Abstract
Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530–580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.
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References
G. Wolf, Lipofuscin and macular degeneration, Nutr. Rev., 2003, 61, 342.
N. Lois, G. E. Holder, C. Bunce, F. W. Fitzke and A. C. Bird, Phenotypic subtypes of Stargardt macular dystrophy-fundus flavimaculatus, Arch. Ophthalmol., 2001, 119, 359.
A. Von Ruckmann, F. W. Fitzke and A. C. Bird, In vivo fundus autofluorescence in macular dystrophies, Arch. Ophthalmol., 1997, 115, 609.
F. G. Holz, M. Fleckenstein, S. Schmitz-Valckenberg and A. C. Bird, in Atlas of fundus autofluorescence imaging, ed. F. G. Holz, S. Schmitz-Valckenberg, R. F. Spaide and A. C. Bird, Springer, Berlin, 2007, p. 71.
F. G. Holz, C. Bellmann, M. Margaritidis, F. Schutt, T. P. Otto and H. E. Volcker, Patterns of increased in vivo fundus autofluorescence in the junctional zone of geographic atrophy of the retinal pigment epithelium associated with age-related macular degeneration, Graefe’s Arch. Clin. Exp. Ophthalmol., 1999, 237, 145.
J. R. Sparrow, Y. Wu, C. Y. Kim and J. Zhou, Phospholipid meets all-trans retinal: the making of RPE bisretinoids, J. Lipid Res., 2009, 51, 247.
L. E. Lamb and J. D. Simon, A2E: a component of ocular lipofuscin, Photochem. Photobiol., 2004, 79, 127.
N. Sakai, J. Decatur, K. Nakanishi and G. E. Eldred, Ocular age pigment “A2E”: an unprecedented pyridinium bisretinoid, J. Am. Chem. Soc., 1996, 118, 1559.
J. R. Sparrow, S. R. Kim, A. M. Cuervo and U. Bandhyopadhyayand, A2E, a pigment of RPE lipofuscin, is generated from the precursor, A2PE by a lysosomal enzyme activity, Adv. Exp. Med. Biol., 2008, 613, 393.
L. B. Avalle, Z. Wang, J. P. Dillon and E. R. Gaillard, Observation of A2E oxidation products in human retinal lipofuscin, Exp. Eye Res., 2004, 78, 895.
J. P. Dillon, Z. Wang, L. B. Avalle and E. R. Gaillard, The photochemical oxidation of A2E results in the formation of a 5,8,5′,8′, -bis-furanoid oxide, Exp. Eye Res., 2004, 79, 537.
T. B. Feldman, M. A. Yakovleva, P. M. Arbukhanova, S. A. Borzenok, A. S. Kononikhin, I. A. Popov, E. N. Nikolaev and M. A. Ostrovsky, Changes in spectral properties and composition of lipofuscin fluorophores from human retinal pigment epithelium with age and pathology, Anal. Bioanal. Chem., 2015, 407, 1075.
K. D. Yoon, K. Yamamoto, K. Ueda, J. Zhou and J. R. Sparrow, A novel source of methylglyoxal and glyoxal in retina: implications for age related macular degeneration, PLoS One, 2012, 7, e41309.
Z. Wang, L. M. M. Keller, J. Dillon and E. R. Gaillard, Oxidation of A2E results in the formation of highly reactive aldehydes and ketones, Photochem. Photobiol., 2006, 82, 1251.
Y. Wu, E. Yanase, X. Feng, M. M. Siegel and J. R. Sparrow, Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 7275.
S. R. Kim, Y. P. Jang, S. Jockusch, N. E. Fishkin, N. J. Turro and J. R. Sparrow, The all-trans-retinal dimer series of lipo-fuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 19273.
J. R. Sparrow, Y. Wu, T. Nagasaki, K. D. Yoon, K. Yamamoto and J. Zhou, Fundus autofluorescence and the bisretinoids of retina, Photochem. Photobiol. Sci., 2010, 9, 1480.
K. Yamamoto, K. D. Yoon, K. Ueda, M. Hashimoto and J. R. Sparrow, A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine, Invest. Ophthalmol. Visual Sci., 2011, 52, 9084.
F. C. Delori, C. K. Dorey, G. Staurenghi, O. Arend, D. G. Goger and J. J. Weiter, In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics, Invest. Ophthalmol. Visual Sci., 1995, 36, 718.
T. B. Feldman, M. A. Yakovleva, A. V. Larichev, P. M. Arbukhanova, A. Sh. Radchenko, S. A. Borzenok, V. A. Kuzmin and M. A. Ostrovsky, Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium, Eye, 2018, 32, 1440.
A. D. Marmorstein, L. Y. Marmorstein, H. Sakaguchi and J. G. Hollyfield, Spectral profiling of autofluorescence associated with lipofuscin, bruch’s membrane, and sub-RPE deposits in normal and AMD eyes, Invest. Ophthalmol. Visual Sci., 2002, 43, 2435.
L. Sauer, R. H. Gensure, K. M. Andersen, L. Kreilkamp, G. S. Hageman, M. Hammer and P. S. Bernstein, Patterns of fundus autofluorescence lifetimes in eyes of individuals with nonexudative agerelated macular degeneration, Invest. Ophthalmol. Visual Sci., 2018, 59, AMD65.
D. Schweitzer, S. Quick, S. Schenke, M. Klemm, S. Gehlert, M. Hammer, S. Jentsch and J. Fischer, Comparison of parameters of time-resolved autofluorescence between healthy subjects and patients suffering from early AMD, Ophthalmology, 2009, 106, 714.
K. M. Andersen, L. Sauer, R. H. Gensure, M. Hammer and P. S. Bernstein, Characterization of retinitis pigmentosa using fluorescence lifetime imaging ophthalmoscopy (FLIO), Transl. Vision Sci. Technol., 2018, 7, 20.
C. Dysli, K. Schürch, E. Pascal, S. Wolf and M. S. Zinkernagel, Fundus autofluorescence lifetime patterns in retinitis pigmentosa, Invest. Ophthalmol. Visual Sci., 2018, 59, 1769.
C. Dysli, S. Wolf, K. Hatz and M. S. Zinkernagel, Fluorescence Lifetime Imaging in Stargardt Disease: Potential Marker for Disease Progression, Invest. Ophthalmol. Visual Sci., 2016, 57, 832.
L. Sauer, R. H. Gensure, M. Hammer and P. S. Bernstein, Fluorescence lifetime imaging ophthalmoscopy: a novel way to assess macular telangiectasia type 2, Ophthalmol. Retina, 2018, 2, 587.
L. Sauer, K. M. Andersen, C. Dysli, M. S. Zinkernagel, P. S. Bernstein and M. Hammer, Review of clinical approaches in fluorescence lifetime imaging ophthal-moscopy, J. Biomed. Opt., 2018, 23, 1.
D. Schweitzer, L. Deutsch, M. Klemm, S. Jentsch, M. Hammer, S. Peters, J. Haueisen, U. A. Muller and J. Dawczynski, Fluorescence lifetime imaging ophthal-moscopy in type 2 diabetic patients who have no signs of diabetic retinopathy, J. Biomed. Opt., 2015, 20, 61106.
S. Jentsch, D. Schweitzer, K. U. Schmidtke, S. Peters, J. Dawczynski, K. J. Bar and M. Hammer, Retinal fluorescence lifetime imaging ophthalmoscopy measures depend on the severity of Alzheimer’s disease. Acta, Ophthalmologica, 2014, 93, 241.
S. R. Sadda, E. Borrelli, W. Fan, A. Ebraheem, K. M. Marion, M. Harrington and S. Kwon, A pilot study of fluorescence lifetime imaging ophthalmoscopy in pre-clinical Alzheimer’s disease, Eye, 2019, 33, 1271.
J. M. Beechem, Global analysis of biochemical and biophysical data, Methods Enzymol., 1992, 210, 37.
M. M. Krishna and N. Periasamy, Spectrally constrained global analysis of fluorescence decays in biomembrane systems, Anal. Biochem., 1997, 253, 1.
J. R. Lakowicz, Principles of fluorescence spectroscopy, Springer, New York, 3rd edn, 2006, pp. 97–149.
T. B. Feldman, M. A. Yakovleva, A. E. Dontsov and M. A. Ostrovsky, Fluorescence emission and excitation spectra of fluorophores of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes, Russ. Chem. Bull. Int. Ed., 2010, 59, 276.
Russian Federation law N 4180-I dated 22.12.1992: On human organs or tissue transplantation (with modifications and additions). http://base.garant.ru/136366/.
J. Folch, M. Lees and G. H. S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 1957, 226, 497.
C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon and J. R. Sparrow, Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 14609.
M. A. Yakovleva, N. L. Sakina, A. S. Kononikhin, T. B. Feldman, E. N. Nikolaev, A. E. Dontsov and M. A. Ostrovsky, Detection and study of the products of photooxidation of N-Retinylidene-N-retinylethanolamine (A2E), the fluorophore of lipofuscin granules from retinal pigment epithelium of human donor eyes, Dokl. Biochem. Biophys., 2006, 409, 223.
L. S. Murdaugh, L. B. Avalle, S. Mandal, A. E. Dill, J. Dillon, J. D. Simond and E. R. Gaillard, Compositional studies of human RPE lipofuscin, Mass. Spectrom., 2010, 45, 1139.
D. B. Gutierrez, L. Blakeley, P. W. Goletz, K. L. Schey, A. Hanneken, Y. Koutalos and R. K. Crouchand, Z. Ablonczy, Mass spectrometry provides accurate and sensitive quantitation of A2E, Photochem. Photobiol. Sci., 2010, 9, 1513.
Y. Miura, G. Huettmann, R. Orzekowsky-Schroeder, P. Steven, M. Szaszák, N. Koop and R. Brinkmann, Two-photon microscopy and fluorescence lifetime imaging of retinal pigment epithelial cells under oxidative stress, Invest. Ophthalmol. Visual Sci., 2013, 54, 3366.
L. Ragauskaite, R. C. Heckathorn and E. R. Gaillard, Environmental effects on the photochemistry of A2E, a component of human retinal lipofuscin, Photochem. Photobiol., 2001, 74, 483.
E. R. Gaillard, S. J. Atherton, G. Eldred and J. Dillon, Photophysical studies on human retinal lipofuscin, Photochem. Photobiol., 1995, 61, 448.
Y. Wu, N. E. Fishkin, A. Pande, J. Pande and J. R. Sparrow, Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease, J. Biol. Chem., 2009, 284, 20155.
F. Docchio, M. Boulton, R. Cubeddu, R. Ramponi and P. D. Barker, Age-related changes in the fluorescence of melanin and lipofuscin granules of the retinal pigment epithelium: a time-resolved fluorescence spectroscopy study, Photochem. Photobiol., 1991, 54, 247.
R. Cubeddu, P. Taroni, D. N. Hu, N. Sakai, K. Nakanishi and J. E. Roberts, Photophysical studies of A2-E, putative precursor of lipofuscin, in human retinal pigment epithelial cells, Photochem. Photobiol., 1999, 70, 172.
D. Schweitzer, S. Schenke, M. Hammer, F. Schweitzer, S. Jentsch, E. Birckner, W. Becker and A. Bergmann, Towards metabolic mapping of the human retina, Microsc. Res. Tech., 2007, 70, 410.
C. Dysli, R. Fink, S. Wolf and M. S. Zinkernagel, Fluorescence Lifetimes of Drusen in Age-Related Macular Degeneration, Invest. Ophthalmol. Visual Sci., 2017, 58, 4857.
M. Hammer, S. Quick, M. Klemm, S. Schenke, N. Mata, A. Eitner and D. Schweitzer, In vivo and in vitro investigations of retinal fluorophores in age-related macular degeneration by fluorescence lifetime imaging, Proc. SPIE, 2009, 7183, 71832S.
C. Dysli, S. Wolf, M. Y. Berezin, L. Sauer, M. Hammer and M. S. Zinkernagel, Fluorescence lifetime imaging ophthalmoscopy, Prog. Retinal Eye Res., 2017, 60, 120.
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Yakovleva, M.A., Radchenko, A.S., Feldman, T.B. et al. Fluorescence characteristics of lipofuscin fluorophores from human retinal pigment epithelium. Photochem Photobiol Sci 19, 920–930 (2020). https://doi.org/10.1039/c9pp00406h
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DOI: https://doi.org/10.1039/c9pp00406h