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Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL and * U.S. Department of Agriculture, Agricultural Research Service, Western Human Nutrition Research Center, University of California-Davis, Davis, CA
3To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • folate • catabolism • para-aminobenzoylglutamate • para-acetamidobenzoylglutamate • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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first reported the
excretion of free and acetylated diazotizable amines after
administration of folic acid or 5-formyltetrahydrofolate in rats.
Subsequent studies indicated that the catabolic process involved
cleavage of the folate C9-N10 bond, with the primary excretory products
identified as various pterins (Krumdieck et al. 1978
), and the folate-derived amines including
para-aminobenzoylglutamate (pABG) and its
N-acetyl derivative,
para-acetamidobenzoylglutamate (ApABG) (Barford et al. 1978
, Murphy et al. 1976
, Murphy and Scott 1979
). The urinary excretion of ApABG greatly exceeds
that of nonacetylated pABG (Caudill et al. 1998
,
Geoghegan et al. 1995
, McNulty et al. 1987
, McPartlin et al. 1992
). Acetylation
appears to be catalyzed by an arylamine N-transferase
isoform with specificity toward pABG (Estrada-Rodgers et al. 1998
, Minchin 1995
).
The relationship between folate nutritional status and the rate of pABG
and ApABG excretion has not been fully established, although excretion
of pABG and/or ApABG is not clearly indicative of folate nutritional
status. In addition, it is unclear which specific form(s) of folate and
body pools are most prone to catabolism. Urinary excretion of folate
catabolites greatly exceeds the excretion of intact folate in rats
(Wang et al.1994
) and humans (Caudill et al. 1998
, Gregory et al. 1998
). At higher intakes of
folate, urinary folate excretion increases to a proportionally greater
extent than does catabolite excretion. Supplementation with folic acid
has little effect on the short-term excretion of folate catabolites
(Kownacki-Brown et al. 1993
, McNulty et al. 1987
). In studies of chronic control of folate intake, the
excretion of folate catabolites is proportional to folate intake
(Caudill et al. 1998
, Gregory et al. 1998
). In a study in which nonpregnant women were chronically
given oral doses of deuterated folic acid, examination of the rate and
extent of labeling of ApABG provided evidence that folates from both
rapid- and slow-turnover pools are subject to catabolism
(Gregory et al. 1998
). For all intakes examined (200,
300 and 400 µg total folate/d), ApABG excretion greatly
exceeded the excretion of intact folate. The sum of pABG and ApABG,
along with concurrently formed pterins, appears to account for the
majority of folate catabolites. Geoghegan et al. (1995)
concluded that the excretion of ApABG is itself a suitable indicator of
folate catabolism. Folate catabolism also may be increased by
anticonvulsant drugs, alcohol and certain cancers (Kelly et al. 1979
and 1981
, Shaw et al. 1989
).
Measurement of total excretion of folate catabolites (i.e., ApABG +
pABG) has been reported in a number of studies. McPartlin et al. (1993)
examined catabolite excretion in free-living
pregnant women and nonpregnant controls. They reported that nonpregnant
controls, postpartum women and women in the first trimester of
pregnancy excreted total catabolites that were the equivalent of 
100
µg of folate/d, which corresponded to 227 nmol/d.
Greater excretion of catabolites was reported during the second and
third trimesters of pregnancy. Long-term dietary control was not
employed in that study. Caudill et al. (1998)
conducted
a similar study except that folate intake was precisely controlled for
12 wk in second trimester pregnant women and nonpregnant controls. In
this protocol, no difference was observed between pregnant and
nonpregnant women consuming the same level of folate (either 450 or 850
µg/d). However, the steady-state excretion of total
pABG + ApABG increased in proportion to folate intake. The study of
Caudill et al. (1998)
showed clearly that at least 4 wk
was required during these relatively high levels of controlled intake.
Little is known about the level of folate catabolism and time required
for catabolite excretion to stabilize during periods of low dietary
folate intake. In this paper, we report the measurement and
interpretation of pABG and ApABG excretion in a study with carefully
controlled dietary intake intended to provide depletion followed by
repletion with folate. This provided an ideal context for examination
of effects of a modest folate deficiency on catabolite excretion.
Detailed analysis of the influence of dietary intake on various
functional indices of folate status has been reported in a separate
publication (Jacob et al. 1998
).
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As described previously (Jacob et al. 1998
), healthy
nonsmoking postmenopausal volunteer women (n = 10),
ages 49–63 y, were admitted to the metabolic unit of the USDA Western
Human Nutrition Research Center (WHNRC) after medical and psychological
screening. Normal renal function was ensured by testing for serum
creatinine, blood urea nitrogen and urinalysis at screening and
biweekly throughout the study. Tests for plasma folate and vitamin
B-12, alcohol, tobacco and drug use were also performed. All subjects
were within 90–130% of desirable weight (Metropolitan Life Insurance
Company, New York, NY), except for one subject who was 145% of
desirable weight. Other criteria for subject selection included the
following: nonsmokers, nonusers of vitamin or other dietary supplements
containing folic acid, hemoglobin > 115 g/L and hematocrit
> 0.34. Three subjects, who were receiving estrogen replacement
therapy when they entered the study, continued the same regimen
throughout. The study protocol and informed consent were approved by
the Human Subjects Review Committee of the University of California,
Davis, and by the Human Studies Review Committee of the Agricultural
Research Service, U. S. Department of Agriculture. Signed informed
consent was obtained from each volunteer. For the duration of the 91-d
study, the subjects lived in and ate all meals in the WHNRC metabolic
unit and were chaperoned at all times when outside the unit.
Throughout the 91-d study, a low folate, low choline diet was fed,
which provided an average of 56 µg/d folate from food
sources (Jacob et al. 1999
). Synthetic folic acid
(pteroylglutamic acid) was added to the diet at various stages of the
protocol to allow precise control of folate intake from 56 to 516
µg/d. Total folate intakes were as follows: 195
µg/d (d 1–5), 56 µg/d (d 6–41), 111
µg/d (d 42–69), 286 µg/d (d 70–80)
and 516 µg/d (d 81–91).
Specimen collections and analytical methods.
Blood samples were obtained weekly, and complete urine collections were
obtained as successive 7-d pools for each subject. All samples were
stored at -70°C until analyzed. Urinary pABG and ApABG were measured
using the procedure of McPartlin et al. (1992)
with
modifications as described by Caudill et al. (1998)
.
Excretion per day of pABG and ApABG was calculated by multiplying the
concentration of each compound in a given pooled urine sample by the
mean daily urine volume. We analyzed four urine pools corresponding to
study d 1–7 (baseline), 36–42, 64–70 and 85–91. Urine collections
at d 36–42 and 64–70 represent the end of each depletion period (56
and 111 µg/d folate intake, respectively), whereas d
85–91 represent the end of the repletion period (11 d at 286
µg/d, then 11 d at 516 µg/d).
However, the folate intakes corresponding to each of the first three
urine pools are not exactly the same as the dietary folate intakes of
the preceding period because the 7-d urine pool periods overlapped the
succeeding dietary folate intake period by 2, 1 and 1 d,
respectively. Hence, the average folate intakes during
each of the first three 7-d urine pools were 155, 58, and 117
µg/d, rather than 195, 56, and 111
µg/d. The average folate intake during the last 7-d
urine pool is the weighted average of 10 d at 286
µg/d and 11 d at 516 µg/d, a
weighted average of 406 µg/d.
Additional measurements reported here were made using methods described
in the previous paper regarding this study (Jacob et al. 1998
), including the following. Plasma homocysteine was
measured by fluorometric HPLC (Araki and Sako 1987
). The
methyl acceptance capacity of lymphocyte DNA was measured by a
modification of the method of Balaghi and Wagner (1993)
,
in which the extent of SssI CpG methylase-catalyzed transfer of
[3H]methyl groups from [methyl-3H]
S-adenosylmethionine is reflective of the relative state of
hypomethylation of available sites. Plasma folate was measured by
competitive protein binding radioassay (Quantaphase II B-12/Folate
Radioassay, BioRad, Hercules, CA). Total folate content of the diet was
determined by microbiological assay as previously described
(Jacob et al. 1998
).
Statistical analysis.
The effects of folate intake on the excretion of each catabolite in the
respective dietary treatment periods were determined by one-way
repeated-measures ANOVA (Glantz 1992
). Further analysis
of data, including molar ratios of ApABG/(pABG + ApABG) and (pABG
+ ApABG)/total folate intake, was also conducted by one-way
repeated-measures ANOVA. Differences were considered significant at
P < 0.05. When significance was observed, multiple
comparisons were conducted using the Student-Newman-Keuls method.
Statistical analyses were conducted using SigmaStat for Windows version
1.0 (Jandel Corporation, San Rafael, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Excretions of pABG and ApABG did not change significantly during the
experiment. Summarizing observations for all samples collected
throughout this protocol, the subjects excreted 308 ± 27.5 nmol
ApABG/d and 25.3 ± 2.48 nmol pABG/d (means ± SEM, n = 40 total observations). Total
folate catabolite excretion (i.e., pABG + ApABG) was 333 ± 28.4 nmol/d (mean ± SEM, n = 40 total
observations), which corresponds to daily elimination of 147 ± 12.5 µg folic acid equivalents through catabolism
throughout this study. The excretion of ApABG constituted 89.9 ± 1.50% of total pABG + ApABG excretion (mean ± SEM,
n = 40 total observations), and this value did not
change significantly throughout the various dietary periods of the
study.
|
. This molar ratio rose from 1.05 ± 0.17
during the initial dietary period (155 µg/d folate intake)
to 3.0 ± 0.55 during the 58 µg/d depletion period
(means ± SEM, P < 0.0001). In other
words, folate catabolite excretion exceeded folate intake by a factor
of 3 during wk 5 of 58 µg/d folate intake. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reported data from this study
indicating that plasma homocysteine and methylation status of
lymphocyte DNA underwent significant changes during the course of the
dietary manipulations of this protocol. These findings contrast with
the observations reported here, indicating that folate catabolism is
only weakly influenced by short-term changes of folate intake.
Thus, we infer that folate pools that serve as precursors of folate
catabolites differ from those directly involved in cellular acquisition
and generation of one-carbon units, as 5-methyl-tetrahydrofolate,
destined for homocysteine remethylation and formation of
S-adenosylmethionine for cellular methylation reactions. The
results of this study are consistent with the findings of
Caudill et al. (1998)
who reported that the excretion of
folate catabolites increased slowly after the initiation of folate
supplementation in pregnant women and nonpregnant controls.
The calculation of the molar ratio of catabolites and folate intake is
not meant to imply that there is a direct temporal linkage between
recently ingested folate and excreted catabolites. By contrast, the
molar ratio of 3 after 5 wk of 58 µg/d folate intake
demonstrates clearly that it was the tissue folate derived from
previous intake that yielded the excreted catabolites. This is
consistent with the observation of Kownacki-Brown et al. (1993)
, who found that only a small percentage of a bolus oral
dose of deuterated folate appeared in short-term collections of
urine. Under the conditions of this protocol, it is likely that the
overall turnover of body folate would occur very slowly, probably with
an overall mean residence time for total body folate of 200 d as
reported by Gregory et al. (1998)
. This is consistent
with the lack of significant change in red cell folate, which is known
to turn over much more slowly than the 5-methyl-tetrahydrofolate pool.
The relatively rapid responsiveness of plasma homocysteine, and
lymphocyte DNA methylation status reported by Jacob et al. (1998)
suggests that 5-methyl-tetrahydrofolates responsible for
these functional changes of one-carbon metabolism are in a much
greater state of flux than the bulk of the body folate pools, which
turn over much more slowly. Several abundant enzymes are known to bind
folate very tightly, which may account for a portion of the large,
relatively refractory component of body folate. Further work is
required to identify such pools and their in vivo behavior. An
additional finding of interest is the fact that the proportion of total
pABG that undergoes acetylation of the aryl amino group is relatively
constant and not influenced by the level of folate intake. It should be
noted that the diet used in this study was low in choline; thus, it is
likely that low choline intake accentuated the methyl group deficiency
caused by folate deficiency (Jacob et al. 1999
). One
should also recognize that not all aspects of methyl group metabolism
are impaired during folate deficiency. In a previous study of
short-term folate depletion coupled with low methionine and choline
intakes in men, plasma homocysteine concentration increased, whereas
methylation of a test dose of nicotinamide (as an indicator of
methylation capacity) was unchanged (Jacob et al. 1995
).
In summary, the findings reported here illustrate clear kinetic differences between folate pools functioning directly in methyl group metabolism and those from which folate catabolites are primarily generated. From the point of view of the design of nutrition intervention studies regarding folate, this study adds to the growing body of evidence that most studies of practical duration do not achieve a steady state of folate status or metabolism. Hence, such intervention studies must be interpreted cautiously regarding effects of relatively short-term dietary treatments. However, these observations also indicate that clear changes in plasma folate concentration and aspects of methyl group metabolism occur well before complete attainment of steady state of all tissue folate pools.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Reference to a company or product name does not
imply approval or recommendation of the product by the U.S. Department
of Agriculture to the exclusion of others that may be suitable.
Manuscript received May 15, 2000. Revision accepted August 31, 2000.
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