Vol. 3, 2018

Original research papers



Jelena S. Stanojević, Dragan J. Cvetković, Jelena B. Zvezdanović, Ljiljana P. Stanojević, Dejan Z. Marković†

Pages: 213–219

DOI: 10.21175/RadProc.2018.45

The aim of the present study was to consider bilirubin (BRB) and riboflavin (RFL) mutual interaction in methanol solution under continuous UV-A and UV-B irradiation regime. Continuous irradiations of samples were performed in a cylindrical photochemical reactor “Rayonet”, with 10 symmetrically placed lamps having the emission maximum at 300 nm (UV-B) and 350 nm (UV-A). The rate of BRB and RFL photodegradation along with simultaneous products formation, as a function of UV exposure time, was followed by combining UV-VIS absorption measurements with RP-HPLC analysis. The compounds were separated by gradient elution with mobile phase A (formic acid, 0.1% water solution) and B (formic acid, 0.1% methanol solution). According to the results obtained, BRB degradation in the absence of RFL was almost 22 times and 9 times slower in comparison to its degradation observed in BRB-RFL mixture under continuous UV-A and UV-B irradiation, respectively. Moreover, BRB degradation in BRB-RFL mixture under anaerobic conditions was almost 24 times and 16 times slower in comparison to the degradation in aerobic conditions under UV-A and UV-B light, respectively. The latter observation suggests that presence of ROS species contributes to UV-induced BRB degradation. These experiments provide the indirect proof of BRB acting as Type II sensitizer because of the fact that 1O2 produced by RFL mediates BRB irreversible degradation giving rise to dipyrrole methanol adducts as typical products obtained via Type II mechanism.
  1. N. Schade, C. Esser, J. Krutmann, “Ultraviolet B radiation-induced immunosuppression: molecular mechanisms and cellular alterations,” Photochem. Photobiol. Sci., vol. 4, no. 9, pp. 699 – 708, Sep. 2005.
    DOI: 10.1039/b418378a
    PMid: 16121280
  2. S. González, M. Fernández-Lorente, Y. Gilaberte-Calzada, “The latest on skin photoprotection,” Clin. Dermatol., vol. 26, no. 6, pp. 614 – 626, Nov-Dec. 2008.
    DOI: 10.1016/j.clindermatol.2007.09.010
    PMid: 18940542
  3. F. Afaq, “Natural agents: cellular and molecular mechanisms of photoprotection,” Arch. Biochem. Biophys., vol. 508, no. 2, pp. 144 – 151, Apr. 2011.
    DOI: 10.1016/j.abb.2010.12.007
    PMid: 21147060
  4. A. R. Young, “Acute effects of UVR on human eyes and skin,” Prog. Biophys. Mol. Biol., vol. 92, no. 1, pp. 80 –85, Sep. 2006.
    DOI: 10.1016/j.pbiomolbio.2006.02.005
    PMid: 16600340
  5. A. Svobodová, J. Psotová, D. Walterová, “Natural phenolics in the prevention of UV-induced skin damage. A review,” Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub., vol. 147, no. 2, pp. 137 – 145, Dec. 2003.
    PMid: 15037894
  6. S. L. Deore, S. Kombade, B. A. Baviskar, S. S. Khadabadi, “Photoprotective antioxidant phytochemicals,” Int. J. Phyto. Pharm., vol. 2 no. 3, pp. 72 – 76, May-Jun. 2012.
    DOI: 10.7439/ijpp.v2i3.501
  7. J. Krutmann, “Ultraviolet A radiation-induced biological effects in human skin: relevance for photoaging and photodermatosis,” J. Dermatol. Sci., vol. 23, suppl. 1, pp. S22 – S26, Mar. 2000.
    PMid: 10764987
  8. F. Menaa, A. Menaa, J. Tréton, “Polyphenols against skin aging,” in Polyphenols in human health and disease, New York (NY), USA: Academic Press, 2013, ch. 63., sec. 6.2, pp. 819 – 830.
    DOI: 10.1016/B978-0-12-398456-2.00063-3
  9. A. Mahns, I. Melchheier, C. V. Suschek, H. Sies, L. O. Klotz, “Irradiation of cells with Ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide,” Free Radic. Res., vol. 37, no. 4, pp. 391 – 397, Apr. 2003.
    PMid: 12747733
  10. T. Herrling, K. Jung, J. Fuchs, “Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin,” Spectrochim. Acta A. Mol. Biomol. Spectrosc., vol. 63, no. 4, pp. 840 – 845, Mar. 2006.
    DOI: 10.1016/j.saa.2005.10.013
    PMid: 16543118
  11. C. A. Brohem et al., “Artificial skin in perspective: concepts and applications,” Pigment Cell Melanoma Res., vol. 24, no. 1, pp. 35 – 50, Feb. 2011.
    DOI: 10.1111/j.1755-148X.2010.00786.x
    PMid: 21029393
  12. G. T. Wondrak, M. K. Jacobson, E. L. Jacobson, “Endogenous UVA photosensitizers: mediators of skin photodamage and novel targets for skin photoprotection,” Photochem. Photobiol. Sci., vol. 5, no. 2, pp: 215 – 237, Feb. 2006.
    DOI: 10.1039/b504573h
    PMid: 16465308
  13. D. Z. Marković, “Kinetika i mehanizam fotosenzibilizovane peroksidacije lipidnih konstituenata biomembrana,” Doktorska disertacija, Univerzitet u Beogradu, Prirodnomatematički fakulteti, Fakultet za fizičku hemiju, Srbija, 1990. (D. Z. Marković, “Kinetics and mechanism of photosensitized peroxidation of lipid constituents of biomembranes,” Ph.D. dissertation, University of Belgrade, Faculties of Sciences and Mathematics, Faculty for Physical Chemistry, Serbia, 1990.)
  14. C. S. Foote, “Photooxidation,” in Phototherapy in the newborn: An overview, Washington (DC), USA: National academy of sciences, 1974, pp. 21 – 33.
    DOI: 10.17226/20078
  15. H. P. Lassalle, “Etude des mécanismes du photoblanchiment de la 5,10,15,20-tetrakis(m-hydroxyphenyl)bactériochlorine, en solution, in vitro et in vivo,” Thèse de doctorat, Université Henri Poincaré-Nancy I, Faculté de Médecine, Nancy, France, 2005. (H. P. Lassalle, “Study of the photobleaching mechanisms of the 5,10,15,20-tetrakis (m-hydroxyphenyl) bacteriochlorin (m-THPBC), in solution, in vitro and in vivo,” Ph.D. dissertation, Henri Poincare University-Nancy I, Faculty of Medicine, Nancy, France, 2005.)
    Retrieved from: https://tel.archives-ouvertes.fr/tel-00378394/document;
    Retrieved on: Jun. 24, 2018
  16. C. S. Foote, “Mechanisms of Photosensitized Oxidation,” Science, vol. 162, no. 3857, pp. 963 – 970, Nov. 1968.
    DOI: 10.1126/science.162.3857.963
  17. S. K. Dey, D. A. Lightner, “Lipid- and water-soluble bilirubins,” Monatsh. Chem., vol. 141, no. 1, pp. 101 – 109, Jan. 2010.
    DOI: 10.1007/s00706-009-0232-5
  18. A. F. McDonagh, “Controversies in bilirubin biochemistry and their clinical relevance,” Semin. Fetal. Neonatal Med., vol. 15, no. 3, pp. 141 – 147, Jun. 2010.
    DOI: 10.1016/j.siny.2009.10.005
    PMid: 19932645
  19. J. Fevery, “Bilirubin in clinical practice: a review,” Liver Int., vol. 28, no. 5, pp. 592 – 605, May. 2008.
    DOI: 10.1111/j.1478-3231.2008.01716.x
    PMid: 18433389
  20. G. F. Combs, Jr., “Riboflavin,” in The vitamins Fundamental aspects in nutrition and health, 3rd ed., Amsterdam, Netherlands: Academic Press, 2012, ch. 3, pp. 53 – 54.
    Retrieved from:;
    Retrieved on: Jun. 24, 2018
  21. H. J. Powers, “Riboflavin (vitamin B-2) and health,” Am. J. Clin. Nutr., vol. 77, no. 6, pp. 1352 – 1360, Jun. 2003.
    DOI: 10.1093/ajcn/77.6.1352
    PMid: 12791609
  22. J. S. Stanojevic, J. B. Zvezdanovic, D. Z. Markovic, “Bilirubin degradation in methanol induced by continuous UV-B irradiation: a UHPLC – ESI-MS study,” Pharmazie, vol. 70, no. 4, pp. 225 – 230, Apr. 2015.
    DOI: 10.1691/ph.2015.4122
    PMid: 26012251
  23. J. S. Stanojevic, J. B. Zvezdanovic, D. Z. Markovic, “Riboflavin degradation in the presence of quercetin in methanol under continuous UV-B irradiation: the ESI–MS–UHPLC analysis,” Monatsh. Chem., vol. 146, no. 11, pp. 1787 – 1794, Nov. 2015.
    DOI: 0.1007/s00706-015-1561-1
  24. J. N. Chacόn, J. McLearie, R. S. Sinclair, “Singlet oxygen yields and radical contributions in the dye-sensitized photo-oxidation in methanol of esters of polyunsatured fatty acids (oleic, linoleic, linolenic and arachidonic),” Photochem Photobiol., vol. 47, no. 5, pp: 647 – 656, May 1988.
  25. J. Koziol, “Studies on flavins in organic solvents-II.* Photodecomposition of riboflavin in the presence of oxygen,” Photochem Photobiol., vol. 5, no. 1, pp. 55 – 62, Jan. 1966.
    DOI: 10.1111/j.1751-1097.1966.tb05760.x
  26. M. A. Sheraz, S. H. Kazi, S. Ahmed, Z. Anwar, I. Ahmad, “Photo, thermal and chemical degradation of riboflavin,” Beilstein J. Org. Chem., vol. 10, pp. 1999 – 2012, Aug. 2014.
    DOI: 10.3762/bjoc.10.208
  27. M. Insińska-Rak, A. Golczak, M. Sikorski, “Photochemistry of riboflavin derivatives in methanolic solutions,” J. Phys. Chem. A., vol. 116, no. 4, pp. 1199 – 1207, Jan. 2012.
    DOI: 10.1021/jp2094593
    PMid: 22217187
  28. I. Ahmad et al., “Solvent effect on the photolysis of riboflavin,” AAPS PharmSciTech., vol. 16, no. 5, pp. 1122 – 1128, Oct. 2015.
    DOI: 10.1208/s12249-015-0304-2
    PMid: 25698084
  29. D. R. Cardoso, S. H. Libardia, L. H. Skibsted, “Riboflavin as a photosensitizer. Effects on human health and food quality,” Food Funct., vol. 3, no. 5, pp. 487 – 502, May 2012.
    DOI: 10.1039/c2fo10246c
    PMid: 22406738
  30. P. F. Heelis, “The photophysical and photochemical properties of flavins (isoalloxazines),” Chem. Soc. Rev., vol. 11, no. 1, pp. 15 – 39, 1982.
    DOI: 10.1039/cs9821100015
  31. C. Y. Lu et al., “Generation and photosensitization properties of the oxidized radical of riboflavin: a laser flash photolysis study,” J. Photochem. Photobiol. B: Biol., vol. 52, no. 1-3, pp. 111 – 116, Oct. 1999.
    DOI: 10.1016/S1011-1344(99)00111-6
  32. M. Boopathi, M. Subbaiyan, “Oxidation and estimation of bilirubin by using carbon microelectrode,” Indian J. Chem. Sec. A., vol. 38A, no. 12, pp. 1239 – 1244, Dec. 1999.
    Retrieved from: http://hdl.handle.net/123456789/16134;
    Retrieved on: Dec. 3, 2018
  33. E. Knobloch, R. Hodr, J. Herzman, V. Houdkova, “Function of flavins in photolysis of bilirubin in vitro,” Collection Czechoslovak Chern. Commun., vol. 47, no. 5, pp. 1514 – 1522, 1982.
    DOI: 10.1135/cccc19821514
  34. R. Bonnett, J. C. M. Stewart, “Photo-oxidation of bilirubin in hydroxylic solvents: propentdyopent adducts as major products,” J. Chem. Soc., Chem. Commun., no. 10, pp. 596 – 597, 1972.
    DOI: 10.1039/C39720000596
  35. G. Agati, F. Fusi, “New trends in photobiology (invited review). Recent advances in bilirubin photophysics,” J. Photochem. Photobiol. B., vol. 7, no. 1. pp. 1 – 14, Sep. 1990.
    PMid: 2125072
  36. C. S. Foote, T. Y. Ching, “Chemistry of singlet oxygen. XXI. Kinetics of bilirubin photooxygenation,” J. Am. Chem. Soc., vol. 97, no. 21, pp. 6209 – 6214, Oct. 1975.
    PMid: 1176727
  37. C. H. Gray, A. Kulczycka, D. C. Nicholson, “The photodecomposition of bilirubin and other bile pigments,” J. Chem. Soc. Perkin 1, vol. 3, pp. 288 – 294, 1972.
    PMid: 5065591
  38. D. A. Lightner, “In vitro photooxidation products of bilirubin,” in Phototherapy in the newborn: An overview, Washington (DC), USA: National academy of sciences, 1974, pp. 34 – 55.
    Retrieved from: https://www.nap.edu/read/20078/chapter/1;
    Retrieved on: Jun. 24, 2018
  39. D. A. Lightner, D. C. Crandall, “Biliverdin photo-oxidation. In vitro formation of methylvinylmaleimide,” FEBS Lett., vol. 20, no. 1, pp. 53 – 56, Jun. 1972.
    PMid: 11946380