Fluoromefric Defermination of Pyridoxal and that of Pyridoxal5' -Phosphote by Cyanide

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Although a number of procedures have been proposed for the quantitative determination of pyridoxal (PAL) and that of pyridoxal 5'-phosphate (PAL-P), it has been required to develop more reliable and simplified methods for the determination in biological materials. Bonavita found that the reaction of PAL and that of PAL-P upon cyanide in an alkaline solution produced highly fluorescent cyanohydrins. The authors studied on the reactions of PAL and PAL-P on cyanide and devised a method for the determination of PAL and PAL-P in biological materials by means of these reactions. Recently, Fukui et al. pointed out that the main reaction product of PAL on cyanide was not PAL-cyanohydrin [(II) in Fig.3] but 4-pyridoxolactone (PiAL) [(VI) in Fig.3] and that of PAL-P was presumed to be 4-pyridoxic acid5'-phosphate.In our own experiments, similar results were obtained as to the reaction products by means of fluorescence and excitation spectra (Fig.1) and thin layer chromatography, and furthermore an intermediate compound [(III) in Fig.3] of the reaction of RAL on cyanide was isolated.The intermediate compound was quantitatively converted to PiAL in an alkaline solution in the presence of oxygen with liberation of cyanide ion. Its structure was determined by X-ray crystallography and the reaction mechanism was presumed to be as shown in Fig. 3. 4-Pyridoxic acid (PiA) is a metabolite of the vitamin B6 group and it interferes with determination of PAL and PAL-P in biological materials by the present method. PAL can be more easily separated from PiA than from PAL-P and fluorescence intensity of the reaction product of PAL was about 5 times as strong as that of PAL-P, therefore PAL-P was conveniently determined as PAL by hydrolysis. Hydrolysis of PAL-P was carried out by acid phosphatase partially purified from potatoes. Determination of PAL Sample was diluted with 0.2 M acetate buffer (pH 4.0) and deproteinized with 20% trichloroacetic acid (TCA). The deproteinized solution was placed on a column of Dowex IX 8 (5×80 mm, acetate form) and any interfering substances were removed by the column.The effluent from the column was passed through a column of Amberlite CG120 (5× 100 mm) which was previously bufferized to pH 4.0 with acetate buffer (pH 4.0). PAL absorbed on the column was eluted with 0.4 M phosphate buffer (pH 7.5)(Fig.4) and treated with KCN. Fluorescence intensity at 430 mμ, excited at 356 mμ, was measured after being made alkaline with 0.4 M Na2CO3, and compared with the standards. Determination of PAL-P Sample was diluted with 0.2 M acetate buffer (pH 4.0) and heated at 80°C for 5 minutes. After cooling, acid phosphatase was added. It was incubated at 37°C for 1 hour and deproteinized with 20% TCA. Then it was treated as in the case of PAL. The amount of PAL-P was calculated by subtracting the amount of PAL from the value obtained as above. In the case of PAL-P, additional tests were carried out simultaneously and the determined value was corrected by recovery. The results of recovery tests are shown in TABLE I. The present method is available to serum, plasma, urine and tissues.
Japan Society of Clinical Chemistryの論文

Fluoromefric Defermination of Pyridoxal and that of Pyridoxal5' -Phosphote by Cyanide

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