Journal of Applied Spectroscopy, Vol. 76, No. 2, 2009 POSSIBLE USE OF PROVITAMIN D3 PHOTOISOMERIZATION FOR SPECTRAL DOSIMETRY OF BIOACTIVE ANTIRACHITIC UV RADIATION T. N. Orlova* and I. P. Terenetskaya UDC 551.586;535.341;543.422.3;544.52;577.161.2 The possible use of a simplified UV absorption spectroscopic method for dosimetry of bioactive antirachitic UV radiation has been analyzed. The method is based on the observation of the phototransformation kinetics of the provitamin D3 primary molecule in ethanol (in vitro vitamin D3 synthesis model) by measuring the decrease in the optical density at a fixed wavelength during UV exposure. The method can be used successfully for artificial UV sources with a constant radiation spectrum. However, such a technique turns out to be inapplicable to solar UV dosimetry in view of the variability of the solar UV spectrum that results in a varying rate of formation of irreversible photoproducts. Key words: UV absorption spectroscopy, spectral kinetics of previtamin D photosynthesis, UV monitoring, biological effects of UV radiation, antirachitic activity of UV sources. Introduction. The personal UV dosimeter industry has recently been rapidly developing because of the biological activity of UV radiation, which makes it necessary to monitor the dose of UV radiation received by man. The change in the thickness of the ozone layer and the amount of industrial atmospheric emissions has a noticeable effect on the spectrum of solar UV radiation. Furthermore, the popularity of tanning salons leads to an additional dose of artificial UV radiation that in general produces a broad variability in the individual UV radiation dose. Despite the variety of biological effects from the action of UV radiation, the erythema UV radiation dose is most commonly measured because it produces an unfavorable photobiological response in man (erythema, pigmentation, burn, aging, and cancer of skin). Polysulfone film is used most often as a personal UV dosimeter. Quantitation of the received UV dose is based on measurement of the absorption changes of this material at a fixed wavelength (330 nm) that are caused by UV radiation [1]. However, it must be recalled that irradiation by UVB light (280–315 nm) has a favorable biological action that initiates the synthesis of vitamin D3 in the outer skin layers (epidermis). It has recently been found that vitamin D3 has the classical actions in man of normalizing homeostasis of calcium and phosphorus in blood and preventing osteoporosis and rickets and also regulates on the genetic level the growth of cells and the functioning of vitally important organs (kidneys, liver, immune system) [2]. Thus, the dose of bioactive antirachitic UV radiation that is beneficial for man must be measured in addition to the erythema UV dose. Obviously the spectral sensitivity of the dosimeter for measuring the antirachitic activity of UV radiation should be identical to the activity spectrum of the examined biological effect, i.e., vitamin D3 synthesis. This reaction is known to occur in two steps [3]. In the first step, provitamin D3 (7-dehydrocholesterol, 7-DHC) is transformed photochemically through the action of UV radiation into previtamin D, from which vitamin D3 is formed in the second step through a thermochemical route (Fig. 1a). Thus, the concentration of previtamin D formed upon UV irradiation is a measure of the antirachitic activity of the UV radiation [4, 5]. Because a multicomponent mixture of photoisomers is formed upon UV irradiation, the concentrations are usually analyzed using high-performance liquid chromatography (HPLC) [4], which is not suitable for in situ measurements. Therefore, an original spectrophotometric method for measuring the concentration of previtamin D was devel∗ To whom correspondence should be addressed. Institute of Physics, National Academy of Sciences of Ukraine, 46 Nauka Ave., Kiev, 03680, Ukraine; e-mail: orlovat@iop.kiev.ua. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 76, No. 2, pp. 256–260, March–April, 2009. Original article submitted August 1, 2008. 240 0021-9037/09/7602-0240 ©2009 Springer Science+Business Media, Inc. Fig. 1. Diagram of vitamin D3 synthesis (a) and UV absorption spectra of principal photoisomers (b): provitamin D3 (1), previtamin D (2), vitamin D3 (3), tachysterol (4), lumisterol (5), toxisterol (6); R = C8H17 for vitamin D3 series; R = C9H17 for vitamin D2 series. Numbers by arrows give quantum yields of phototransformations [3] (a). UV absorption spectra of toxisterols were obtained by irradiation of 7-DHC in ethanol by an XeCl eximer laser at 308 nm [6] (b). oped [7, 8] that takes into account its irreversible phototransformations (photodegradation of the multicomponent mixture). For this, a solution of 7-DHC in ethanol in a square quartz cuvette was treated with UV radiation. The UV absorption spectra (230–330 nm) were recorded before and after exposure. Then, they were analyzed using a specially developed computer program [8] that can determine the quantitative composition of the photoisomeric mixture from the individual UV absorption spectra of the isomers [9] (Fig. 1b). However, specially trained personnel are required to use such dosimetry. Therefore, the question of the possible use of a simplified spectral method similar to that used in UV dosimetry based on polysulfone film is timely. The individual UV absorption spectra of the photoisomers (Fig. 1b) show that previtamin D accumulation causes a noticeable decrease of the optical density at 270–300 nm. This suggests that the method of UV spectral analysis can be simplified, i.e., the decrease of optical density can be observed at a fixed wavelength. This is most conveniently done at the absorption maximum of starting provitamin D3 (282 nm). Herein we report experiments on the confirmation of this approach. Experimental. Field tests of a dosimeter of the antirachitic activity of solar UV radiation (Greece) were analyzed. The daily accumulation of previtamin D was measured using spectrophotometric analysis [7]. For this, two separate cuvettes with 7-DHC solution in ethanol were simultaneously subjected to the action of solar radiation for one day under clear skies. UV absorption spectra were recorded before the experiment and after each hour of irradiation (or 30 min during midday) (Fig. 2a). The decrease of optical density at λ = 282 nm as a function of the concentration of accumulated previtamin D (Fig. 2b) was plotted. It can be seen that this function is described rather well by a linear approximation. We propose that the resulting linear function can be used as a calibration curve and that the concentration of t 0 previtamin D can be calculated from the optical density D at λ = 282 nm before (D282) and after (D282) UV irradiation using the formula: t 0 CPre (%) = (1 − D282 ⁄ D282) ⁄ 0.01 . (1) o o The proposed simplified method for UV dosimetry was tested in Kiev (50 23′N, 30 23′E) from April to September 2005 [10]. For this, two quartz cuvettes (0.5 cm thick) with 7-DHC (Fluka) in ethanol solution (C = 20 µg/mL) were irradiated by solar radiation for 3 h (from 11:30 to 14:30 local time). Solar rays were kept normal 241 Fig. 2. Transformation of absorption spectrum of provitamin D3 by the action of solar radiation (July 20, 1997, Nea Michaniona, Greece) under clear skies (a); optical density at λ = 282 nm as a function of concentration of accumulated previtamin D in vitro (b), Y = A + Bx, A = 1, B = –0.00959, R = −0.99589, SD = 0.00687, 䉱 and 䉮 correspond to two different cuvettes. Fig. 3. Concentrations of photosynthesized previtamin D obtained (in vitro) in Kiev using spectrophotometric analysis (a) and calculated from data for the decrease of optical density at λ = 282 nm (b) (2005); correlation of these values and linear approximation using the Origin program Y = BX, B = 0.098925, R = 0.7711, SD = 3.2983, dashed lines are upper and lower 90% limits. 242 to the input face of the cuvette by using a specially constructed servo-mechanism for automatic tracking of the solar zenith angle. UV absorption spectra of the solutions before and after exposure were recorded using a Perkin–Elmer Lambda 25 UV-VIS spectrophotometer in the range 230–330 nm in 1-nm steps. Then, spectrophotometric analysis was performed and the concentration of previtamin D formed during the irradiation was determined. Figure 3a shows that seasonal changes and weather conditions have a substantial effect on the concentration of accumulated previtamin D, i.e., on the antirachitic activity of solar UV radiation. The change of optical density at λ = 282 nm was determined for each measurement. The concentration of previtamin D was calculated using Eq. (1) (Fig. 3b). The results were compared with the value obtained using spectrophotometric analysis (Fig. 3c). It can be seen that the coefficient B = 0.98925 in the linear approximation Y = BX is close to unity. The correlation coefficient R = 0.7711 is consistent with a strong correlation. However, a direct examination of the data distribution shows that the uncertainty in the determination of the previtamin D concentration is significantly greater than that given in Fig. 2b and in many instances is comparable with the concentration. Discussion. Let us examine in more detail the photoreaction of provitamin D3 in order to explain the reasons for such significant uncertainties in determining the concentration of previtamin D from the decrease of optical density at λ = 282 nm (Fig. 3c). Figure 1b shows that previtamin D absorbs in the same spectral region as starting provitamin D3. Therefore, it undergoes several phototransformations upon UV irradiation, the most effective of which is cis-transisomerization into tachysterol (Fig. 1a) [3]. However, a substantial accumulation of tachysterol in the photoisomeric mixture that is accompanied by an increase of optical density at λ = 282 nm is observed only for short-wavelength UV (254 nm) irradiation [3]. The reverse reactions of closing the diene ring to form provitamin D3 and its diastereomer lumisterol occur with low quantum yield that is an order of magnitude less than that of the forward reaction to form previtamin D from 7-DHC. Thus, the optical density at λ = 282 nm decreases upon long-wavelength UV irradiation (313 nm) as a result of the predominance in the photoisomeric mixture of previtamin D and also its irreversible phototransformations into toxisterols. The rate of formation of toxisterols is known to increase sharply upon UV irradiation at λ > 300 nm [6]. This sets up a competition between formation of previtamin D and its irreversible phototransformations. The maximum accumulation of previtamin D depends sharply on the spectral position of the short-wavelength cutoff of the solar spectrum [7, 8]. The decrease of optical density at λ = 282 nm is limited when primarily previtamin D accumulates in solution. The optical density drops all the way to zero if toxisterols are formed effectively because toxisterols do not absorb at this wavelength (Fig. 1b). o o Obviously differences in the latitudes of Kiev (50 23′N) and Nea Michaniona (40 47′N) and the variable weather conditions have an effect on the solar spectrum of UV radiation, shifting the edge of the UV spectrum to λ = 285–300 nm [11]. The decrease of optical density at λ = 282 nm is not absolutely related to the concentration of previtamin D that was formed because of the sensitivity of the rate of formation of toxisterols to the UV wavelength. In our opinion, this causes the rather large scatter in the Kiev data compared with those from Greece. Thus, the variability of the short-wavelength solar spectrum does not enable the amount of accumulated previtamin D to be determined unambiguously from the decrease of optical density at λ = 282 nm alone. It is noteworthy that the simplified method of spectral analysis can be used for artificial UV light sources with a constant radiation spectrum after performing a calibration procedure. Obviously, the calibration should be carried out anew for each source with its unique UV radiation spectrum. Finally, the simplified spectrophotometric method can be used to estimate the antirachitic activity of solar UV radiation if a medium in which the effectiveness of the irreversible phototransformations would be increased and would not depend on the wavelength of the UV radiation is selected. Further research will be dedicated to a search for such a medium. Conclusion. Modern research demonstrating the significant role of vitamin D3 for human health proves that the antirachitic activity of solar UV radiation must be monitored in addition to it erythema activity. Possible use of a simplified method of absorption spectroscopy, namely monitoring of the decrease of optical density at a fixed wavelength that is used for chemical UV dosimeters, for dosimetry of bioactive antirachitic UV radiation is examined. The application of this method for artificial UV sources with a constant radiation spectrum requires measurements of an individual calibration curve for each source. Also, a more suitable reaction medium must be sought for monitoring solar antirachitic UV radiation considering the variability of the solar UV spectrum. 243 Acknowledgments. We thank the Ukraine Scientific Technical Center for support of the research (Projects Gr50 and P344). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 244 P. Knuschke, J. Barth. Akt. Dermatol., 19, 238–240 (1993). Encyclopedia of Human Biology, Academic Press, New York (1991), pp. 859–871. H. J. C. Jacobs and E. Havinga, Adv. Photochem., 11, 305–373 (1979). A. R. Webb, L. W. Kline, and M. F. Holick, J. Clin. 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