4-Pyridoxic acid is the major metabolite of all forms of
vitamin B₆ in mammalian species. The determination of 4-pyridoxic
acid, therefore, is important in the investigation of vitamin B₆
metabolism and for the establishment of human requirements for the
vitamin.
Although 4-pyridoxic acid is itself fluorescent, Huff and
Perlzweig (1944) developed a classic method for its determination
based on the conversion of the metabolite to the even more highly
fluorescent lactone.
Sarett in 1951 recognized that there are many substances in
urine other than 4-pyridoxic acid which are fluorescent and which
may interfere with its determination. His attempts to remove the
extraneous fluorescent compounds with Decalso or with charcoal were not entirely satisfactory. He proposed, therefore, the administration
of a test dose of 4-pyridoxic acid to subjects whose urine was
to be analyzed for the metabolite of vitamin B₆, thus rendering the
foreign fluorescence less significant by dilution. In 1955, Fujita and
associates employed ion exchange chromatography as a means of
separating 4-pyridoxic acid from other fluorescent compounds in
urine and in 1958 Reddy, Reynolds and Price developed a chromatographic
method using Dowex 1 (Cl⁻), a strongly basic anion exchange
resin and Dowex 5OW (H⁺), a strongly acidic cation exchange resin
for the separation of 4-pyridoxic acid from extraneous fluorescent
materials in urine. Eluates from the latter of the two columns,
used in sequence, were subjected to a chemical procedure to
oxidize the 4-pyridoxic acid to the more highly fluorescent lactone
form. The Reddy, Reynolds and Price procedure has provided the
means for achieving more valid results than had been possible
previously.
Because of the importance of this procedure, the dynamics of
the ion exchange method of Reddy and associates was investigated.
Standards of 4-pyridoxic acid, or 4-pyridoxic acid lactone, and of
urine were subjected to ion exchange chromatography using their
procedure. Conversion of the 4-pyridoxic acid to the lactone was
accomplished by means of the microprocedure of Woodring, Fisher
and Storvick (1964). Effluent, wash, and eluate fractions were collected to determine the pattern of elution of the 4-pyridoxic acid,
as well as to determine the effect of interfering fluorescence from
the resins and from reagents.
Eluates of urine from the second in the series of two Dowex
ion exchange resins were subjected to paper and thin layer chromatography
along with standards of 4-pyridoxic acid and the lactone
of 4-pyridoxic acid. Eluates from influents of hydrolyzed urine
produced highly fluorescent zones which did not correspond to those
of standards of 4-pyridoxic acid or its lactone.
A tryptophan load test is often used to diagnose vitamin B₆
deficiency but some of the metabolites of tryptophan are highly
fluorescent and may interfere with the fluorometric measurement
of 4-pyridoxic acid even after its conversion to the more fluorescent
lactone. To study the effect of the presence of tryptophan metabolites
on the determination of 4-pyridoxic acid in urine, a test dose of
L-tryptophan was administered to one subject and the subsequent
24-hour urine collection was treated according to the chromatographic
procedure of Reddy et al. The final eluate was analyzed for 4-
pyridoxic acid using the raicroprocedure of Woodring and associates.
Readings of fluorescence indicated that much foreign fluorescence
remained even after the lactonization procedure. Recovery of 4-
pyridoxic acid was 65.5 percent. Therefore, for the determination of
4-pyridoxic acid following a test dose of L-tryptophan, a recovery curve would have to be used for calculation of 4-pyridoxic acid
excretion.
It was made emphatically clear that there are highly fluorescent
compounds present in the resin itself, even after extensive treatment,
which cannot be removed. The results of this study emphasize the
great importance of maintaining a constant rate of flow, and thus a
constant rate of leaching, during chromatography to minimize the
effect of the interfering fluorescence.
Dowex 5OW (H⁺) will not retain 4-pyridoxic acid well unless
the resin has been rendered strongly acidic. It was ascertained that
this procedure should be accomplished just before use.
4-Pyridoxic acid and the lactone of 4-pyridoxic acid are eluted
almost at once from the Dowex 1 resin but are released in the middle
fractions during elution from freshly activated Dowex 50W, with
50 ml. of elutriant.
It was determined that chromatography could be undertaken at
a relatively rapid flow rate, thus allowing the entire chromatogtaphic
procedure and analysis of eluates to be completed in one day. / Graduation date: 1967
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/26794 |
| Date | 27 July 1966 |
| Creators | Rohrer, Martha Jane |
| Contributors | Storvick, Clara A. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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