Problems in the microbiological determination of vitamin B₆ in blood

Graduation date: 1964The initial purpose of this study was to develop a reliable method\ud for the determination of vitamin B₆ in blood. At the present time\ud microbiological assays have demonstrated greater sensitivity and\ud specificity than other methods for the of vitamin B₆ in\ud pure solution and many foodstuffs. However, adetermination pplication of a\ud differential microbiological assay to blood revealed the presence of a\ud substance(s) which invalidated the assay. The differential technique\ud employs three organisms whose differences in response to the three\ud components of the vitamin should permit an accurate measurement of\ud total vitamin B₆ plus a differential estimation of the vitamin B₆\ud components. Saccharomyces carlsbergensis responds to pyridoxine,\ud pyridoxal and pyridoxamine; Lactobacillus casei responds to pyridoxal\ud and Streptococcus faecium 51 responds to pyridoxal and pyridoxamine.\ud Surprisingly, the values obtained for blood with L. casei and\ud S. faecium were 20-50 times greater than that given by S. carlsbergensis,\ud which should show a response at least equal to those of the other two\ud organisms.\ud Since the various forms of vitamin B₆ exist in biological\ud materials as phosphorylated and protein-bound complexes, hydrolytic\ud extraction to liberate the vitamin in a measurable state is a\ud necessary preliminary to analysis. Therefore, the investigation was\ud expanded to a consideration of acid and enzymatic hydrolytic\ud techniques, since inadequacies in this area seemed the most logical\ud explanation for the discrepant values obtained with blood. At the\ud same time attempts were made to characterize the unknown growth-promoting\ud factor in blood.\ud The hydrolysis studies revealed that a greater response to blood\ud could be obtained from the yeast by using either a combination of\ud acid and enzymatic hydrolysis or by incubating an acid hydrolysate\ud with a cell-free extract of S. faecium. This seemed to indicate that\ud currently accepted vitamin B₆-extraction procedures are not adequate\ud for blood and that the S. carlsbergensis values in the literature for\ud total vitamin B₆ may be questionable. Furthermore, the results\ud indicated additional enzyme capacity for S. faecium, presumably for\ud bound forms of the vitamin. It was also observed that with prolonged\ud heating in the presence of protein, there was a loss in vitamin B₆\ud activity which appeared to be due to rebinding and condensation\ud processes rather than outright destruction. An additional reason for\ud the discrepant values may have been demonstrated by column\ud chromatography experiments which suggested that blood contains\ud something inhibitory to the yeast.\ud Characterization of the unknown factor reveals that it is found\ud in the protein fraction of blood and is formed or released upon acid\ud hydrolysis. It can be separated from blood by paper chromatography as indicated by bioautography with the lactic acid bacteria. Its\ud activation and fluorescence peaks approximate those of pyridoxa1.\ud It appears to be used in the same way as pyridoxal by S. faecium\ud according to the results of a dilution study combining pyridoxal\ud and blood hydrolysate. Unlike free vitamin B₆ it is stable to\ud ultraviolet and gamma irradiation, suggesting that the reactive sites\ud on the vitamin may be protected, possibly by binding to proteins\ud Continuing research is needed to develop a hydrolytic technique\ud that will free the vitamin from its bound forms and at the same time\ud avoid,to the extent possible, its condensation with other constituents\ud of the hydrolysate