Retinoids pp 229-245 | Cite as

Molecular Biology and Analytical Chemistry Methods Used to Probe the Retinoid Cycle

  • Marcin Golczak
  • Grzegorz Bereta
  • Akiko Maeda
  • Krzysztof Palczewski
Part of the Methods in Molecular Biology book series (MIMB, volume 652)


The retinoid (visual) cycle is a complex enzymatic pathway essential for regeneration of the visual chromophore, 11-cis-retinal, a component of rhodopsin that undergoes activation by light in vertebrate eyes. Pathogenic mutations within genes encoding proteins involved in the retinoid cycle lead to abnormalities in retinoid homeostasis and numerous congenital blinding diseases of humans. Thus, elucidation of disease-specific changes in enzymatic activities and retinoid content of the retina can provide important insights into the mechanisms of disease initiation and progression. Here, we use the protein RPE65 as an example to describe generally applicable methods for determining the stability and enzymatic activity of proteins and their mutants involved in retinoid metabolism. Additionally, we introduce a range of analytical techniques involving high-performance liquid chromatography and mass spectrometry to detect and quantify retinoids and their derivatives in eye extracts. Biochemical protocols combined with advanced mass spectrometry should facilitate fundamental biological studies of vision.

Key words

RPE65 retinoid isomerization mass spectrometry A2E retinal dimer 



This research was supported in part by grants EY009339 and P30 EY11373 from the National Institutes of Health and the Foundation Fighting Blindness.


  1. 1.
    Palczewski, K. (2006) G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75, 743–767.PubMedCrossRefGoogle Scholar
  2. 2.
    Travis, G.H., Golczak, M., Moise, A.R., Palczewski, K. (2007) Diseases caused by defects in the visual cycle: Retinoids as potential therapeutic agents. Annu. Rev. Pharmacol. Toxicol. 47, 469–512.PubMedCrossRefGoogle Scholar
  3. 3.
    Jacobson, S.G., Aleman, T.S., Cideciyan, A.V., Heon, E., Golczak, M., Beltran, W.A., Sumaroka, A., Schwartz, S.B., Roman, A.J., Windsor, E.A., Wilson, J.M., Aguirre, G.D., Stone, E.M., Palczewski, K. (2007) Human cone photoreceptor dependence on RPE65 isomerase. Proc. Natl. Acad. Sci. USA 104, 15123–15128.PubMedCrossRefGoogle Scholar
  4. 4.
    Thompson, D.A., Gal, A. (2003) Vitamin A metabolism in the retinal pigment epithelium: Genes, mutations, and diseases. Prog. Retin. Eye Res. 22, 683–703.PubMedCrossRefGoogle Scholar
  5. 5.
    McBee, J.K., Palczewski, K., Baehr, W., Pepperberg, D.R. (2001) Confronting complexity: The interlink of phototransduction and retinoid metabolism in the vertebrate retina. Prog. Retin. Eye Res. 20, 469–529.PubMedCrossRefGoogle Scholar
  6. 6.
    Gu, S.M., Thompson, D.A., Srikumari, C.R., Lorenz, B., Finckh, U., Nicoletti, A., Murthy, K.R., Rathmann, M., Kumaramanickavel, G., Denton, M.J., Gal, A. (1997) Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nat. Genet. 17, 194–197.PubMedCrossRefGoogle Scholar
  7. 7.
    Morimura, H., Fishman, G.A., Grover, S.A., Fulton, A.B., Berson, E.L., Dryja, T.P. (1998) Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis. Proc. Natl. Acad. Sci. USA 95, 3088–3093.PubMedCrossRefGoogle Scholar
  8. 8.
    Bereta, G., Kiser, P.D., Golczak, M., Sun, W., Heon, E., Saperstein, D.A., Palczewski, K. (2008) Impact of retinal disease-associated RPE65 mutations on retinoid isomerization. Biochemistry47, 9856–9865.Google Scholar
  9. 9.
    Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Leowski, C., Ducroq, D., Souied, E., Dufier, J.L., Munnich, A., Kaplan, J. (1999) Leber congenital amaurosis. Mol. Genet. Metab. 68, 200–208.PubMedCrossRefGoogle Scholar
  10. 10.
    Kitamura, T., Koshino, Y., Shibata, F., Oki, T., Nakajima, H., Nosaka, T., Kumagai, H. (2003) Retrovirus-mediated gene transfer and expression cloning: Powerful tools in functional genomics. Exp. Hematol. 31, 1007–1014.PubMedGoogle Scholar
  11. 11.
    Redmond, T.M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J.X., Crouch, R.K., Pfeifer, K. (1998) Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20, 344–351.PubMedCrossRefGoogle Scholar
  12. 12.
    Imanishi, Y., Batten, M.L., Piston, D.W., Baehr, W., Palczewski, K. (2004) Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye. J. Cell. Biol. 164, 373–383.PubMedCrossRefGoogle Scholar
  13. 13.
    Batten, M.L., Imanishi, Y., Tu, D., Doan, T., Zhu, L., Pang, J., Glushakova, L., Moise, A.R., Baehr, W., Van Gelder, R.N., Hauswirth, W.W., Rieke, F., Palczewski, K. (2005) Pharmacological and rAAV gene therapy rescue of visual functions in a blind mouse model of Leber congenital amaurosis. PLoS Med. 2, e333.PubMedCrossRefGoogle Scholar
  14. 14.
    Batten, M.L., Imanishi, Y., Maeda, T., Tu, D.C., Moise, A.R., Bronson, D., Possin, D., Van Gelder, R.N., Baehr, W., Palczewski, K. (2004) Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J. Biol. Chem. 279, 10422–10432.PubMedCrossRefGoogle Scholar
  15. 15.
    Driessen, C.A., Winkens, H.J., Hoffmann, K., Kuhlmann, L.D., Janssen, B.P., Van Vugt, A.H., Van Hooser, J.P., Wieringa, B.E., Deutman, A.F., Palczewski, K., Ruether, K., Janssen, J.J. (2000) Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters. Mol. Cell Biol. 20, 4275–4287.PubMedCrossRefGoogle Scholar
  16. 16.
    Weng, J., Mata, N.L., Azarian, S.M., Tzekov, R.T., Birch, D.G., Travis, G.H. (1999) Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice. Cell 98, 13–23.PubMedCrossRefGoogle Scholar
  17. 17.
    Maeda, A., Maeda, T., Golczak, M., Palczewski, K. (2008) Retinopathy in mice induced by disrupted all-trans-retinal clearance. J Biol Chem 283, 26684–26693.PubMedCrossRefGoogle Scholar
  18. 18.
    Saari, J. C., Nawrot, M., Kennedy, B.N., Garwin, G.G., Hurley, J.B., Huang, J., Possin, D.E., Crabb, J.W. (2001) Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation. Neuron 29, 739–748.PubMedCrossRefGoogle Scholar
  19. 19.
    McCollum, E.V., Davis, M. (1913) The necessity of certain lipins in the diet during growth. J. Biol. Chem. 15, 167–175.Google Scholar
  20. 20.
    Vecchi, J., Vesely, J., Oesterhelt, G. (1973) Applications of high-pressure liquid chromatography and gas chromatography to problems in vitamin A analysis. J. Chromatogr. 83, 447–453.PubMedCrossRefGoogle Scholar
  21. 21.
    Rotmans, J.P., Kropf, A. (1975) The analysis of retinal isomers by high speed liquid chromatography. Vision Res. 15, 1301–1302.PubMedCrossRefGoogle Scholar
  22. 22.
    Kane, M.A., Folias, A.E., Napoli, J.L. (2008) HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues. Anal. Biochem. 378, 71–79.PubMedCrossRefGoogle Scholar
  23. 23.
    Kane, M.A., Folias, A.E., Wang, C., Napoli, J.L. (2008) Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry. Anal. Chem. 80, 1702–1708.PubMedCrossRefGoogle Scholar
  24. 24.
    Van Hooser, J.P., Liang, Y., Maeda, T., Kuksa, V., Jang, G.F., He, Y.G., Rieke, F., Fong, H.K., Detwiler, P.B., Palczewski, K. (2002) Recovery of visual functions in a mouse model of Leber congenital amaurosis. J. Biol. Chem. 277, 19173–19182.PubMedCrossRefGoogle Scholar
  25. 25.
    Van Hooser, J.P., Garwin, G.G., Saari, J.C. (2000) Analysis of visual cycle in normal and transgenic mice. Methods Enzymol. 316, 565–575.PubMedCrossRefGoogle Scholar
  26. 26.
    Mears, A.J., Kondo, M., Swain, P.K., Takada, Y., Bush, R.A., Saunders, T.L., Sieving, P.A., Swaroop, A. (2001) Nrl is required for rod photoreceptor development. Nat. Genet. 29, 447–452.PubMedCrossRefGoogle Scholar
  27. 27.
    Fishkin, N.E., Sparrow, J.R., Allikmets, R., Nakanishi, K. (2005) Isolation and characterization of a retinal pigment epithelial cell fluorophore: An all-trans-retinal dimer conjugate. Proc. Natl. Acad. Sci. USA 102, 7091–7096.PubMedCrossRefGoogle Scholar
  28. 28.
    Golczak, M., Maeda, A., Bereta, G., Maeda, T., Kiser, P.D., Hunzelmann, S., von Lintig, J., Blaner, W.S., Palczewski, K. (2008) Metabolic basis of visual cycle inhibition by retinoid and nonretinoid compounds in the vertebrate retina. J. Biol. Chem. 283, 9543–9554.PubMedCrossRefGoogle Scholar
  29. 29.
    van Breemen, R.B., Nikolic, D., Xu, X., Xiong, Y., van Lieshout, M., West, C.E., Schilling, A.B. (1998) Development of a method for quantitation of retinol and retinyl palmitate in human serum using high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. J. Chromatogr. A 794, 245–251.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Marcin Golczak
    • 1
  • Grzegorz Bereta
    • 1
  • Akiko Maeda
    • 1
  • Krzysztof Palczewski
    • 1
  1. 1.Department of Pharmacology, School of MedicineCase Western Reserve UniversityClevelandUSA

Personalised recommendations