Quantitative Analyses of Naturally Occurring Retinoids

  • Joseph L. Napoli
  • Ronald L. Horst
Part of the Methods in Molecular Biology book series (MIMB, volume 89)

Abstract

It is widely believed that the concentrations of retinoids that activate the two classes of nuclear-retinoid receptors are crucial to the effecis of the receptors, and ultimately the actions of the retinoid-humoral system. It is therefore truly important to know the precise concentrations and exact nature of the retinoids present at definite times and specific loci during development, and indeed, during any event mediated by retinoids. To determine conclusively the mechanisms of retinoid action, it is not sufficient to localize only the receptors; eventually we must establish which retinoids are present and the concentrations in which they are present, because several naturally occurring retinoids can stimulate receptor action, albeit with different ED50 values. For example, retinoids that can activate retinoic acid (RA) receptors (RARs) include, in addition to all-trans RA and 9-cis RA, 4-hydroxy RA, 4-oxo RA, and 1%hydroxy RA. 13-cis RA also binds to RARs, but with a K d value higher than the former group. Retinol can also induce responses in RA-sensitive systems; generally at doses ~200-fold higher than RA. In RA-dependent responses, retinol probably functions through conversion into RA (both enzymatic and artifactual conversion may contribute, depending on the circumstances). For example, uses of exogenous retinol, especially in higher concentrations (mM) could generate small amounts of retinoids by oxidation or by the actions of enzymes that normally are denied access to retinol in vivo. The techniques described in this chapter offer sensitive (<2 pmol), specific quantification of a variety of retinoids. A gas chromatography/mass spectrometrc (GC-MS) system has been described previously with greater sensitivity (~0.25 pmol), which can be coupled with high-performance liquid chromatography (HPLC) to enhance specificity (1, 2, 3). Additional HPLC systems for quantifying RA in blood have been summarized previously (4, 5).

Keywords

Vortex HPLC Hexane Hydrocarbon Chlorinate 

References

  1. 1.
    Napoli, J. L., Pramanik, B. C, Willlams, J. B, Dawson, M. I., and Hobbs, P D. (1985) Quantification of retinoic acid by gas-liquid chromatography/mass spectrometry: total vs. all-trans-retinoic acid in human plasma. J Lipid. Res 26, 387–392.PubMedGoogle Scholar
  2. 2.
    Napoli, J. L (1986) Quantification of physiological levels of retinoic acid. Methods Enzymol 123, 112–124PubMedCrossRefGoogle Scholar
  3. 3.
    DeLeenheer, A. P., and Lambert, W. E. (1990) Mass spectrometry of methyl ester of retinoic acid. Methods Enzymol. 189, 104–111CrossRefGoogle Scholar
  4. 4.
    DeLeenheer, A. P., and Nelis, H J. (1990) High-performance liquid chromatograhy of retinoids in blood. Methods Enzymol. 189, 50–59.CrossRefGoogle Scholar
  5. 5.
    Napoli, J. L. (1990) Quantification and charactertstics of retinoid synthesis from retinol and b-carotene in tissue fractions and established cell lines. Methods in Enzymol. 189, 410–482.Google Scholar
  6. 6.
    Patel, P, Hanning, R M., Atkinson, S. A., Dent, P. B, and Dolovitch, J. (1988) Intoxication from vitamin A in an asthmatic child. Can. Med. Assoc J. 139 755–756Google Scholar
  7. 7.
    Horst, R. L., Remhardt, T A., Goff, J. P., Nonnecke, B. J., Gambhir, V. K., Fiorella, P. D., and Napoli, J. L (1995) Identification of 9-cis, 13-cis-retinoic acid as a major circulating retinoid in plasma. Biochemistry 34, 1203–1209PubMedCrossRefGoogle Scholar
  8. 8.
    Horst, R. L., Reinhardt, T. A, Goff, J. P., Koszewski, N J., and Napoli, J L. (1995) Retinoic acid is the major circulating geometric isomer of retinoic acid during the periparturient period. Arch. Biochem. Biophys 322, 235–239PubMedCrossRefGoogle Scholar
  9. 9.
    Boerman, M H. E. M and Napoli, J L. (1995) Characterization of a microsomal retinol dehydrogenase: a short-chain alcohol dehydrogenase with integral and peripheral membrane forms that interacis with holo-CRBP (type I) Biochemistry 34, 7027–7037.PubMedCrossRefGoogle Scholar
  10. 10.
    Williams, J. B., Pramamk, B C, and Napoli, J. L. (1984) Vitamin A metabolism: analysis of steady-state neutral metabolites in rat tissues J. Lipid Res. 25, 638–645.PubMedGoogle Scholar
  11. 11.
    Boerman, M H. E M., and Napoli, J L. (1996) Cellular retinol-binding protein-supported retinol dehydrogenation in cytosol and microsomes. relative roles in retinoic acid synthesis J Biol. Chem 271, 5610–5616.PubMedCrossRefGoogle Scholar
  12. 12.
    El Akawi, Z., and Napoli, J. L (1994) Rat liver cytosolic retinal dehydrogenase. comparison of 13-cis-, 9-cis-, and all-trans-retinal as substrates and effecis of cellular retinoid-binding proteins and retinoic acid on activity. Biochemistry 33, 1938–1943PubMedCrossRefGoogle Scholar
  13. 13.
    Napoli, J L (1993) Prostaglandin E and phorbol diester are negative modulators of retinoic acid synthesis. Arch. Biochem Biophys. 300, 577–581.PubMedCrossRefGoogle Scholar
  14. 14.
    Posch, K. C, Burns, R B., and Napoli, J L. (1992) Biosynthesis of all-trans-retinoic acid from retinal recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosohc dehydrogenase J Biol Chem. 267, 19,676–19,682PubMedGoogle Scholar
  15. 15.
    Posch, K C and Napoli, J L. (1992) Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice Biochem Pharmacol 43, 2296–2298PubMedCrossRefGoogle Scholar
  16. 16.
    Posch, K C., Enright, W. E, and Napoli, J. L (1989) The synthesis of retinoic acid from retinol by cytosol from alcohol dehydrogenase negative deermice Arch Biochem Biophys. 274, 171–178.PubMedCrossRefGoogle Scholar
  17. 17.
    McCaffery, P, Posch, K C, Napoli, J L., Gudas, L, and Drager, U C. (1993) Changing patterns of the retinoic acid system in the developing retina. Dev. Biol. 158, 390–399CrossRefGoogle Scholar
  18. 18.
    Napoli, J L (1986) Retinol metabolism in LLC-PK1 cells characterization of retinoic acid synthesis by an established mammalian cell line J. Biol Chem. 261, 13,592–13,597PubMedGoogle Scholar

Copyright information

© Humana Press Inc, Totowa, NJ 1998

Authors and Affiliations

  • Joseph L. Napoli
    • 1
  • Ronald L. Horst
    • 2
  1. 1.Department of Biochemistry, School of Medicine and Biomedical SciencesState University of New York at Buffalo
  2. 2.Metabolic Diseases and Immunobiology Research Unit, Agricultural Research ServicesNational Animal Disease Center, USDAAmes

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