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Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • 10-formyl-7,8-dihydrofolic acid • 10-formyl-folic acid • bioactivity in human leukemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Using chromatography followed by
differential microbiological assay, Butterworth et al. (1963)
identified both 5-formyl-tetrahydrofolic acid and 10-formyl-folic acid
in mixed diets. Recently, using HPLC, Pfeiffer et al. (1997)
have
identified 5-formyl-tetrahydrofolic acid, 10-formyl-folic acid, and
10-formyl-dihydrofolic acid in cereal-grain food products.
All of the above substances support the growth of the
folate-requiring bacteria L. casei and E.
hirae (formerly S. faecalis) (Johns and Bertino 1965
; Rabinowitz 1960
). Because the above
bacteria must readily metabolize these folates, it was assumed that
mammalian cells would have similar metabolic pathways available. The
bioactivities of 5-formyl-5,6,7,8-tetrahydrofolic acid
(5-HCO-H4folic
acid),2
as a control, 10-formyl-7,8,-dihydrofolic acid
(10-HCO-H2folic acid); and 10-formyl-folic acid
(10-HCO-folic acid) were determined in mammalian cells in a
bacteria-free environment. We report here that excess
10-HCO-H2folic acid, but not 10-HCO-folic acid,
supports the growth of human leukemia cells (CCRF-CEM cells) grown in a
culture medium containing methotrexate (MTX).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sources and preparation of aminoimidazole carboxamide ribotide (AICAR)
(6S) 5-HCO-H4folic acid and (6R) 10-formyl-5,6,7,8
tetrahydrofolic acid (10-HCO-H4folic acid) were described
previously (Baggott et al. 1995
). NADPH and ascorbic
acid were purchased from Sigma Chemical (St. Louis, MO).
10-HCO-H2folic acid was prepared by air oxidation of (6R)
10-HCO-H4folic acid as previously described (Baggott et al. 1995
). 10-HCO-folic acid was prepared by dissolving
folic acid in 98% formic acid for several days and recrystallized from
hot water until a constant UV spectrum was obtained. Stock solutions of
the above folates were made 0.5 to 1 x 10-3 mol/L in
0.01 mol/L Tris buffer, pH 8.0. The UV spectra of 10-HCO-folic acid and
10-HCO-H2folic acid are shown in Figure 1
A and
B, respectively.
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in L/mol x cm), at pH 7.0:
folic acid, 282 = 2.7 x 104;
10-HCO-folic acid, 271 = 2.7 x 104;
10-HCO-H2folic acid, 234 = 3.4 x
104; 5-HCO-H4folic acid, 287 =
3.2 x 104; and at pH 1.0:
10-HCO-H4folic acid as 5,10-CH = H4folic
acid, 356 = 2.5 x 104 (Baggott et al. 1995
, Rabinowitz 1960
, Temple and Montgomery 1984
). The neutral pH UV spectrum of 10-HCO-folic
acid is shown in Rabinowitz (1960)
as having ( max)
270 = 2.1 x 104, but is reported
by Temple and Montgomery (1984)
as having ( inflection point)
263 = 2.6 x 104. Thus our molar
extinction coefficient for 10-HCO-folic acid at 271 nm agrees better
with that reported by the latter investigators. To ensure that no
10-HCO-H4folic acid remained in the preparation of
10-HCO-H2folic acid, the latter was over-oxidized,
which changed its UV spectra as shown in Figure 1C
. Cell culture experiments.
The human acute lymphoblastic leukemia cell line CCRF-CEM was
obtained from the American Type Culture Collection (Rockville, MD; ATCC
# CCL 119). Cells were routinely grown at 37°C in a humidified
atmosphere of 5% CO2 in RPMI medium 1640 (without
added folic acid) (Gibco BRL, Gaithersburg, MD), 20% dialyzed fetal
bovine serum (HyClone Laboratories, Logan, UT), and an antibiotic
mixture of 1.0 x 105 IU penicillin/L, 100 mg
streptomycin/L, and 0.25 mg fungizone/L (Irvine Scientific, Santa Ana,
CA). Folates were added in the concentrations indicated in Table 1
at the beginning of the cell culture experiments. The experiments were
conducted in 24-well plates seeded with 1 x 108
cells/L.
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5 cell count determinations was
used. The percentage viability was determined by trypan blue exclusion.
An average of 4 microscope fields was used to determine percentage
viability. Enzyme assays.
A previously described colorimetric assay (the change in
absorbance at 552 nm, 
A552) of AICAR Transformylase
(AICAR Tase) was performed by using initial concentrations of 5 x
10-4 mol AICAR/L, 5 x 10-4 mol of the
folate/L, 1 x 10-3 mol ascorbate/L, and 0.1 mol K
phosphate buffer/L (pH 7.0) at 23°C (Baggott et al. 1995
). Each assay contained 1.4 mg protein of CCRF-CEM
extract per mL. Blank assays contained all components except the
folate.
A spectrophotometric assay of dihydrofolate reductase was performed by
using concentrations of 5 x 10-4 mol NADPH/L, 5 x
10-4 mol of the folate/L, 1 x 10-3 mol
ascorbate/L, and 0.1 mol K phosphate/L (pH 7.0) at 23°C. Each assay
contained 0.8 mg protein of CCRF-CEM extract/mL. Aliquots of the
assay solution were diluted 1:10 into the phosphate-ascorbate
buffer before measuring the change in absorbance at 340 nm
(A340). Blank assays contained all components except the
folate.
Extracts of CCRF-CEM cells were made by freezing and thawing, three times, in the above phosphate buffer. The absorbance at 280 nm (A280) was used to measure CCRF-CEM protein concentration; an A280 = 1 was assumed to be 1 g protein/L.
Statistics.
Differences in mean fractional change in cell growth were detected by the t-test. Square root transformations of the data were used to normalize the data.
Differences in mean enzyme activity (A552 or
A340) at the final assay point (~280 min for
A340 and 24 h for A552) were
detected by ANOVA followed by Scheffe's test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At the 25 and 100 nmol/L concentrations of MTX, both of these
compounds showed a dose dependency in the stimulation of cell growth
above control levels and in percentage viability. These folates were
also capable of preventing net cell death, which occurred in the
controls with the highest concentration of MTX. In contrast, in the
vast majority of experiments using 10-HCO-folic acid, there was either
no cell growth stimulation above control levels or there was actual
cell growth inhibition. Means of fractional change in cell growth were
0.0 and 0.7 using 10-HCO-folic acid and
10-HCO-H2folic acid, respectively (P
= 0.001).
The substrate activity of these folates was assayed by using two folate
metabolizing enzymes. Dihydrofolate reductase, from CCRF-CEM cells,
readily utilized both 10-HCO-H2folic acid and
folic acid (a control), but had little or no activity with 10-HCO-folic
acid (Fig. 2
A). All three substrates had different activities (P < 0.05). AICAR Tase, from CCRF-CEM cells, was active with
10-HCO-H2folic acid and
10-HCO-H4folic acid (a control), but had little
or no activity with 10-HCO-folic acid (Fig. 2B)
. Both the tetra and
dihydro folates had higher activities than did 10-HCO-folic acid
(P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
latter fact indicates that their growth is dependent upon bioactive
folates. We elected to use MTX in the media to minimize the effect of
the folates in the seed cells and the folates which cannot be removed
from the culture medium. MTX and its polyglutamates are well
known inhibitors of many folate-utilizing enzymes, and therefore,
MTX treatment will stress cellular folate metabolism and suppress cell
growth. This antifolate-induced cell growth suppression can be
reversed by adding bioactive folates. A variety of cell culture times
and concentrations of the folate and MTX concentrations were used to
increase the chances of finding this reversal of antifolate-induced
cell growth suppression. To our knowledge, this is the first report of
the use of 10-HCO-H2folic acid and 10-HCO-folic
acid in mammalian cell culture.
The cell culture experiments used 5-HCO-H4folic
acid as a control and it reversed the effects of MTX. Our cell culture
experiments indicate that 10-HCO-H2folic acid
reverses the antifolate effects of MTX, but was less potent than
5-HCO-H4folic acid. The fact that the initial
preparation of 10-HCO-H2folic acid was
overoxidized and contained some 10-HCO-folic acid may partially account
for its reduced potency. Some oxidation of
10-HCO-H2folic acid to 10-HCO-folic acid must
also have occurred during the 4–6 d of cell culture (Baggott et al. 1995
). We found little or no evidence for 10-HCO-folic acid
being a bioactive folate by reversing the effect of
antifolate-induced cell growth suppression in this cell line.
Although our results apply strictly to human leukemia, it is likely
that 10-HCO-folic acid is metabolically inert for normal mammalian and
vertebrate cells. Our results are consistent with several other lines
of evidence, which are discussed below.
Human experiments indicate that 10-HCO-folate has relatively low
bioactivity. In an early report by Spies et al. (1948)
, 20 mg/d oral
doses of 10-HCO-folic acid for 10 d produced a much weaker
reticulocyte response than did 10 mg/d oral doses of folic acid for
10 d in a patient with nutritional macrocytic anemia and in a
patient with pernicious anemia. The peak reticulocyte response occurred
at 10–13 d after the initiation of 10-HCO-folic acid therapy versus
only 7–8 d after the initiation of folic acid therapy. In a 12-h
experiment in humans, 20–40% of a 5 mg oral dose of radiolabeled
10-HCO-folic acid was excreted, unmetabolized, in the urine. Only
~1% of this dose was identified in urine as 5-methyl-tetrahydrofolic
acid (5-CH3-H4folic acid)
(Saleh et al. 1982
). Ratanasthien et al. (1974)
reported
that 10-HCO-folic acid appeared in plasma, unmetabolized, up to 3 h after a 5 mg oral dose. In contrast, 25–30% of the total increase
in plasma folates was
5-CH3-H4folic acid within
3 h after an oral dose of 5 mg of folic acid. Thus, in this
short-term experiment, substantial metabolism of folic acid had
occurred, whereas 10-HCO-folic acid was not metabolized.
Our enzyme studies also suggest that 10-HCO-folic acid is metabolically
inert. CCRF-CEM AICAR Tase utilized both
10-HCO-H4folic acid and
10-HCO-H2folic acid but not 10-HCO-folic acid
which is in agreement with reports using chicken liver AICAR Tase and
mammalian AICAR Tase (Baggott et al. 1986 and 1995
).
CCRF-CEM dihydrofolate reductase utilized both folic acid and
10-HCO-H2folic acid, but not 10-HCO-folic acid.
This was expected because 10-HCO-folic acid is known to be an inhibitor
of (not a substrate for) mammalian dihydrofolate reductase
(Bertino et al. 1965
; Whitburn et al. 1983
). Indeed, 10-HCO-folic acid has very little activity with
chicken liver glycinamide ribotide transformylase (Smith et al. 1981
), and the reversible reaction catalyzed by bacterial
10-formyl-tetrahydrofolic acid synthetase does not utilize folic acid,
and thus 10-HCO-folic acid cannot be converted to folic acid by this
synthetase (Buttlaire 1980
). We are unaware of any
mammalian enzyme that effectively metabolizes 10-HCO-folic
acid.
10-HCO-H2folic acid was reported to be
inactive with mammalian dihydrofolate reductase (Bertino et al. 1965
). This previous report used dihydrofolic acid, which is
known to be much more active than folic acid for mammalian
dihydrofolate reductase (Coward et al. 1974
), as a
positive control. Also, relatively short incubation times
(i.e., 4 min) were used. Using folic acid as a positive control, and
the longer incubation times, substantially increased the sensitivity of
our dihydrofolate reductase assay.
There is no question that relatively long-term feeding of
10-HCO-folic acid does produce bioactive folates, which supports the
growth of chickens (Gregory et al. 1984
) and produces a
relatively weak hematological response in humans (Spies et al. 1948
). Also, ~15% of an oral dose of radiolabeled
10-HCO-folic acid is retained and metabolized to tetrahydro compounds
in rats in 24 h (Gregory et al. 1984
). In light of
our failure to demonstrate bioactivity of 10-HCO-folic acid in a
bacteria-free environment, a logical explanation for its
bioactivity in chickens and humans and metabolism in rats would be that
endogenous bacteria metabolized 10-HCO-folic acid to a form that is
bioactive for vertebrates. A logical metabolic pathway for bacteria
involves the dihydrofolate reductase catalyzed reduction of
10-HCO-folic acid to 10-HCO-H4folic acid.
Remarkably, bacterial dihydrofolate reductase (in dramatic contrast to
mammalian dihydrofolate reductase) is much more active with
10-HCO-folic acid than with folic acid (Dann et al. 1976
; McIntyre and Harding 1977
; Whitburn et al. 1983
). The 10-HCO-H4folic acid
produced by the bacteria would then be bioactive for the host. For
example, S. faecalis (now E. hirae) is a very
common bacteria of the human bowel and found in relatively high amounts
in feces (Noble 1978
). 10-HCO-folic acid supports the
growth of this bacteria (Johns and Bertino 1965
). The
metabolism of 10-HCO-folic acid in rats after 24 h (Gregory et al. 1984
) might be explained by the 5-h mouth-to-cecum
transit time in these animals (Perry et al. 1993
). Thus,
in 5 h, any unabsorbed 10-HCO-folic acid would be available to the
enteric bacteria of the rat.
In conclusion, 10-HCO-H2folic acid should be considered a bioactive folate for human leukemia cells because it substantially reversed MTX-induced growth suppression, and it served as a substrate for folate metabolizing enzymes. On the other hand, 10-HCO-folic acid had little or no effect in reversing MTX-induced growth suppression in this cell line. Its bioactivity is, therefore, quite low, and the metabolism (if any) of this folate by mammalian cells remains unclear. We propose that 10-HCO-folic acid be classified as a conditionally bioactive folate for vertebrates. Thus, 10-HCO-folic acid requires enteric bacteria to metabolize it to a bioactive form(s), which then can be utilized by the host.
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FOOTNOTES |
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Manuscript received December 1, 1998. Initial review completed December 29, 1998. Revision accepted March 23, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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