Simple Assay for Femtomoles of Pyridoxal and Pyridoxamine Phosphates

  • Bob Inyu Yang
  • Ashok K. Sawhney
  • Ronnie C. Pitchlyn
  • Peter M. Peer


In recent years, there has been a renewed interest in vitamin B-6, sparked in part by the discoveries that several population groups are only marginally sufficient in the vitamin. These include alcoholics (1), pregnant women (2,3), females on oral contraceptive agents (3,4), and senior citizens (1,5-7). Of the various methods devised to indicate the nutritional status of vitamin B-6, measurement of plasma levels of pyridoxal 5’-P is being employed with increasing frequency. Pyridoxal 5’-P concentration in human blood correlates with the urinary excretion of xanthurenic acid (8), and enzyme activity and coenzyme saturation of erythrocyte aspartate aminotransferase (9). In rats, plasma pyridoxal 5’-P bears close relation to pyridoxine intake, growth, erythrocyte aspartate, and alanine aminotransferase activities and muscle store of the vitamer (10). Assay of pyridoxal 5’-P is best carried out by enzymatic means because they offer high specificity and sensitivity. Currently available protocols involve the use of the apoenzyme of tyrosine decarboxylase (11-13), tryptophanase (14,15), wheat germ and yeast aspartate aminotransferase (6,16), D-serine dehydratase (17), phosphorylase b (18), and y-cyanoaminobutyric acid synthetase (19).


Tris Buffer Aspartate Aminotransferase Residual Activity Sodium Hydroxide Aminotransferase Activity 
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.

Literature Cited

  1. 1.
    Lumeng, L. Li, T.-K. (1974) Vitamin B-6 metabolism in chronic alcohol abuse: Pyridoxal phosphate levels in plasma and the effects of acetaldehyde on pyridoxal phosphate synthesis and degradation in human erythrocytes. J. Clin. Invest. 53, 693–704.CrossRefGoogle Scholar
  2. 2.
    Shane, B. Contractor, S. F. (1975) Assessment of vitamin B-6 status. Studies on pregnant women and oral contraceptive users. Am. J. Clin. Nutr. 28, 739–747.Google Scholar
  3. 3.
    Lumeng, L., Cleary, R. E., Wagner, R., Yu, P.-L. Li, T.-K. (1976) Adequacy of vitamin B-6 supplementation during pregnancy: A prospective study. Am. J. Clin. Nutr. 29, 1376–1383.Google Scholar
  4. 4.
    Lumeng, L., Cleary, R. E. Li, T.-K. (1974) Effect of oral contraceptives on the plasma concentrations of pyridoxal phosphate. Am. J. Clin. Nutr. 27, 326–333.Google Scholar
  5. 5.
    Hamfelt, A. (1964) Age variation of vitamin B-6 metabolism in man. Clin. Chim. Acta 10, 48–54.Google Scholar
  6. 6.
    Walsh, M. P. (1966) Determination of plasma pyridoxal phosphate with wheat germ glutamic-aspartic apotransaminase. Am. J. Clin. Path. 36, 282–285.Google Scholar
  7. 7.
    Jacobs, A., Cavill, I.A.J. Hughes, J.N.P. (1968) Erythro- cyte transaminase activity: Effect of age, sex, and vitamin B-6 supplementation. Am. J. Clin. Nutr. 21, 502–507.Google Scholar
  8. 8.
    Hamfelt, A. (1967) Enzymatic determination of pyridoxal phosphate in plasma by decarboxylation of L-tyrosine-14C (U) and a comparison with the tryptophan load test. Scan. J. Clin. Lab. Invest. 20, 1–10.Google Scholar
  9. 9.
    Hamfelt, A. (1967) Pyridoxal phosphate concentration and aminotransferase activity in human blood cells. Clin. Chim. Acta 16, 19–28.Google Scholar
  10. 10.
    Lumeng, L., Ryan, M. P. Li, T.-K. (1978) Validation of the diagnostic value of plasma pyridoxal 5’-phosphate measurements in vitamin B-6 nutrition of the rat. J. Nutr. 108, 545–553.Google Scholar
  11. 11.
    Chabner, B. Livingston, D. (1970) A simple enzymatic assay for pyridoxal phosphate. Anal. Biochem. 34, 413–423.Google Scholar
  12. 12.
    Sundaresan, P. R. Coursin, D. B. (1971~~ Microassay of pyridoxal phosphate using L-tyrosine-1-I4 C and tyrosine apodecarboxylase. Methods Enzymol. 18A, 509–512.Google Scholar
  13. 13.
    Bhagavan, H. N., Koogler, J. M., Jr. Coursin, D. B. (19N) Enzymatic nicroassay of pyridoxal-5’-phosphate using L-(1-14C) tyrosine. Internat. J. Vit. Nutr. Res. 46, 160–164.Google Scholar
  14. 14.
    Haskell, B. E. Snell, E. E. (1972) An improved apotryptophanase assay for pyridoxal phosphate. Anal. Biochem. 45, 567–576.Google Scholar
  15. 15.
    Suelter, C. H., Wang, J. Snell, E. E. (1976) Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5’-phosphates. Anal. Biochem. 76, 221–232.Google Scholar
  16. 16.
    Holzer, H. Gerlach, U. (1963) Pyridoxal phosphate determination with apotransaminase from Brewers yeast. In: Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 606–609, Academic Press, New York.Google Scholar
  17. 17.
    Worland, S. T. Shafer, J. A. (1980) A convenient lactic dehydrogenase-coupled assay for determing pyridoxal 5’-phosphate in plasma. Anal. Biochem. 103, 323–330.Google Scholar
  18. 18.
    Hines, J. D. Love, D. S. (1969) Determination of serum and blood pyridoxal phosphate concentrations with purified rabbit skeletal muscle apophosphorylase. J. Lab. Clin. Med. 73, 343–349.Google Scholar
  19. 19.
    Abe, K. Ressler, C. (1979) Enzymatic determination of pyridoxal 5’-phosphate with y-cyanoaminobutyric acid aposynthase. Internat. J. Peptide Protein Res. 13, 102–105.CrossRefGoogle Scholar
  20. 20.
    Newton, W. A., Morino, Y. Snell, E. E. (1965) Properties of crystalline tryptophanase. J. Biol. Chem. 240, 1211–1218.Google Scholar
  21. 21.
    Huntly, T. E. Metzler, D. E. (1968) The reaction of a-methylglutamate with glutamic acid decarboxylase. In: Symposium on Pyridoxal Enzymes, pp. 81–84. Maruzen, Tokyo.Google Scholar
  22. 22.
    Yang, B. I. Metzler, D. E. (1979) Pyridoxal 5’-phosphate and analogs as probes of coenzyme-protein interaction. Methods Enzymol. 62D, 528–551.CrossRefGoogle Scholar
  23. 23.
    Jenkins, W. T., Yphantis, D. A. Sizer, I. W. (1959) Glutamic aspartic transaminase: Assay, purification and general properties. J. Biol. Chem. 234, 51–57.Google Scholar
  24. 24.
    Martinez-Carrion, M., Turano, D., Chiancone, E. Bossa, F., Giartosio, A., Riva, F. Fasella, P. (1967) Isolation and characterization of multiple forms of glutamate-aspartate aminotransferase from pig heart. J. Biol. Chem. 242, 23972409.Google Scholar
  25. 25.
    Scardi, V., Scotto, P., Saccarino, M. Scarano, E. (1963) The binding of pyridoxal 5’-phosphate to aspartate aminotransferase of pig heart. Biochem. J. 88, 172–175.Google Scholar
  26. 26.
    Furbish, F. S., Fonda, M. L. Metzler, D. E. (1969) Reactions of apoaspartate aminotransferase with analogs of pyridoxal phosphate. Biochemistry 8, 5169–5180.CrossRefGoogle Scholar
  27. 27.
    Okamoto, M. Morino, Y. (1973) Affinity labeling of aspartate aminotransferase isozymes by bromopyruvate. J. Biol. Chem. 248, 82–90.Google Scholar
  28. 28.
    Ovchinnikov, Yu. A., Egorov, C. A., Adlanova, N. A., Feigina, M. Yu., Lipkin, V. M., Abdulaev, N. G., Grishin, E. V., Kiselev, A. P., Modyanov, N. N., Braunstein, A. E., Polyanovsky, 0. L. Nosikov, V. V. (1973) The complete amino acid sequence of cytoplasmic aspartate aminotransferase from pig heart. FEBS Lett. 29, 31–34.Google Scholar
  29. 29.
    Peterson, E.A. Sober, H. A. (1954) Preparation of crystalline phosphorylated derivatives of vitamin B-6. J. Am. Chem. Soc. 76, 169–175.CrossRefGoogle Scholar
  30. 30.
    Jentoft, N. Dearborn, D. G. (1979) Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 254, 4359–4365.Google Scholar
  31. 31.
    Ritchey, J. M., Gibbons, I. Schachman, H. K. (1977) Reactivation of enzymes by light-stimulated cleavage of reduced pyridoxal 5’-phosphate-enzyme complexes. Biochemistry 16, 4584–4590.CrossRefGoogle Scholar
  32. 32.
    Uchida, T. O’Brian, R. D. (1964) The effects of hydrazines on pyridoxal phosphate in rat brain. Biochem. Pharmacol. 13, 1143–1150.Google Scholar

Copyright information

© Springer Science+Business Media New York 1981

Authors and Affiliations

  • Bob Inyu Yang
    • 1
  • Ashok K. Sawhney
    • 1
  • Ronnie C. Pitchlyn
    • 1
  • Peter M. Peer
    • 1
  1. 1.Department of Chemistry, College of Arts & Sciences and the School of MedicineUniversity of Missouri-Kansas CityKansas CityUSA

Personalised recommendations