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Clinical Chemistry 48: 1560-1564, 2002;
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(Clinical Chemistry. 2002;48:1560-1564.)
© 2002 American Association for Clinical Chemistry, Inc.

Homogeneous, Nonradioactive, Enzymatic Assay for Plasma Pyridoxal 5-Phosphate

Qinghong Han1, Mingxu Xu1, Li Tang1, Xuezhong Tan1, Xiuying Tan1, Yuying Tan1 and Robert M. Hoffmana1

1 A/C Diagnostics, 7917 Ostrow St., San Diego, CA 92111.

aAuthor for correspondence. Fax 858-268-4175; e-mail all{at}anticancer.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{alpha},{gamma}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pyridoxal 5'-phosphate (PLP)1 is the biologically active form of vitamin B6 and is involved in numerous metabolic pathways as an enzyme cofactor (1)(2)(3). Homocysteine (HCY), a risk factor for cardiovascular and other diseases, including Alzheimer disease (4)(5), is converted to cysteine by PLP-dependent transsulfuration enzymes, which condense HCY with serine to form cystathionine and subsequently convert it to cysteine (6). A major cause of homocysteinemia is insufficient intake of vitamin B6, vitamin B12, and folic acid, which are also necessary for HCY metabolism (7). Clinical studies suggest that vitamin B6 is independently associated with an increased risk for cardiovascular disease (8)(9)(10), and recent studies have shown that plasma PLP concentrations are significantly decreased in other pathologic conditions, including rheumatoid arthritis (11). High total HCY (tHCY) and low vitamin B6 plasma concentrations are associated with an increased risk for deep venous thrombosis independent of established risk factors for deep venous thrombosis. The association of low vitamin B6 concentrations with deep venous thrombosis is also independent of the tHCY concentration (12).

Several methods have been developed for vitamin B6 measurement. The most common assay for PLP uses activated apo-tyrosine decarboxylase, which requires PLP to convert [1-14C]tyrosine to 14CO2, which is then trapped and counted for radioactivity. Other methods include the vitamin B6 3H radioenzymatic assay (REA), which is also based on PLP-dependent tyrosine decarboxylase (13)(14)(15) and HPLC (16)(17). These methods are relatively complex and nonhomogeneous and require specialized equipment and materials, including radioactive reagents.

We describe in this report a single-enzyme, nonradioactive, homogeneous assay for PLP that uses the apo form of PLP-dependent, recombinant homocysteine {alpha},{gamma}-lyase (rHCYase) cloned from Trichomonas vaginalis (18)(19). The PLP enzymatic assay is simple, rapid, sensitive, and specific and requires only a small volume of plasma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The following materials were obtained from Sigma–Aldrich: pyridoxal phosphate, pyridoxal, pyridoxamine, pyridoxine, dithiothreitol, sodium bisulfate, D,L-HCY, potassium ferricyanide, and Triton X-100. N,N-Dibutylphenylenediamine (DBPDA) was synthesized in our laboratory. rHCYase was produced in our laboratory (19). The vitamin B6 3H REA reagent set was purchased from American Laboratory Products Co., Ltd.

preparation of apo-rHCYase
Hydroxylamine is a carbonyl-specific reagent that reversibly dissociates PLP from the holoenzyme. Holo-rHCYase was treated with 10 mmol/L hydroxylamine in 1.8 mL containing 3.75 g/L protein, 40 µL of 100 mmol/L dithiothreitol, and 200 µL of 100 mmol/L hydroxylamine phosphate and incubated overnight at 4 °C. The PLP-depleted apoenzyme was precipitated with 450 g/L ammonium sulfate and centrifuged at 10 000g for 10 min. The supernatant was discarded, and the pellet was dissolved in 1.0 mL of 20 mmol/L potassium phosphate buffer (pH 7.6) containing 1 mmol/L EDTA and 1 mmol/L dithiothreitol. Gel filtration was then performed with 1.0 mL of the aforementioned solution loaded onto a 1.0 x 10 cm G-25 column. The apoenzyme was eluted with 20 mmol/L potassium phosphate buffer (pH 7.6), and the protein peak was collected as measured at the absorbance at 280 nm. This protein peak contained PLP-depleted rHCYase at 0.1 g/L protein. Trehalose at a final concentration of 1.0 g/L was added as a preservative to the apoenzyme solution, which was stored at -80 °C.

determination of plp content in holo- and apo-rHCYase
The PLP content of holo- and apo-HCYase was determined fluorometrically with a modification of a published procedure (20). In brief, 0.2 mL each of holoenzyme (3.75 g/L) and apoenzyme (1.0 g/L) was added to 0.2 mL of 0.8 mol/L perchloric acid. The samples were then centrifuged at 10 000g for 5 min. The pellet was discarded, and the supernatant was used for HPLC analysis. The mobile phase, consisting of 0.1 mol/L sodium perchlorate and 0.5 g/L sodium bisulfite, was adjusted to pH 7.0 by addition of phosphoric acid and pumped at a flow rate of 1.0 mL/min through a LC-18-DB column. The eluate was assessed for PLP content with a spectrofluorophotometer at an excitation wavelength of 300 nm and an emission wavelength of 400 nm. The detection limit for PLP was 0.5 pmol (20).

reconstitution of holo-rHCYase
Apo-rHCYase (0.1 g/L) was preincubated at 37 °C for 60 min in phosphate buffer (pH 7.6) with 50 µmol/L PLP (final concentration). Ten microliters of the sample was diluted in 290 µL of phosphate buffer (pH 7.6), and 10 µL was used for the enzyme activity assay. Ten microliters of the holo-rHcyase (1.0 g/L) was diluted in 290 µL of phosphate buffer (pH 7.6), and 10 µL was used for the enzyme activity assay. Enzyme activity was measured with a 3-methyl-2-benzothiazoline hydrazone assay for the keto product of the {alpha},{gamma}-elimination reaction (19). The assay was performed in 1 mL of 50 mmol/L phosphate buffer (pH 8.0) and 5 mmol/L D,L-HCY for 10 min at 37 °C with various amounts of enzyme. The reaction was stopped by addition of 0.5 mL of 45 mL/L trichloroacetic acid. Supernatant (0.5 mL) was added to 0.5 mL of 0.5 mL/L 3-methyl-2-benzothiazoline hydrazone in 1.0 mL of 1.0 mol/L sodium acetate (pH 5.2) and incubated for 30 min at 50 °C. The product was determined spectrophotometrically by the absorbance at 335 nm.

plasma sample preparation
Blood was collected by venipuncture into a Vacutainer Tube (Becton-Dickinson) containing EDTA. After centrifugation, the plasma sample was stored at -80 °C until the time of assay.

plp enzymatic assay protocol
Binding step.
Five microliters of plasma or calibrators and 20 µL of apo-rHCYase (0.1 g/L) were added to 475 µL of binding buffer [10 mmol/L citrate-phosphate buffer (pH 5.3) containing 1 mmol/L EDTA] and mixed well. The binding step was performed for 60 min at 37 °C. All samples were assayed in duplicate. An apoenzyme blank was also included in each assay to correct for endogenous PLP that might be present in the apoenzyme extract.

Enzymatic reaction step.
Five hundred microliters of assay buffer containing 200 mmol/L potassium phosphate buffer (pH 8.3), 2.5 mmol/L D,L-HCY, 1 mmol/L EDTA, 0.2 mmol/L dithiothreitol, and 2 g/L Triton X-100 were added to each tube. The reaction was carried out at 37 °C for 20 min.

Chromogenic step.
The enzymatic reaction was stopped by addition of 50 µL of chromophore reagent (40 mmol/L DBPDA in 6 mol/L HCl), followed by addition of 50 µL of oxidizing agent (40 mmol/L potassium ferricyanide in 20 mmol/L potassium phosphate buffer, pH 8.3). The chromogenic reaction was allowed to progress for 10 min at 37 °C. The resulting absorbance was measured at 675 nm.

vitamin b6 3h rea method
PLP was also determined using the vitamin B6 3H REA (American Laboratory Products Co., Ltd.). In this method, [3H]tyrosine is decarboxylated to [3H]tyramine by the PLP-dependent enzyme, tyrosine decarboxylase (13)(14). The activity of the apo form of tyrosine decarboxylase is linearly dependent on PLP concentration. The [3H]tyramine product of this reaction is selectively extracted in a scintillation mixture, with the unreacted [3H]tyrosine remaining in the aqueous phase.

specificity for plp
To determine the specificity of the PLP enzymatic assay, apo-rHCYase was first incubated with pyridoxal phosphate, pyridoxal, pyridoxamine, and pyridoxine, each at a concentration of 100 nmol/L. The PLP enzymatic assay was then performed with HCY as described above.

reconstitution stability of apo-rHCYase
Twenty microliters of apo-rHCYase (0.1 g/L), stored at 4 °C, and 5 µL of a 50 nmol/L PLP calibrator were added to 475 µL of binding buffer (pH 5.3). After a 60-min preincubation at 37 °C, 500 µL of assay buffer (pH 8.3) was added to each tube, vortex-mixed, and incubated for 20 min at 37 °C. The reconstituted holoenzyme was then used in the PLP enzymatic assay with HCY as described above.

interference
Interference in the PLP enzymatic assay was determined by addition of the following substances: bilirubin (ICN), to achieve plasma concentrations of 125 and 250 µmol/L; hemoglobin (Sigma) at 2500 and 5000 mg/L; and triglycerides (Sigma) at 3.32 and 6.65 mmol/L. All plasma samples were obtained from three healthy individuals (Table 3 ).


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  		Table 3. Interference from bilirubin, hemoglobin, and triglycerides added to plasma samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
principle of the assay
PLP is removed from the rHCYase holoenzyme by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme depends on the amount of PLP bound to rHCYase (Reaction 1):


characterization of apo-rHCYase
PLP was removed from holo-rHCYase by reaction with hydroxylamine phosphate for 12 h at 4 °C. The PLP was derivatized with sodium bisulfite. The PLP content of holo- and of apo-rHCYase was 3.8 ± 0.4 and 0.072 ± 0.008 per tetramer, respectively. The hydroxylamine-treated apo-rHCYase retained residual enzyme activity (0.46 U/mg of protein). Addition of 50 µmol/L (final concentration) PLP to apo-rHCYase produced a reconstituted activity of 16.1 U/mg of protein.

specificity for plp reconstitution
We confirmed the absolute dependence of HCYase activity on PLP. Other vitamin B6 compounds, including pyridoxal, pyridoxamine, and pyridoxine, did not restore any detectable activity to apo-rHCYase.

stability of apo-rHCYase
The apo-rHCYase reconstituted with PLP in 20 mmol/L potassium phosphate buffer (pH 7.6) was stable at 4 °C for at least 1 month. The apoenzyme was stable at -80 °C for 1 year.

evaluation of the assay
Linear response.
To determine the linear response of the assay, we assayed five different PLP concentrations, 5, 10, 50, 100, and 200 nmol/L, by the present method. As shown in Fig. 1 , the calibration was linear between 5 and 200 nmol/L.



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  		Figure 1. Linearity of the calibration curve for the PLP enzymatic assay.

Abs, absorbance. Equation for the line: y = 0.0006x + 0.0036 (R2 = 0.9987).

Analytic recovery.
The analytic recoveries for PLP added to human pooled serum are shown in Table 1 . Recoveries were 103% at 10 nmol/L, 96% at 50 nmol/L, and 94% at 100 nmol/L, with a mean recovery of 98%.


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  		Table 1. Recovery of PLP added to plasma.1

Precision.
The within-assay variation was estimated after three samples, containing 22.4, 77.2, and 125.1 nmol/L PLP, were analyzed in 20 parallel determinations. Each sample was assayed in duplicate. The between-assay variation was determined from 20 successive analytic set-ups that analyzed aliquots from a single sample on 10 different days over a 1-month period. The within- and between-assay mean CVs were 9.6% and 12%, respectively (Table 2 ).


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  		Table 2. Within- and between-run imprecision of the PLP enzymatic assay.1

Correlation.
The concentration of PLP in 45 different human serum samples was measured by the present method and the vitamin B6 3H REA method. As shown in Fig. 2 , excellent agreement was observed between the methods by linear regression analysis (Fig. 2A ), which yielded the equation y = 0.9367x + 10.569 (R2 = 0.9201), and by a Bland–Altman plot (21) of the difference between the results obtained by the two methods as a function of their mean value ± 2 SD (Fig. 2B ).



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  		Figure 2. Correlation between the PLP enzymatic assay (y) and vitamin B6 3H REA (x).

(A), linear regression analysis: y = 0.9367x + 10.569 (R2 = 0.9201; n = 45). (B), Bland–Altman plot of the difference in PLP values between the two methods as a function of their mean value ± 2 SD (21).

interference
Bilirubin at 125 and 250 µmol/L had no appreciable effect on the assay (Table 3Up ). Hemoglobin at 2500 mg/L had no appreciable effect, but at 5000 mg/L produced a -13% change in the result (Table 3Up ). Triglycerides at 3.32 and 6.65 mmol/L had a negligible effect on the assay (Table 3Up ).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the first homogeneous, nonradioactive assay for determining vitamin B6 (PLP) in plasma. Only a single enzyme was necessary in this assay. We used a three-step procedure to prepare apo-rHCYase by use of hydroxylamine phosphate, a carbonyl-specific reagent known to reversibly dissociate PLP from PLP-dependent enzymes (22)(23)(24)(25). To prevent the apoenzyme from rebinding free PLP, the enzyme was precipitated rapidly with ammonium sulfate to remove free PLP and hydroxylamine phosphate.

We observed that 10 mmol/L citric phosphate buffer (pH 5.3) was more effective for dissociation of PLP and the subsequent apoenzyme reconstitution. The apo-rHCYase typically retained 0.5–1.0% of the original enzyme activity, which indicates that a small, residual amount of PLP remained bound to the PLP-free enzyme (26). The apo-rHCYase, however, had lower residual enzyme activity and a high recovery of enzyme activity with PLP.

We previously described a simple, single-enzyme assay method for measurement of HCY by modified methylene blue determination of H2S (27). The PLP method uses the amplification principle of the HCY enzymatic assay, which allows nanomolar concentrations of PLP to be measured, with the signal conversion of millimolar concentrations of HCY to H2S. Amplification of the PLP signal is on the order of 106, which enables PLP concentrations as low as 5 nmol/L to be measured.

We observed an excellent correlation between our new method and the vitamin B6 3H REA method. The new analytic method may be useful in the diagnosis of vitamin B6 deficiency in large, population-based studies because PLP appears to be an independent risk factor for cardiovascular and other diseases. We are now developing a fully automated method based on this assay system. A main goal will be to shorten the reconstitution time to further reduce the total assay time.


   Acknowledgments
 
This research was partially funded by National Heart, Lung, and Blood Institute Grant 2 R44 HL63263-02.


   Footnotes
 
1 Nonstandard abbreviations: PLP, pyridoxal 5'-phosphate; tHCY, total homocysteine; REA, radioenzymatic assay; rHCYase, recombinant homocysteine {alpha},{gamma}-lyase; and DBPDA, N,N-dibutylphenylenediamine.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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