. 19 (1999) 269–275. [5] M.K. Off, A.E. Steindal, A.C. Porojnicu, A. Juzeniene, A. Vorobey, A. Johnsson, J. Moan, Ultraviolet photodegradation of folic acid, J. Photochem. Photobiol. B 80 (2005) 47–55. [6] T. Fukuwatari, M. Fujita, K. Shibata, Effects of UVA irradiation on the concentration of folate in human blood, Biosci. Biotechnol. Biochem. 73 (2009) 322–327. [7] M.L. Dantola, M.P. Denofrio, B. Zurbano, C.S. Gimenez, P.R. Ogilby, C. Lorente, A.H. Thomas, Mechanism of photooxidation of folic acid sensitized by unconjugated pterins, Photochem. Photobiol. Sci. 9 (2010) 1604–1612. [8] D.C. Borradale, M.G. Kimlin, Folate degradation due to ultraviolet radiation: possible implications for human health and nutrition, Nutr. Rev. 70 (2012) 414–422. [9] R.F. Branda, J.W. Eaton, Skin color and nutrient photolysis: an evolutionary hypothesis, Science 201 (1978) 625–626. [10] M. Der-Petrossian, M. Fodinger, R. Knobler, H. Honigsmann, F. Trautinger, Photodegradation of folic acid during extracorporeal photopheresis, Br. J. Dermatol. 156 (2007) 117–121. [11] J.D. Williams, M.K. Jacobson, Photobiological implications of folate depletion and repletion in cultured human keratinocytes, J. Photochem. Photobiol. B 99 (2010) 49–61. [12] T. Gambichler, A. Bader, K. Sauermann, P. Altmeyer, K. Hoffmann, Serum folate levels after UVA exposure: a two-group parallel randomised controlled trial, BMC Dermatol. 1 (2001) 8. [13] A. Osmancevic, L.T. Nilsen, K. Landin-Wilhelmsen, E. Soyland, T.P. Abusdal, T.A. Hagve, M.S. Nenseter, A.L. Krogstad, Effect of climate therapy at Gran Canaria on vitamin D production, blood glucose and lipids in patients with psoriasis, J. Eur. Acad. Dermatol. Venereol. 23 (2009) 1133–1140. [14] E. Cicarma, C. Mork, A.C. Porojnicu, A. Juzeniene, T.T. Tam, A. Dahlback, J. Moan, Influence of narrowband UVB phototherapy on vitamin D and folate status, Exp. Dermatol. 19 (2010) e67–e72. [15] A. Juzeniene, K.T. Stokke, P. Thune, J. Moan, Pilot study of folate status in healthy volunteers and in patients with psoriasis before and after UV exposure, J. Photochem. Photobiol. B 101 (2010) 111–116. [16] P. Vorobey, A.E. Steindal, M.K. Off, A. Vorobey, J. Moan, Influence of human serum albumin on photodegradation of folic acid in solution, Photochem. Photobiol. 82 (2006) 817–822. [17] A.H. Thomas, C. Lorente, A.L. Capparelli, C.G. Martinez, A.M. Braun, E. Oliveros, Singlet oxygen (1deltag) production by pterin derivatives in aqueous solutions, Photochem. Photobiol. Sci. 2 (2003) 245–250. [18] A.H. Thomas, C. Lorente, A.L. Capparelli, M.R. Pokhrel, A.M. Braun, E. Oliveros, Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in aqueous solution: pH effects, Photochem. Photobiol. Sci. 1 (2002) 421–426. [19] C. Lorente, A.H. Thomas, Photophysics and photochemistry of pterins in aqueous solution, Acc. Chem. Res. 39 (2006) 395–402. [20] T. Offer, B.N. Ames, S.W. Bailey, E.A. Sabens, M. Nozawa, J.E. Ayling, 5Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA, FASEB J. 21 (2007) 2101–2107. [21] J. Arcot, A. Shrestha, Folate: methods of analysis, Trends Food Sci. Technol. 16 (2005) 253–266. [22] M.J. Akhtar, M.A. Khan, I. Ahmad, Identification of photoproducts of folic acid and its degradation pathways in aqueous solution, J. Pharm. Biomed. Anal. 31 (2003) 579–588. [23] A.H. Thomas, G. Suarez, F.M. Cabrerizo, F.S.G. Einschlag, R. Martino, C. Baiocchi, E. Pramauro, A.L. Capparelli, Photochemical behavior of folic acid in alkaline aqueous solutions and evolution of its photoproducts, Helv. Chim. Acta 85 (2002) 2300–2315.
[24] M.J. Akhtar, M.A. Khan, I. Ahmad, Effect of riboflavin on the photolysis of folic acid in aqueous solution, J. Pharm. Biomed. Anal. 23 (2000) 1039–1044. [25] C. Lorente, G. Petroselli, M.L. Dantola, E. Oliveros, A.H. Thomas, Electron transfer initiated reactions photoinduced by pterins, Pteridines 22 (2011) 111–119. [26] A.A. Shaw, L.A. Wainschel, M.D. Shetlar, The photochemistry of paminobenzoic acid, Photochem. Photobiol. 55 (1992) 647–656. [27] G. Suarez, F.M. Cabrerizo, C. Lorente, A.H. Thomas, A.L. Capparelli, Study of the photolysis of 6-carboxypterin in acid and alkaline aqueous solutions, J. Photochem. Photobiol. B 132 (2000) 53–57. [28] A.H. Thomas, G. Suarez, F.M. Cabrerizo, R.l. Martino, A.L. Capparelli, Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions, J. Photochem. Photobiol. A 135 (2000) 147–154. [29] K. Hirakawa, H. Suzuki, S. Oikawa, S. Kawanishi, Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product, Arch. Biochem. Biophys. 410 (2003) 261–268. [30] M. Soniat, C.B. Martin, Theoretical study on the relative energies of anionic pterin tautomers, Pteridines 20 (2009) 124–128. [31] F.M. Cabrerizo, G. Petroselli, C. Lorente, A.L. Capparelli, A.H. Thomas, A.M. Braun, E. Oliveros, Substituent effects on the photophysical properties of pterin derivatives in acidic and alkaline aqueous solutions, Photochem. Photobiol. 81 (2005) 1234–1240. [32] A.D. Smith, Y.I. Kim, H. Refsum, Is folic acid good for everyone?, Am J. Clin. Nutr. 87 (2008) 517–533. [33] S.W. Bailey, J.E. Ayling, The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake, Proc. Natl. Acad. Sci. USA 106 (2009) 15424–15429. [34] P. Kelly, J. McPartlin, M. Goggins, D.G. Weir, J.M. Scott, Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements, Am. J. Clin. Nutr. 65 (1997) 1790–1795. [35] M.R. Sweeney, J. McPartlin, J. Scott, Folic acid fortification and public health: report on threshold doses above which unmetabolised folic acid appear in serum, BMC Public Health 7 (2007) 41. [36] R.L. Bailey, J.L. Mills, E.A. Yetley, J.J. Gahche, C.M. Pfeiffer, J.T. Dwyer, K.W. Dodd, C.T. Sempos, J.M. Betz, M.F. Picciano, Unmetabolized serum folic acid and its relation to folic acid intake from diet and supplements in a nationally representative sample of adults aged > or =60 y in the United States, Am. J. Clin. Nutr. 92 (2010) 383–389. [37] R.L. Bailey, J.L. Mills, E.A. Yetley, J.J. Gahche, C.M. Pfeiffer, J.T. Dwyer, K.W. Dodd, C.T. Sempos, J.M. Betz, M.F. Picciano, Serum unmetabolized folic acid in a nationally representative sample of adults >/=60 years in the United States, 2001–2002, Food Nutr. Res. 56 (2012). [38] A. Boilson, A. Staines, C.C. Kelleher, L. Daly, I. Shirley, A. Shrivastava, S.W. Bailey, P.B. Alverson, J.E. Ayling, A.P. McDermott, A. Maccooey, J.M. Scott, M.R. Sweeney, Unmetabolized folic acid prevalence is widespread in the older Irish population despite the lack of a mandatory fortification program, Am. J. Clin. Nutr. 96 (2012) 613–621. [39] A.H. Steindal, T.T. Tam, X.Y. Lu, A. Juzeniene, J. Moan, 5-Methyltetrahydrofolate is photosensitive in the presence of riboflavin, Photochem. Photobiol. Sci. 7 (2008) 814–818. [40] A. Juzeniene, T.T. Thu Tam, V. Iani, J. Moan, 5-Methyltetrahydrofolate can be photodegraded by endogenous photosensitizers, Free Radical Biol. Med. 47 (2009) 1199–1204. [41] T.T. Thu Tam, A. Juzeniene, A.H. Steindal, V. Iani, J. Moan, Photodegradation of 5-methyltetrahydrofolate in the presence of Uroporphyrin, J. Photochem. Photobiol. B 94 (2009) 201–204. [42] A.H. Steindal, A. Juzeniene, A. Johnsson, J. Moan, Photodegradation of 5methyltetrahydrofolate: biophysical aspects, Photochem. Photobiol. 82 (2006) 1651–1655. [43] G.T. Wondrak, M.K. Jacobson, E.L. Jacobson, Endogenous UVAphotosensitizers: mediators of skin photodamage and novel targets for skin photoprotection, Photochem. Photobiol. Sci. 5 (2006) 215–237. [44] R. Joshi, S. Adhikari, B.S. Patro, S. Chattopadhyay, T. Mukherjee, Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity, Free Radical Biol. Med. 30 (2001) 1390–1399. [45] T.N. Wien, E. Pike, T. Wisloff, A. Staff, S. Smeland, M. Klemp, Cancer risk with folic acid supplements: a systematic review and meta-analysis, BMJ Open 2 (2012) e000653.
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
The action spectrum for folic acid photodegradation in aqueous solutions Asta Juzeniene a,⇑, Tran Thi Thu Tam a, Vladimir Iani a, Johan Moan a,b a b
Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310 Oslo, Norway Department of Physics, University of Oslo, N-0316 Oslo, Norway
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 8 May 2013 Accepted 29 May 2013 Available online 7 June 2013 Keywords: Folic acid Action spectrum UV radiation
a b s t r a c t Folate is essential for cell division and growth. Deficiency is linked to birth defects, magaloblastic anaemia, cardiovascular disease, etc. Folic acid is a synthetic form of folate and is used to fortify food and in supplements. In aqueous solutions, in blood and even in human skin, folic acid may be degraded by ultraviolet radiation. Consequently, photodegradation of folic acid in human blood may lead to folate deficiency. However, the degree and the health consequences of such photodegradation are unknown. It is not clear which spectral region plays the most important role in the photodegradation of folic acid. In this study the photodegradation of folic acid in aqueous solution under different wavelengths of ultraviolet radiation (260–400 nm) was investigated by fluorescence spectroscopy. The photodegradation rate of folic acid was dependent on wavelength. Action spectrum for 1 lM folic acid photodegradation was determined. Its action spectrum is not identical to its absorption or fluorescence excitation spectra. The action spectrum demonstrated that UVB and UVA degrade folic acid. Protecting skin against UVB and UVA radiation by sunscreens may help to protect folic acid in human blood under intense solar radiation. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Folate, a water-soluble B vitamin, is involved in the synthesis, methylation and repair of DNA [1,2]. Folate deficiency may lead to magaloblastic anaemia, adverse pregnancy outcomes (neural tube defects), fertility problems and neuropsychiatric disorders [1,2]. Mammals cannot produce folate and must obtain it from their diet. Folic acid (FA), a synthetic folate form, is used for food fortification and nutritional supplements. The World Health Organization recommends that all women of reproductive age daily take FA supplements to prevent neural tubes defects in babies [3]. FA undergoes photodegradation in aqueous solutions, in blood irradiated in test tubes and in blood in healthy humans exposed to ultraviolet radiation (UV) from artificial sources and from sunlight [4–8]. Some studies have claimed that solar or artificial UV radiation can also deplete natural folates in human blood and skin [6,9–11], while other studies have claimed that UV radiation has no effect on folate levels [6,12–14]. Discrepancies in the different studies may be due to differences in used UV sources, fluence, skin condition (healthy, vitiligo, psoriasis, etc.), and methods for the measurements of folates in blood [15]. Recently, it was demonstrated by Fukuwatari et al. [6] that FA is more photolabile in blood than natural forms of folate. It seems that the presence of folic acid may stimulate photodegradation of the natural forms of folates. This means that photodegradation of FA in human blood may lead ⇑ Corresponding author. Tel.: +47 22934260; fax: +47 22781207. E-mail address: [email protected] (A. Juzeniene). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.05.011
to folate deficiency. However, the degree, the mechanism, and the health consequences of this photodegradation are unknown. FA has peaks in the UVB and UVA regions (Fig. 2). Due to this, broadband UV sources were used to study FA photodegradation [5,7,16]. It is not clear which wavelength plays the most important role in the initiation of the photodegradation of FA. The action spectrum for FA will show which wavelengths are most efficient in degrading FA molecules. It may help to understand the mechanisms of the photodegradation of FA, not only in aqueous solutions, but also in blood, and to predict its influence on human health. In the present work this action spectrum for FA has been determined by fluorescence spectroscopy. During UV exposure FA is converted into p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-formylpterin (FPT), which is then further oxidized to 6-carboxypterin (CPT) (Fig. 1). Based on our earlier work [5] we have chosen to measure only linear increase of initial fluorescence emission intensity. The formation of FPT is responsible for the increase of fluorescence intensities under UV exposure. The increase of fluorescence intensity by 40% was chosen due to statistical considerations. 2. Materials and methods 2.1. Chemicals Folic acid (FA, catalog number F7876, Lot. Number 081M1546V) and p-aminobenzoyl-L-glutamic acid (PABA-Glu) were purchased from Sigma–Aldrich Norway AS (Oslo, Norway), and both compounds were used without further purification. Dulbecco’s
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phosphate buffered saline (PBS) was obtained from PAA laboratories GmbH (Pasching, Austria). 6-formylpterin (FPT) was obtained from Schircks Laboratories (Jona, Switzerland). Before each experiment stock solutions of 100 mM FA, PABA-Glu or FPT were prepared in 1 M NaOH. Afterwards, they were diluted in PBS (pH = 7.4) to the required concentrations. pH 7.4 was chosen to simulate physiological media, because normal blood pH is also around 7.4. 2.2. Absorption and fluorescence measurements Absorption spectra were registered with a Perkin-Elmer Lambda 40 UV/VIS spectrophotometer (Norwalk, CT, USA). Fluorescence emission and excitation spectra were registered with a Perkin-Elmer LS50B luminescence spectrometer (Norwalk, CT, USA). Fluorescence emission spectra were measured in the range of 300–540 nm. Excitation wavelength was set at 270 nm. For fluorescence excitation spectroscopy, the emission wavelength was set at 445 nm. The complete excitation spectra were recorded in the range of 220–420 nm. 2.3. Photolysis A set-up with a high pressure Xenon arc lamp 900 W (Oriel, Stamford, CT) and a monochromator MODEL (Jarrell Ash, Boston, MA, USA) were used during the experiments. The monochromator was set with relatively wide slits resulting in Gaussian shaped peaks with 12 nm bandwidth. A power meter SOLO 2 with Gentec-EO PH photodetector PH100-SiUV (Gentec-EO, Québec, Canada) was used to measure the fluence rates (U) at the monochromator exit slit at different wavelengths (Fig. 3). FA samples (0.7 ml) in opened quartz cuvettes were exposed to UV radiation in the spectral range from 260 nm to
Fig. 2. Absorption spectra of 1 lM FA, p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-formylpterin (FPT) in PBS.
400 nm. The same quartz cuvette with 10 mm length and 4 mm inside width (Fig. 4) was used for absorption and fluorescence measurements and for UV exposure. The fluorescence excitation wavelength was set at 270 nm and the photodegradation of FA was measured by determining the increase of fluorescence of photoproducts at 445 nm. This increase of fluorescence (data not shown) is due to the photoproducts of FA [5,17]. FA has a low fluorescence quantum yield (<0.005) [18]. However, when the molecule is exposed to UV radiation, the bond between the 6-methylpterin and the p-aminobenzoic acid (Fig. 1) is broken and the fluorescence of the solution increases [5,18,19]. The obtained fluorescence emission values at 445 nm were normalized to 1 at the initial time point for all concentrations. The temperatures of the samples were approximately 22–25 °C. All experiments were performed in constant dim light in order to avoid external, uncontrolled light exposure.
Fig. 1. The structural formula of folic acid (FA), 6-formylpterin (FPT), p-aminobenzoyl-L-glutamic acid (PABA-Glu) and 6-carboxypterin (CPT). Schematic photodegradation of FA.
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Fig. 3. Measured fluence rates of the lamp in the region from 260 to 400 nm.
Fig. 5. (A) Absorption and fluorescence emission spectra of 0.26, and 1 lM FA in PBS. (B) Normalized absorption, fluorescence emission and excitation spectra of 0.26 and 1 lM FA in PBS. The excitation wavelength was set at 270 nm for the fluorescence emission spectra and the emission wavelength was set at 445 nm for the fluorescence excitation spectra. Fig. 4. Experimental setup of the cuvette in the holder, where ‘1 = 0.2 cm, ‘2 = 0.4 cm and ‘3 = 1.0 cm. For UV exposure 1 cm path length of the cuvette was used.
2.4. Construction of action spectra for FA photogeneration An action spectrum is a plot of a response per number of incident photons, against wavelength. The total number of incident photons hitting the sample (ni) to give the same effect at all wavelengths is given by:
ni ¼
Utka hc
(data not shown). Not more than 5% of FA was photooxidized during given UV exposures [5,20]. FA has absorption peaks at around 280 nm and 350 nm (Figs. 2 and 5). For this reason, these two wavelengths were chosen for the demonstration of FA photodegradation and its photoproduct formation (Fig. 6). From these results the rates of photoproducts formation were determined as well as the fluences needed for a fluorescence intensity increase by 40% at 445 nm of 0.26 and 1 lM FA (Figs. 6 and 7). Photoproducts formation rates were similar (P > 0.05) at 0.26 lM and 1 lM at 280 and 350 nm (Fig. 6).
;
where U is the fluence rate (mW/cm2), t is the radiation exposure time (s) needed for a fluorescence intensity increase by 40% at 445 nm, k is the radiation wavelength (nm), a is the exposed area on the cuvette (0.7 cm2), h is the Planck constant (6.626 1034 J s), and c is the speed of light (3 108 m/s). 2.5. Data analysis Data are represented as mean values and standard errors of the mean of at least three independent experiments are given. The data were also analyzed using the linear fitting routines of SigmaPlot 11.0 from Systat Software Inc. (Richmond, CA, USA). 3. Results Aqueous solutions of 0.26 and 1 lM FA were exposed to UV radiation at every 10 nm in the range between 260 nm and 400 nm with total fluences up to 2 J/cm2. The fluorescence emission spectra and its kinetics at 445 nm were recorded (data not shown). This led to increase in fluorescence intensity by more than 40% as measured at 445 nm. No apparent changes were observed in the absorption spectra of 0.26 and 1 lM FA after UV exposure
Fig. 6. Kinetics of the increase of the fluorescent products resulting from exposure of 0.26 lM (A) and 1 lM (B) FA to radiation at 280 nm and 350 nm. The fluorescence excitation wavelength was set at 270 nm.
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Fig. 7. Fluence doses needed for a fluorescence intensity increase by 40% at 445 nm of 0.26 lM and 1 lM FA. FA was exposed to radiation at 280 and 350 nm.
FA absorbs around 3.8-fold stronger at 280 nm than at 350 nm (Fig. 5B). Correspondingly, the fluence needed for a fluorescence intensity increase by 40% was approximately 4-folds higher at 350 nm than at 280 nm at a concentrations of 0.26 lM and 1 lM FA (Fig. 7). The action spectrum for FA photodegradation showed that UVB quanta were much more efficient to degrade FA than UVA quanta at 1 lM (Fig. 8). The action spectrum is not identical to its absorption or fluorescence excitation spectra (Fig. 8). The absorption spectrum of FA has peak at around 280 nm, while its action spectrum has peak at around 270 nm, at the same wavelength as its fluorescence excitation peak (Fig. 8). While in the UVA region the action spectrum is lower in comparison to the absorption spectrum of 1 lM FA (Fig. 8).
4. Discussion Due to the biological importance of FA, many scientists have studied its photodegradation mechanism in aqueous solutions [4,5,7,16,20–22]. The photolysis of FA has been studied mostly under broadband UVA, UVC (which is absorbed by the ozone layer and does not reach the Earth’s surface) or visible light exposure [4,5,7,23,24]. No data have been published about the photolysis of FA under UVB exposure, and, consequently, the action spectrum for FA degradation has been unknown until now. Photooxidation of FA is initiated by excited states of FA itself [7], and it is usually divided into three phases [5,7,23]. In the first phase, formation of the photoproducts PABA-Glu and FPT occurs and obeys a zero order rate law. In the second phase the presence of FPT sensitizes the degradation of FA, and its degradation process
Fig. 8. The action spectra for 1 lM photodegradation, normalized to maxima fluorescence excitation and absorption spectra of FA.
is accelerating [7,25]. In the third phase the degradation of FPT to CPT is the dominating process, and this reaction follows a first order kinetics. In the present study only the first phase (initial) of the photodegradation of commercially available FA was studied. PABA-Glu, FPT and CPT are not photostable [26–28]. Their formation is dominant in the beginning of the photolysis of FA, while later their degradation becomes dominant. Additionally, FPT and CPT, but not PABA-Glu, act as photosensitizers [5,7,17,20,23]. Furthermore, they act differently at different wavelengths, due to differences in their absorption characteristics (Fig. 2): PABA-Glu absorbs mostly in the UVB region, while FPT and CPT absorb both in the UVB and in the UVA regions [5]. All these factors influence the photobleaching rates at different wavelengths [26–28]. PABA-Glu undergoes photolysis under UVB exposure, but above 320 nm, PABA-Glu does not absorb, then it is photostable. CPT is not photostable under UVB and UVA exposure, but its quantum yield of photodegradation is much lower than those of FA and FPT [27,28]. Besides singlet oxygen production by energy transfer of the triplet excited states of pterins to oxygen, electron transfer processes have been shown to play a dominant role in the formation of other reactive oxygen species and in the p the autocatalytic photooxidation of FA [7,25,29,30]. It has been found that FA is photolabile only in the presence of oxygen [7,28]. Moreover, many endogenous or exogenous photosensitizers might cause the degradation of FA, under UV or even visible light exposure [7,25]. The action spectrum of 1 lM photodegradation is not identical to its absorption and fluorescence excitation spectra (Fig. 8). At the same time fluorescence excitation spectrum is not similar to the absorption spectrum of FA (Figs. 5 and 8). FA has fluorescence excitation peaks at around 270 nm and 350 nm, while absorbance peaks at around 280 nm and 350 nm (Figs. 5 and 8). Thomas et al. [18] reported that fluorescence excitation spectrum of FA obtained by excitation at 450 nm was different from the absorption spectrum. In their case, fluorescence excitation peaks were around 270 nm and 370 nm. The ratio between the fluorescence excitation bands at 370 nm and 270 nm was around 0.3. They did not mention which concentration of FA was measured, but in order to avoid inner filter effect, they kept the absorption of FA solution below 0.1. This means that the concentration of FA was below 4 lM. In our case the ratio between the fluorescence excitation bands at 350 nm and 270 nm was around 1.3 (Fig. 5). We could not find any other study, which would describe the fluorescence excitation spectra of FA. So big discrepancy between our and Thomas et al. fluorescence excitation spectra may be due to impurities of FA itself (different producers) or solution in which it was prepared, aggregation of FA, or differences in used solvents. We were using commercially available FA without further purification, which may contain some impurities. Thomas et al. [18] calculated that the fluorescence quantum yields for the high energy bands are lower by a factor of at least 3 than those corresponding to the low energy bands [18]. This means that only a fraction of the energy of the upper excited state(s) (S2,Sn) is dissipated through internal conversion to the lowest singlet excited state (S1) [18]. Consequently, a photophysical (intersystem crossing to the triplet manifold) or/and a photochemical process should occur from an upper singlet excited state [18,19]. The relatively long chain substituent of FA might act as an ‘‘internal fluorescence quencher,’’ enhancing the radiationless deactivation of the singlet excited state [18,31]. Due to this reason FA does not produce singlet oxygen [17]. The quantum yield of singlet oxygen production is very low for FA (60.02). At the very early stage of the photoconversion (below 10%) the light absorption by the photoproducts can be neglected and the rate is proportional to the quantum yield for FA phototransformation. The quantum yield associated with the disappearance of 600 lM
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FA under UVA exposure was measured by Thomas et al. [28] and a value of 0.025 was observed. On the other hand, the same value was also obtained for the quantum yield of FPT formation. Therefore, consumption of FA to lead FPT is the only process that takes place at very early stage of FA photodegradation [28]. A higher photoreaction quantum yield in the UVB would be consistent with the fluorescence excitation spectra, indicating that some upper excited state does not undergo quantitative internal conversion to the lowest lying (fluorescent) state. FA is used for supplementation and food fortification. It lacks coenzyme activity and must be reduced to dihydrofolate and then to tetrahydrofolate after it enters body [32]. The same enzyme, dihydrofolate reductase, catalyzes both these reactions. The activity of dihydrofolate reductase is much lower in humans than in animals, and it varies greatly between individuals [32,33]. In humans, increased FA intake (over 200 lg) leads to elevated blood concentrations of unmetabolized FA [33–35]. Unmetabolized serum FA was detected in 38% of the older population (aged P 60 years) in the United States (mandatory folic acid fortification) with a mean concentration of 1.2 nM (range 0–273 nM), accounting for 2.25% of total plasma folate [36,37]. In the older Irish population (aged P 60 years) despite the lack of a mandatory fortification program unmetabolized FA was detected in 94.1% of the cohort with a mean concentration of 0.39 nM (range 0–1.59 nM), accounting for 1.3% of total plasma folate [38]. Spending time in the sun or in sunbeds shortly after taking FA supplementation may lead to FA degradation. In healthy volunteers plasma folate levels decreased by 26% only in the group of volunteers who took FA before a single exposure of 2 h to sunlight [6]. The dose of UVA in sunlight was about 12 J/cm2. The natural folate derivative, 5-methyltetrahydrofolate, may also degrade under UV exposure [39–41], but 5-methyltetrahydrofolate is more stable than FA, because it 5-methyltetrahydrofolate almost does not absorb in the UVA region [42]. Earlier studies have demonstrated that FA photodegradation is concentration dependent [5,20]. Due to too low sensitivity of absorption and fluorescence spectroscopy we were not able to study degradation of physiologically relevant concentrations (<2 nM FA, [36–38]) and have chosen to study degradation of 0.26 and 1 lM FA. Solutions of 0.26 lM and 1 lM FA has the same absorbance at 280 and 350 nm, respectively (Fig. 5). It has been reported that a UVA dose of 2 J/cm2 from a broadband lamp degrades 10 and 85 lM FA completely in aqueous solutions in a cuvette (1 cm optical path), while much higher UVA doses are needed to destroy lower concentration of FA [5,20]. While 1 lM FA degrades completely only after UVA dose of 30 J/ cm2 [5]. It will take 5 and 85 min, respectively, to obtain UVA doses of 2 or 30 J/cm2, from the sun at noon under the Equator (where fluence rate is around 6 mW/cm2). In blood much higher UVA doses will be needed to degrade FA. Under physiological conditions (<2 nM FA) the first phase of the degradation of FA will be dominant. However, most studies have been performed using much higher FA concentrations (600 lM FA [28], 450 lM FA [28], 85 lM FA [20], 20–1000 lM FA [7]) where the first phase of FA degradation is rapidly changed into the second and third phases. To obtain conditions more physiologically relevant during the initial stage of FA photodegradation, much lower concentrations of FA must be studied. Additionally, the mechanisms involved in the photoooxidation of FA under UV exposure strongly depend on the pH [4,19]. Excitation of the acidic form of FA in the presence of oxygen leads to cleavage and oxidation of the molecules, yielding FPT and PABAGlu [19]. In the photooxidation of the basic form of FA, an additional reaction pathway exists, leading to an unknown compound with a molecular weight higher than that of FA [19].
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The present results demonstrate that in aqueous solutions UVB radiation is more efficient to degrade FA of the same concentration than UVA radiation. Only a small fraction of UVB radiation reaches blood vessels, and it is unlikely that direct photodegradation of FA takes place in human skin during exposure to UVB radiation. However, the presence of photosensitizers, such as riboflavin, significantly increases the photolysis rates of FA under UVA radiation [24]. In this case the photolysis of FA is mostly mediated via photosensitization rather than via direct photolysis. Exposure of humans taking FA supplementation to solar radiation both processes may be involved in the photolysis of FA, because blood and skin contains many endogenous photosensitizers [43]. Williams and Jacobson [11] have demonstrated that folate depleted human keratinocytes exposed to UV radiation (0.2 J/cm2 UVB and 4 J/cm2 UVA) were unable to repair DNA damage, while non-depleted control keratinocytes exposed to the same UV dose showed a nearly complete removal of DNA strand breaks to reach a level of non-exposed control cells after 24 h. Thus, chronic folate deficiency (due to low intake of natural folates and FA or due to photodegradation of folates and FA) may lead to skin photocarcinogenesis. Additionally, high concentrations of the photoproducts of FA may induce DNA damage under UV radiation and lead to the initiation of skin cancer [20,29]. At the same time, FA is an effective singlet oxygen and free radical (peroxyl, azide, sulfate, hydroxyl, thiyl) scavenger [17,44]. Thus, it may protect human skin and other organs from DNA damage mediated by reactive oxygen and nitrogen species. However, a meta-analysis of six randomized controlled trials showed that folic acid supplementation may boost prostate cancer risk [45]. Further studies are needed to understand a possible mechanism of FA photodegradation in human body. 5. Conclusions The photodegradation rates of FA are wavelength, but not concentration, dependent. The action spectrum for 1 lM FA in aqueous solutions shows that quanta in the UVB region are more efficient than quanta in the UVA region to degrade FA. The action spectrum for 1 lM FA photodegradation has maximum in the UVB region around 267 nm, in the same position as in the fluorescence excitation spectrum, but it is shifted to around 280 nm in the absorption spectrum. The peak in the UVA region of the action spectrum, fluorescence emission and absorption spectra is in the same position at around 350 nm. The action spectrum of 1 lM FA resembles its absorption spectrum, but it is blue shifted. Our experiments indicate that FA is the main chromophore in the initial degradation of FA. 6. Abbreviations
FA FPT CPT PABA-Glu UVA UVB
folic acid 6-formylpterin 6-carboxypterin p-aminobenzoyl-L-glutamic acid ultraviolet A (400–315 nm) ultraviolet B (280–315 nm)
Acknowledgments The present work was supported by the Research Foundation of the Norwegian Radium Hospital and the South-Eastern Norway Regional Health Authority. The authors thank Armend Håti for his help in performing fluorescence spectroscopy.
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References [1] M. Lucock, I. Daskalakis, New perspectives on folate status: a differential role for the vitamin in cardiovascular disease, birth defects and other conditions, Br. J. Biomed. Sci. 57 (2000) 254–260. [2] N.A. Wani, A. Hamid, J. Kaur, Folate status in various pathophysiological conditions, IUBMB Life 60 (2008) 834–842. [3] L.M. De-Regil, A.C. Fernandez-Gaxiola, T. Dowswell, J.P. Pena-Rosas, Effects and safety of periconceptional folate supplementation for preventing birth defects, Cochrane Database Syst. Rev. 6 (2010). CD007950. [4] M.J. Akhtar, M.A. Khan, I. Ahmad, Photodegradation of folic acid in aqueous solution, J. Pharm. Biomed. Anal. 19 (1999) 269–275. [5] M.K. Off, A.E. Steindal, A.C. Porojnicu, A. Juzeniene, A. Vorobey, A. Johnsson, J. Moan, Ultraviolet photodegradation of folic acid, J. Photochem. Photobiol. B 80 (2005) 47–55. [6] T. Fukuwatari, M. Fujita, K. Shibata, Effects of UVA irradiation on the concentration of folate in human blood, Biosci. Biotechnol. Biochem. 73 (2009) 322–327. [7] M.L. Dantola, M.P. Denofrio, B. Zurbano, C.S. Gimenez, P.R. Ogilby, C. Lorente, A.H. Thomas, Mechanism of photooxidation of folic acid sensitized by unconjugated pterins, Photochem. Photobiol. Sci. 9 (2010) 1604–1612. [8] D.C. Borradale, M.G. Kimlin, Folate degradation due to ultraviolet radiation: possible implications for human health and nutrition, Nutr. Rev. 70 (2012) 414–422. [9] R.F. Branda, J.W. Eaton, Skin color and nutrient photolysis: an evolutionary hypothesis, Science 201 (1978) 625–626. [10] M. Der-Petrossian, M. Fodinger, R. Knobler, H. Honigsmann, F. Trautinger, Photodegradation of folic acid during extracorporeal photopheresis, Br. J. Dermatol. 156 (2007) 117–121. [11] J.D. Williams, M.K. Jacobson, Photobiological implications of folate depletion and repletion in cultured human keratinocytes, J. Photochem. Photobiol. B 99 (2010) 49–61. [12] T. Gambichler, A. Bader, K. Sauermann, P. Altmeyer, K. Hoffmann, Serum folate levels after UVA exposure: a two-group parallel randomised controlled trial, BMC Dermatol. 1 (2001) 8. [13] A. Osmancevic, L.T. Nilsen, K. Landin-Wilhelmsen, E. Soyland, T.P. Abusdal, T.A. Hagve, M.S. Nenseter, A.L. Krogstad, Effect of climate therapy at Gran Canaria on vitamin D production, blood glucose and lipids in patients with psoriasis, J. Eur. Acad. Dermatol. Venereol. 23 (2009) 1133–1140. [14] E. Cicarma, C. Mork, A.C. Porojnicu, A. Juzeniene, T.T. Tam, A. Dahlback, J. Moan, Influence of narrowband UVB phototherapy on vitamin D and folate status, Exp. Dermatol. 19 (2010) e67–e72. [15] A. Juzeniene, K.T. Stokke, P. Thune, J. Moan, Pilot study of folate status in healthy volunteers and in patients with psoriasis before and after UV exposure, J. Photochem. Photobiol. B 101 (2010) 111–116. [16] P. Vorobey, A.E. Steindal, M.K. Off, A. Vorobey, J. Moan, Influence of human serum albumin on photodegradation of folic acid in solution, Photochem. Photobiol. 82 (2006) 817–822. [17] A.H. Thomas, C. Lorente, A.L. Capparelli, C.G. Martinez, A.M. Braun, E. Oliveros, Singlet oxygen (1deltag) production by pterin derivatives in aqueous solutions, Photochem. Photobiol. Sci. 2 (2003) 245–250. [18] A.H. Thomas, C. Lorente, A.L. Capparelli, M.R. Pokhrel, A.M. Braun, E. Oliveros, Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in aqueous solution: pH effects, Photochem. Photobiol. Sci. 1 (2002) 421–426. [19] C. Lorente, A.H. Thomas, Photophysics and photochemistry of pterins in aqueous solution, Acc. Chem. Res. 39 (2006) 395–402. [20] T. Offer, B.N. Ames, S.W. Bailey, E.A. Sabens, M. Nozawa, J.E. Ayling, 5Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA, FASEB J. 21 (2007) 2101–2107. [21] J. Arcot, A. Shrestha, Folate: methods of analysis, Trends Food Sci. Technol. 16 (2005) 253–266. [22] M.J. Akhtar, M.A. Khan, I. Ahmad, Identification of photoproducts of folic acid and its degradation pathways in aqueous solution, J. Pharm. Biomed. Anal. 31 (2003) 579–588. [23] A.H. Thomas, G. Suarez, F.M. Cabrerizo, F.S.G. Einschlag, R. Martino, C. Baiocchi, E. Pramauro, A.L. Capparelli, Photochemical behavior of folic acid in alkaline aqueous solutions and evolution of its photoproducts, Helv. Chim. Acta 85 (2002) 2300–2315.
[24] M.J. Akhtar, M.A. Khan, I. Ahmad, Effect of riboflavin on the photolysis of folic acid in aqueous solution, J. Pharm. Biomed. Anal. 23 (2000) 1039–1044. [25] C. Lorente, G. Petroselli, M.L. Dantola, E. Oliveros, A.H. Thomas, Electron transfer initiated reactions photoinduced by pterins, Pteridines 22 (2011) 111–119. [26] A.A. Shaw, L.A. Wainschel, M.D. Shetlar, The photochemistry of paminobenzoic acid, Photochem. Photobiol. 55 (1992) 647–656. [27] G. Suarez, F.M. Cabrerizo, C. Lorente, A.H. Thomas, A.L. Capparelli, Study of the photolysis of 6-carboxypterin in acid and alkaline aqueous solutions, J. Photochem. Photobiol. B 132 (2000) 53–57. [28] A.H. Thomas, G. Suarez, F.M. Cabrerizo, R.l. Martino, A.L. Capparelli, Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions, J. Photochem. Photobiol. A 135 (2000) 147–154. [29] K. Hirakawa, H. Suzuki, S. Oikawa, S. Kawanishi, Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product, Arch. Biochem. Biophys. 410 (2003) 261–268. [30] M. Soniat, C.B. Martin, Theoretical study on the relative energies of anionic pterin tautomers, Pteridines 20 (2009) 124–128. [31] F.M. Cabrerizo, G. Petroselli, C. Lorente, A.L. Capparelli, A.H. Thomas, A.M. Braun, E. Oliveros, Substituent effects on the photophysical properties of pterin derivatives in acidic and alkaline aqueous solutions, Photochem. Photobiol. 81 (2005) 1234–1240. [32] A.D. Smith, Y.I. Kim, H. Refsum, Is folic acid good for everyone?, Am J. Clin. Nutr. 87 (2008) 517–533. [33] S.W. Bailey, J.E. Ayling, The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake, Proc. Natl. Acad. Sci. USA 106 (2009) 15424–15429. [34] P. Kelly, J. McPartlin, M. Goggins, D.G. Weir, J.M. Scott, Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements, Am. J. Clin. Nutr. 65 (1997) 1790–1795. [35] M.R. Sweeney, J. McPartlin, J. Scott, Folic acid fortification and public health: report on threshold doses above which unmetabolised folic acid appear in serum, BMC Public Health 7 (2007) 41. [36] R.L. Bailey, J.L. Mills, E.A. Yetley, J.J. Gahche, C.M. Pfeiffer, J.T. Dwyer, K.W. Dodd, C.T. Sempos, J.M. Betz, M.F. Picciano, Unmetabolized serum folic acid and its relation to folic acid intake from diet and supplements in a nationally representative sample of adults aged > or =60 y in the United States, Am. J. Clin. Nutr. 92 (2010) 383–389. [37] R.L. Bailey, J.L. Mills, E.A. Yetley, J.J. Gahche, C.M. Pfeiffer, J.T. Dwyer, K.W. Dodd, C.T. Sempos, J.M. Betz, M.F. Picciano, Serum unmetabolized folic acid in a nationally representative sample of adults >/=60 years in the United States, 2001–2002, Food Nutr. Res. 56 (2012). [38] A. Boilson, A. Staines, C.C. Kelleher, L. Daly, I. Shirley, A. Shrivastava, S.W. Bailey, P.B. Alverson, J.E. Ayling, A.P. McDermott, A. Maccooey, J.M. Scott, M.R. Sweeney, Unmetabolized folic acid prevalence is widespread in the older Irish population despite the lack of a mandatory fortification program, Am. J. Clin. Nutr. 96 (2012) 613–621. [39] A.H. Steindal, T.T. Tam, X.Y. Lu, A. Juzeniene, J. Moan, 5-Methyltetrahydrofolate is photosensitive in the presence of riboflavin, Photochem. Photobiol. Sci. 7 (2008) 814–818. [40] A. Juzeniene, T.T. Thu Tam, V. Iani, J. Moan, 5-Methyltetrahydrofolate can be photodegraded by endogenous photosensitizers, Free Radical Biol. Med. 47 (2009) 1199–1204. [41] T.T. Thu Tam, A. Juzeniene, A.H. Steindal, V. Iani, J. Moan, Photodegradation of 5-methyltetrahydrofolate in the presence of Uroporphyrin, J. Photochem. Photobiol. B 94 (2009) 201–204. [42] A.H. Steindal, A. Juzeniene, A. Johnsson, J. Moan, Photodegradation of 5methyltetrahydrofolate: biophysical aspects, Photochem. Photobiol. 82 (2006) 1651–1655. [43] G.T. Wondrak, M.K. Jacobson, E.L. Jacobson, Endogenous UVAphotosensitizers: mediators of skin photodamage and novel targets for skin photoprotection, Photochem. Photobiol. Sci. 5 (2006) 215–237. [44] R. Joshi, S. Adhikari, B.S. Patro, S. Chattopadhyay, T. Mukherjee, Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity, Free Radical Biol. Med. 30 (2001) 1390–1399. [45] T.N. Wien, E. Pike, T. Wisloff, A. Staff, S. Smeland, M. Klemp, Cancer risk with folic acid supplements: a systematic review and meta-analysis, BMJ Open 2 (2012) e000653.