The Vitamins 175methylation of CpG islands in DNA, and there is
evidence that some cancers (and especially colorectal
cancer) are associated with under-methylation of
CpG islands as a result of low folate status. A number
of small studies have suggested that folate supple-
ments may be protective against colorectal cancer, but
no results from large-scale randomized controlled
trials have yet been reported, and to date there is no
evidence of a decrease in colorectal cancer in coun-
tries where folate enrichment of fl our is mandatory.
Folate defi ciency: megaloblastic anemia
Dietary defi ciency of folic acid is not uncommon and,
as noted above, defi ciency of vitamin B 12 also leads to
functional folic acid defi ciency. In either case, it is cells
that are dividing rapidly, and therefore have a large
requirement for thymidine for DNA synthesis, that
are most severely affected. These are the cells of the
bone marrow that form red blood cells, the cells of
the intestinal mucosa and the hair follicles. Clinically,
folate defi ciency leads to megaloblastic anemia, the
release into the circulation of immature precursors of
red blood cells.
Megaloblastic anemia is also seen in vitamin B 12
defi ciency, where it is due to functional folate defi -
ciency as a result of trapping folate as methyl-tetra-
hydrofolate. However, the neurological degeneration
of pernicious anemia is rarely seen in folate defi ciency,
and indeed a high intake of folate can mask the devel-
opment of megaloblastic anemia in vitamin B 12 defi -
ciency, so that the presenting sign is irreversible nerve
damage.
Folate requirements
Depletion/repletion studies to determine folate
requirements using folate monoglutamate suggest
a requirement of the order of 80–100 μg (170–
220 nmol)/day. The total body pool of folate in adults
is some 17 μmol (7.5 mg), with a biological half-life
of 101 days. This suggests a minimum requirement
for replacement of 37 μg (85 nmol)/day. Studies of
the urinary excretion of folate metabolites in subjects
maintained on folate-free diets suggest that there is
catabolism of some 80 μg (170 nmol) of folate/day.
Because of the problems in determining the bio-
logical availability of the various folate polyglutamate
conjugates found in foods, reference intakes allow a
wide margin of safety, and are based on an allowance
of 3 μg (6.8 nmol)/kg body weight.
Assessment of folate status
Measurement of the serum or red blood cell concen-
tration of folate is the method of choice, and several
simple and reliable radioligand binding assays have
been developed. There are problems involved in
radioligand binding assays for folate, and in some
centers microbiological determination of plasma or
whole blood folates is the preferred technique. Serum
folate below 7 nmol/l or erythrocyte folate below
320 nmol/l indicates negative folate balance and early
depletion of body reserves. At this stage the fi rst bone
marrow changes are detectable.Histidine metabolism:
the formiminoglutamate test
The ability to metabolize a test dose of histidine pro-
vides a sensitive functional test of folate nutritional
status; formiminoglutamate (FIGLU) is an interme-
diate in histidine catabolism, and is metabolized by
the folate-dependent enzyme formiminoglutamate
formiminotransferase. In folate defi ciency the activity
of this enzyme is impaired, and FIGLU accumulates
and is excreted in the urine, especially after a test dose
of histidine: the so-called FIGLU test.
Although the FIGLU test depends on folate nutri-
tional status, the metabolism of histidine will also be
impaired, and hence a positive result obtained, in
vitamin B 12 defi ciency, because of the secondary
defi ciency of free folate. About 60% of vitamin B 12 -
defi cient subjects show increased FIGLU excretion
after a histidine load.The dUMP suppression test
Rapidly dividing cells can either use preformed TMP
for DNA synthesis, or synthesize it de novo from
dUMP. Stimulated lymphocytes incubated with [^3 H]-
TMP will incorporate the label into DNA. In the
presence of adequate amounts of methylene-tetrahy-
drofolate, the addition of dUMP as a substrate for
thymidylate synthetase reduces the incorporation of
[^3 H]-TMP as a result of dilution of the pool of labeled
material by newly synthesized TMP and inhibition of
thymidylate kinase by thymidine triphosphate.
In normal cells the incorporation of [^3 H]-thymi-
dine into DNA after preincubation with dUMP is
1.4–1.8% of that without preincubation. By contrast,
cells that are defi cient in folate form little or no thy-
midine from dUMP, and hence incorporate nearly as