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1 A/C Diagnostics, 7917 Ostrow St., San Diego, CA 92111.
aAuthor for correspondence. Fax 858-268-4175; e-mail all{at}anticancer.com.
Background: Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6. Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely diagnosed because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the PLP-dependent recombinant enzyme, homocysteine-
,
-lyase (rHCYase).
Methods: PLP was removed from holoenzyme rHCYase by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme was linearly related to the amount of PLP bound to the enzyme. The amplification principle of the assay allowed nanomolar concentrations of PLP to be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine to H2S. N,N-Dibutylphenylenediamine (DBPDA) was used for determination of H2S, the combination of which forms a chromophore with high absorbance. The assay was initiated by incubation of 5 µL of plasma with apo-rHCYase in a binding buffer for 60 min at 37 °C. Homocysteine (2.5 mmol/L) was added to the assay buffer and incubated at 37 °C for 20 min. The DBPDA reaction was allowed to progress for 10 min and then read at 675 nm.
Results: The PLP enzymatic assay has a lower limit of detection of 5 nmol/L and is linear to 200 nmol/L. The recovery of PLP was 98%. The mean within- and between-run CVs were 9.6% and 12%, respectively. Correlation of 45 samples in the PLP enzymatic assay and the B6 3H radioenzymatic assay (American Laboratory Products Co., Ltd.) yielded: y = 0.9367x + 10.569 (R2 = 0.9201).
Conclusions: This new PLP assay is the first homogeneous, nonradioactive, vitamin B6 diagnostic method. The assay is applicable to chemistry automated analyzers and may have wide clinical use.
The following articles in journals at HighWire Press have cited this article:
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  M. S. Morris, L. Sakakeeny, P. F. Jacques, M. F. Picciano, and J. Selhub Vitamin B-6 Intake Is Inversely Related to, and the Requirement Is Affected by, Inflammation Status J. Nutr., January 1, 2010; 140(1): 103 - 110. [Abstract] [Full Text] [PDF]
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 M. S. Morris, M. F. Picciano, P. F Jacques, and J. Selhub Plasma pyridoxal 5'-phosphate in the US population: the National Health and Nutrition Examination Survey, 2003-2004 Am. J. Clinical Nutrition, May 1, 2008; 87(5): 1446 - 1454. [Abstract] [Full Text] [PDF]
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 M. E. Rybak, R. B. Jain, and C. M. Pfeiffer Clinical Vitamin B6 Analysis: An Interlaboratory Comparison of Pyridoxal 5'-Phosphate Measurements in Serum Clin. Chem., July 1, 2005; 51(7): 1223 - 1231. [Abstract] [Full Text] [PDF]
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 Z. Yang, X. Sun, S. Li, Y. Tan, X. Wang, N. Zhang, S. Yagi, T. Takakura, Y. Kobayashi, A. Takimoto, et al. Circulating Half-Life of PEGylated Recombinant Methioninase Holoenzyme Is Highly Dose Dependent on Cofactor Pyridoxal-5'-Phosphate Cancer Res., August 15, 2004; 64(16): 5775 - 5778. [Abstract] [Full Text] [PDF]
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Q. Han, M. Xu, L. Tang, X. Sun, N. Zhang, X. Tan, X. Tan, Y. Tan, and R. M. Hoffman Homogeneous Enzymatic Colorimetric Assay for Total Cysteine Clin. Chem., July 1, 2004; 50(7): 1229 - 1231. [Full Text] [PDF]
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X. Sun, Z. Yang, S. Li, Y. Tan, N. Zhang, X. Wang, S. Yagi, T. Yoshioka, A. Takimoto, K. Mitsushima, et al. In Vivo Efficacy of Recombinant Methioninase Is Enhanced by the Combination of Polyethylene Glycol Conjugation and Pyridoxal 5'-Phosphate Supplementation Cancer Res., December 1, 2003; 63(23): 8377 - 8383. [Abstract] [Full Text] [PDF]
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