Graduate Thesis Or Dissertation
 

Determination of pyridoxal phosphate and pyridoxal by the cyanohydrin method

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  • Pyridoxal phosphate is a coenzyme in about 50 known enzymatic reactions. A simple and accurate method for the determination of pyridoxal phosphate would be desirable because it could provide a means to assess the nutritional status of vitamin B₆ in the human. The cyanohydrin methods to determine pyridoxal phosphate appear to be simple and promising. Cyanohydrin methods have been devised by Bonavita and Scardi, and Bonavita, and applied to biological materials by Yamada et al. The cyanohydrin procedure of Yamada et al. was investigated. In this procedure, the pyridoxal phosphate and pyridoxal in a deproteinized sample are separated with the use of a column of SM-cellulose (1 gm., equilibrated with 0.01 N acetic acid). Pyridoxal phosphate is eluted from SM-cellulose with 0.01 N acetic acid, and pyridoxal is eluted with 0.1 M sodium phosphate buffer, pH 7.4. Pyridoxal phosphate and pyridoxal are converted to their respective cyanohydrin derivatives by reaction with potassium cyanide. These cyanohydrin derivatives are measured fluorometrically at their activation and fluorescence maxima. In preliminary studies on the procedure by Yamada et al., the activation and fluorescence spectra of the cyanohydrin derivatives of pyridoxal phosphate and pyridoxal were obtained to determine the appropriate activating and fluorescent wavelength settings to use for subsequent fluorometric analyses. Pyridoxal phosphate cyanohydrin at pH 3.8 in 0.2 M sodium phosphate buffer had an activation maximum at 325 mμ and a fluorescence maximum at 415 mμ; and pyridoxal cyanohydrin at pH 10 in 0.2 M sodium phosphate buffer had an activation maximum at 355 mμ and a fluorescence maximum at 435 mμ. To obtain maximum fluorescence of the cyanohydrin derivatives, pyridoxal phosphate had to be reacted with potassium cyanide at 50°C for 60 minutes, and pyridoxal had to be reacted for 150 minutes. Following these preliminary studies, the elution pattern of pyridoxal phosphate and pyridoxal from a column of SM-cellulose was investigated. Pyridoxal phosphate was eluted with 0.01 N acetic acid; and pyridoxal, with both 0.01 N acetic acid and 0.1 M sodium phosphate buffer, pH 7.4. The recovery of pyridoxal phosphate from SM-cellulose was 93.5% when pyridoxal phosphate alone was applied to the column, and that of pyridoxal was 108.8% when pyridoxal alone was applied. When a mixture of pyridoxal phosphate and pyridoxal was applied to SM-cellulose, the recovery of pyridoxal phosphate was 105.5% and that of pyridoxal was only 59.8%. When either standard alone was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 85.0%, and that of pyridoxal was only 29.1%. When a mixture of pyridoxal phosphate and pyridoxal was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 62.6%, and that of pyridoxal was 52.1%. This lower recovery of pyridoxal phosphate in blood was due mainly to the high readings of the blanks. This higher recovery of pyridoxal phosphate in blood may be explained by the low concentration of pyridoxal in the buffer fractions from a column of SM-cellulose to which a mixture of pyridoxal phosphate and pyridoxal had been applied that was used to calculate the recovery. Determining the recovery of standards added to the supernatant after the precipitation of the proteins in blood, rather than to the hemolyzed blood before precipitation, would indicate whether pyridoxal phosphate and pyridoxal were lost by adsorption on the protein precipitate. The modified procedure of Yamada et al. is not sensitive enough to determine the pyridoxal phosphate and pyridoxal content of human blood.
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