25-Hydroxycholecalciferol: High Affinity Substrate for Hepatic Cytochrome P-450

  • Dominick L. Cinti
  • Francis H. Glorieux
  • Edgard E. Delvin
  • Ellis E. Golub
  • Felix Bronner
Part of the Advances in Experimental Medicine and Biology book series (AEMB)


In vitro studies with hepatic microsomes showed that 25-hydroxy-vitamin D3 (25-OH-D3) is bound tightly by the microsomal cytochrome P-450 system, with the spectral dissociation constant of 84 nM the lowest reported to-date for a natural or xenobiotic compound. Vitamin D2 and dihydrotachysterol also were bound, but their dissociation constants were 200–300 times higher. Aminopyrine demethyl-ation was competitively inhibited in the presence of 25-OH-D3 and NADPH cytochrome. P-450 reductase activity was doubled by 25-OH-D3 addition. Taken together these findings suggest that liver microsomes are involved in the transformation and degradation of 25-OH-D3 and other vitamin D congeners. However, the enzyme systems are not vitamin D-dependent. Moreover, even though phenobarbital treatment led to a doubling of enzyme activity and, in animals undergoing vitamin D depletion, to a 40% faster time-dependent drop of 25-OH-D3 plasma levels, as compared to untreated controls, the two groups had the same levels of intestinal calcium-binding protein (CaBP). If CaBP is one expression of the active metabolite, 1,25-dihydroxy vitamin D3 (1,25-(OH) 2-D3), then alterations of a substrate (25-OH-D 3)-product (1,25-(OH)2-D3) relationship at a single control point (liver), brought about by phenobarbital treatment, did not lead to detectable changes in hormone expression. The experiments underscore the stability of the calcium regulating system and point to the existence of multiple control loops.


Liver Microsome Maximal Binding Capacity Plasma Calcium Level Phenobarbital Treatment High Affinity Substrate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Norman, A.W. and Henry, H. 1,25-dihydroxycholecalciferol — a hormonally active form of vitamin D3. Recent Progr. Hormone Res. 30:431–480, 1974.PubMedGoogle Scholar
  2. 2.
    Hahn, T.J., Birge, S.J., Scharp, C.R. and Avioli, L.R. Pheno-barbital-induced alterations in vitamin D metabolism. J. Clin. Invest. 51:741–748, 1972.PubMedCrossRefGoogle Scholar
  3. 3.
    Hahn, T.J., Hendin, B.A., Scharp, C.R., and Haddad, J.G. Effect of chronic anticonvulsant therapy on serum 25-hydroxy-calciferol levels in adults. New Engl. J. Med. 287:900–904, 1972.PubMedCrossRefGoogle Scholar
  4. 4.
    Bhattacharyya, M. and DeLuca, H.F. The regulation of rat liver calciferol hydroxylase. J. Biol. Chem. 248:2969–2973, 1973.PubMedGoogle Scholar
  5. 5.
    DeLuca, H.F. Metabolism of vitamin D: current status. Am. J. Clin. Nutr. 1976 (in press).Google Scholar
  6. 6.
    Hurwitz, S., Stacey, R.E. and Bronner, F. Role of vitamin D in plasma calcium regulation. Am. J. Physiol. 216:254–262, 1969.PubMedGoogle Scholar
  7. 7.
    Schenkman, J.F. and Cinti, D.L. Hepatic mixed function oxidase activity in rapidly prepared microsomes. Life Sci. 11:247–257, 1972.CrossRefGoogle Scholar
  8. 8.
    Cinti, D.L. and Ozols, J. Binding of homogeneous cytochrome b5 to rat liver microsomes: Effect on N-demethylation reactions. Biochim. Biophys. Acta 410:32–44, 1975.PubMedCrossRefGoogle Scholar
  9. 9.
    Haddad, J.G. and Chyu, K.J. Competitive protein-binding radioassay for 25-hydroxycholecalciferol. J. Clin. Endocr. 33:992, 1971.PubMedCrossRefGoogle Scholar
  10. 10.
    Ellingboe, J., Nyström, E. and Sjövall, J. Liquid-gel chromatography on lipophilic hydrophobic Sephadex derivatives, J. Lipid Res. 11:266, 1970.PubMedGoogle Scholar
  11. 11.
    Freund, Thomas and Bronner, Felix. Regulation of intestinal calcium-binding protein by calcium intake in the rat. Am. J. Physiol. 228:861–869, 1975.PubMedGoogle Scholar
  12. 12.
    Bronner, Felix and Thomas Freund, Intestinal CaBP: a new quantitative index of vitamin D deficiency in the rat. Am. J. Physiol. 229:689–694, 1975.PubMedGoogle Scholar
  13. 13.
    Gigon, P.L., Gram, T.E. and Gillette, J.R. Studies on the rate of reduction of hepatic microsomal cytochrome P-450 by reduced nicotinamide adenine dinucleotide phosphate. Effect of drug substrates. Molec. Pharmacol. 5:109–122, 1969.Google Scholar
  14. 14.
    Wilcoxon, Frank. Individual comparisons by ranking methods Biometrics 1:80–82, 1945.CrossRefGoogle Scholar
  15. 15.
    Orrenius, S. and Ernster, L. Microsomal cytochrome P-450-linked monooxygenase systems in mammalian tissue. In: Molecular Arrangements of Oxygen Activation, ed. O. Hayaishi. Academic Press, New York, 1974, pp. 215–244.Google Scholar
  16. 16.
    Lu, A., Strobel, H. and Coon, M. Properties of a solubilized form of the cytochrome P-450-containing mixed function oxidase of liver microsomes. Molec. Pharmacol. 6:213–220, 1970.Google Scholar
  17. 17.
    Gunsalus, I.C. Soluble methylene hydroxylase system: Structure and role of cytochrome P-450 and iron-sulfur protein components. Hoppe-Seyler’s Z. Physiol. Chem. 349:1610–1613, 1968.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1977

Authors and Affiliations

  • Dominick L. Cinti
    • 1
  • Francis H. Glorieux
    • 2
  • Edgard E. Delvin
    • 2
  • Ellis E. Golub
    • 3
  • Felix Bronner
    • 3
  1. 1.Department of PharmacologyThe University of Connecticut Health CenterFarmingtonUSA
  2. 2.Department of Experimental SurgeryMcGill University and The Shriner’s HospitalMontrealCanada
  3. 3.Department of Oral BiologyThe University of Connecticut Health CenterFarmingtonUSA

Personalised recommendations