The initial purpose of this study was to develop a reliable method
for the determination of vitamin B₆ in blood. At the present time
microbiological assays have demonstrated greater sensitivity and
specificity than other methods for the of vitamin B₆ in
pure solution and many foodstuffs. However, adetermination pplication of a
differential microbiological assay to blood revealed the presence of a
substance(s) which invalidated the assay. The differential technique
employs three organisms whose differences in response to the three
components of the vitamin should permit an accurate measurement of
total vitamin B₆ plus a differential estimation of the vitamin B₆
components. Saccharomyces carlsbergensis responds to pyridoxine,
pyridoxal and pyridoxamine; Lactobacillus casei responds to pyridoxal
and Streptococcus faecium 51 responds to pyridoxal and pyridoxamine.
Surprisingly, the values obtained for blood with L. casei and
S. faecium were 20-50 times greater than that given by S. carlsbergensis,
which should show a response at least equal to those of the other two
organisms.
Since the various forms of vitamin B₆ exist in biological
materials as phosphorylated and protein-bound complexes, hydrolytic
extraction to liberate the vitamin in a measurable state is a
necessary preliminary to analysis. Therefore, the investigation was
expanded to a consideration of acid and enzymatic hydrolytic
techniques, since inadequacies in this area seemed the most logical
explanation for the discrepant values obtained with blood. At the
same time attempts were made to characterize the unknown growth-promoting
factor in blood.
The hydrolysis studies revealed that a greater response to blood
could be obtained from the yeast by using either a combination of
acid and enzymatic hydrolysis or by incubating an acid hydrolysate
with a cell-free extract of S. faecium. This seemed to indicate that
currently accepted vitamin B₆-extraction procedures are not adequate
for blood and that the S. carlsbergensis values in the literature for
total vitamin B₆ may be questionable. Furthermore, the results
indicated additional enzyme capacity for S. faecium, presumably for
bound forms of the vitamin. It was also observed that with prolonged
heating in the presence of protein, there was a loss in vitamin B₆
activity which appeared to be due to rebinding and condensation
processes rather than outright destruction. An additional reason for
the discrepant values may have been demonstrated by column
chromatography experiments which suggested that blood contains
something inhibitory to the yeast.
Characterization of the unknown factor reveals that it is found
in the protein fraction of blood and is formed or released upon acid
hydrolysis. It can be separated from blood by paper chromatography as indicated by bioautography with the lactic acid bacteria. Its
activation and fluorescence peaks approximate those of pyridoxa1.
It appears to be used in the same way as pyridoxal by S. faecium
according to the results of a dilution study combining pyridoxal
and blood hydrolysate. Unlike free vitamin B₆ it is stable to
ultraviolet and gamma irradiation, suggesting that the reactive sites
on the vitamin may be protected, possibly by binding to proteins
Continuing research is needed to develop a hydrolytic technique
that will free the vitamin from its bound forms and at the same time
avoid,to the extent possible, its condensation with other constituents
of the hydrolysate. / Graduation date: 1964
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/28173 |
| Date | 22 August 1964 |
| Creators | Peters, Jean McLeod |
| Contributors | Storvick, Clara |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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