Aspects of Folate and Tetrahydrofolate Chemistry and Metabolism in the Rat

Abstract

Most of the work in the literature dealing with
folate metabolism has been done using unlabelled folates,
analysis being by microbiological assay. This method is
not highly specific or quantitatively accurate: this
thesis reports results on the synthesis of 14 c- labelled
folates, and the use of these compounds in investigation
of folate metabolism in the rat.

The synthesis of (±)-5-formyltetrahydrofolic
acid -(2-14C), (±)-5,10-methenyltetrahydrofolic acid
-(2-14C), (±)-10-formyltetrahydrofolic acid -(2-14C), and
10-formylfolic acid -(2-14C) from folic acid -(2-14C) is
described.

These compounds, and commercial 5-( CH, )-
methyltetrahydrofolic acid, were given orally to rats at
doses of 3 and 30 μg/ke. Quantitative and qualitative
assay of urine, liver, and faeces showed that
5-formyltetrahydrofolate was largely unmetabolised: this
compound was also given i.v., and showed even less
metabolism. 5,10-methenyltetrahydrofolate was more
extensively metabolised, but depended on dose, being less
for small doses. 10-formyltetrahydrofolate was almost
completely metabolised, both diastereoisomers apparently
being utilised: this was true also for 5-(14C-methyl)
tetrahydrofolic acid, the methyl group being rapidly lost
and a small amount exhaled as 14CO2. 10-formylfolate was not metabolised; it gave a large amount of excreted
pteridines, not observed with the other compounds, It
inhibited folic acid metabolism in humans, but was
ineffective as a growth inhibitor of R1 lymphoma in mice.

Folic acid -(2-14C) given to tumour-bearing
animals was metabolised more than in normals: absorption
of the vitamin at low doses was impaired iin some cases,

An unknown folate found in rat urine by
previous work (Dransfield 1972) was not identified.

Rat livers were extracted after folate
administration to investigate any conversion into
polyglutamates,. Complex and variable chromatograms
were obtained with established extraction procedures,
the results being interpreted as artifact formation
during the extraction process, Elevated L. casei
assays after treatment of monoglutamate with conjugase
were found in one case,

Aspects of the in vitro chemistry of folates
which are important in metabolic studies, and which
are ill-defined in the literature, were also studied.

Folic acid, 5-methyltetrahydrofolate,
5-formyltetrahydrofolate and 5,10-methenyltetrahydrofolate
were autoclaved under conditions commonly used for
extraction of folate from natural material. In
presence of antioxidant, little decomposition was found
apart from the expected formyltetrahydrofolate
interconversions.

Folic acid and 5-methyltetrahydrofolate were
photolysed in the presence and absence of riboflavin. An unusual decomposition of 5-methyltetrahydrofolate
to folic acid in presence of riboflavin was found.

Formyltetrahydrofolates were subjected to
differing pH values, and their rates of isomerisation
studied. 5- and 10-formyltetrahydrofolates were stable
above pH 4; below this value, 10-formyltetrahydrofolate
was cyclised more rapidly than 5-formyltetrahydrofolate.
5,10-methenyltetrahydrofolate was hydrolysed above pH 5,
the rate depending on buffer species and the concentration.
The results are discussed in terms of the pH changes in
the alimentary canal.

Date of Award	1973
Original language	English
Awarding Institution	Aston University