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Human Nutrition and Food Science Department, Cal Poly Pomona University, Pomona, CA 91768 and * Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370
2To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate • para-aminobenzoylglutamate • catabolism • stable isotope • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–6
) and involves cleavage of the C9-N10 bond producing a pterin and para-aminobenzoylglutamate (pABG)3
. The majority of pABG (
85%) is N-acetylated in the cytosol by arylamine N-acetyltransferases to produce para-acetamidobenzoylglutamate (apABG) (7
,8
) and is excreted in the urine (1
,2
,5
,9
–11
). It was proposed that nonacetylated or free urinary pABG was an artifact of folate breakdown either in circulation, in the bladder or in the collection container (5
,6
). However, new data suggest that pABG is derived from intracellular catabolism, not oxidative degradation of urinary folate (12
). Furthermore, free pABG appears to arise largely from different tissue folate pools than apABG (12
). It is likely that folate catabolism occurs in part through intracellular nonenzymatic oxidative degradation of labile tetrahydrofolates (13
,14
). More recent data suggest that folate catabolism also occurs through a regulated process that is linked to iron metabolism (15
). The results of in vitro and in vivo experiments suggest that the iron storage protein, ferritin, catalyzes folate turnover and may be an important factor in regulating intracellular folate concentrations (15
,16
).
Urinary excretion of folate catabolites is affected by folate intake (10
), but often to a smaller extent than urinary intact folate excretion and other measurements of folate status (10
,11
,17
–19
). Kinetic analyses of folate metabolism suggest that the rate of folate catabolite excretion is related mainly to masses of slow-turnover folate pools governed by long-term folate intake (11
). Because folate catabolism represents an irreversible loss of folate, the urinary excretion of folate catabolites has been proposed to be a minimal indicator of folate requirements (9
,20
).
One factor limiting the accuracy of the estimation of folate requirements based on catabolite excretion is the variable and potentially large fecal excretion of folates from endogenous pools, as discussed in a recent review (21
). Another uncertainty is the fact that the excretion of folate catabolites can not be adequately interpreted without considering the potential absorption and fate of pABG formed through the breakdown of dietary folate. Food processing and storage inevitably leads to some loss of folates, at least some of which occurs through cleavage of the C9-N10 bond (22
). It also has been reported that folate degradation, probably through C9-N10 bond cleavage, also can occur under the conditions of the gastrointestinal tract before absorption (23
). The metabolic fate and physiologic processing of such dietary pABG and pABG that may arise in the gastrointestinal tract have not been determined. At present, several unanswered questions exist regarding the fate of exogenous pABG including the efficiency of its absorption, the extent to which it undergoes postabsorptive acetylation, whether it may be used in folate synthesis by intestinal microorganisms and the rate at which absorbed pABG and derived apABG are excreted in urine. Until the in vivo fate of dietary pABG is understood, it is not possible to interpret fully the data regarding urinary catabolite excretion and its relation to human folate nutriture.
The study reported here was conducted to evaluate the extent to which orally ingested pABG contributes to the urinary excretion of pABG and apABG. An oral dose of 376 nmol (100 µg) [13C5]pABG was ingested by pregnant women and nonpregnant controls consuming controlled folate intakes of 450 or 850 µg/d. Detailed analyses of the influence of pregnancy as well as two different levels of folate intake, 450 and 850 µg/d, on various folate indices including catabolite excretion and kinetics have been reported in separate publications (10
,12
,19
,24
).
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As described previously (19
), healthy, nonsmoking pregnant women (initially 14 wk gestation; n = 12) and nonpregnant controls (n = 12) ages 18–45 y participated in an 84-d controlled folate feeding study. All participants had normal blood chemistry profiles, folate concentrations > 6.8 and 320 nmol/L for serum and red cell folate, respectively, no history of chronic disease or other medical problems as determined by medical histories and did not habitually consume drugs (including oral contraceptives, folate antagonists and alcohol). Approval of the study protocol by the Institutional Review Board of the University of Florida and signed informed consents by participants were obtained.
The subjects were randomly assigned to consume a combination of dietary folate (120 µg) and synthetic folic acid (330 or 730 µg) for total folate intakes of 450 or 850 µg/d or 681 and 1191 µg dietary folate equivalents (DFE), where DFE = µg dietary folate + (1.7 x µg synthetic folic acid). Approximately 15% of the folate intake consisted of [3',5'-2H2]folic acid during d 1–41 of the study, whereas the administered folic acid was unlabeled during d 42–84. On d 42, 4 subjects (2 nonpregnant and 2 pregnant) consumed an oral dose of [13C5]pABG (376 nmol or 100 µg) dissolved in 40 mL apple juice. Four consecutive 24-h urine collections were obtained immediately after consumption of the [13C5]pABG dose. Urine was collected into 2-L opaque brown plastic bottles containing 3 g sodium ascorbate and stored refrigerated during collection periods. For each subject, the urine was pooled, measured, portioned (200 mL) and transferred to plastic containers for storage at -20°C.
Synthetic folates.
[13C5]folic acid was synthesized by coupling nonlabeled pteroic acid with L-[13C5]glutamic acid according to the method described by Pfeiffer and Gregory (25
). Once synthesized, [13C5]folic acid was subjected to chemical cleavage at the C9-N10 bond and the sample containing both the pteridine and pABG fragments was applied to a size exclusion gel (Bio-Gel P2, BioRad Laboratory, Hercules CA). The fragments were eluted with water, and small fractions (1–2 mL) were collected and analyzed by HPLC to identify fractions containing pABG. A pABG standard made from cleaved folic acid (Sigma Chemical, St. Louis, MO) was used to identify the retention time of pABG. The pABG fractions were pooled and the concentration of the solution was determined by HPLC relative to a pABG standard curve. Nonlabeled folic acid used for the preparation of folate supplements was obtained commercially (Sigma Chemical), and [3',5'-2H2]folic acid used as a stable isotopically labeled tracer was synthesized as described previously (26
). Each was analyzed before use by HPLC, proton nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (GC-MS) to verify purity and identity (26
). The concentration of stock solutions of labeled and unlabeled folic acid was determined spectrophotometrically using published molar absorptivities (27
).
Analytical methods.
Four consecutive 24-h pooled urine collections (0–96 h) for each subject (n = 4) were analyzed for [13C5]pABG by GC-MS (18
). Preparation for GC-MS included cleavage of the 9C-N10 bond (intact folate molecule only), deacetylation of apABG, isolation of the pABG by HPLC and derivatization with combined trifluoroacetic anhydride and trifluoroethanol (28
) to form N-trifluoroacetyl-p-aminobenzoylglutamate lactam 
-trifluoroethyl ester. GC-MS analysis of derivatized pABG (derived from urinary folate, pABG or apABG) was performed in electron-capture negative ionization mode with selected-ion monitoring at mass-to-charge ratios (m/z) 426 and 431 (28
). This analysis permitted unambiguous and specific measurement of the 2H2 and 13C5 forms of this pABG derivative. The isotopic enrichment of each analyte (i.e., molar ratio of labeled and nonlabeled species corrected for natural abundance of stable isotopes) was calculated by solving simultaneous equations in a procedure that corrected for natural abundance (29
). Isotopic enrichment values are expressed as mole percent excess (mol % excess) above the natural abundance of each isotopomer. The absolute excretion of [13C5]pABG and [13C5]apABG was calculated from values for total excretion of pABG and apABG reported previously (10
) and their isotopic enrichments as described herein.
Statistical analysis.
No statistical analyses were conducted because of the small subject number.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Of the labeled pABG excreted, the majority (86%) was excreted as [13C5]apABG and appeared within the first 48 h. No [13C5]folate was detected in the urine at any time point. Nonpregnant women excreted at least three times more of the administered dose than pregnant women (Table 2
). Mean urinary excretion of [13C5]total was 27.6 and 7.4 nmol for nonpregnant and pregnant women, respectively, which corresponded to 7.3 and 2.0% of the tracer dose. For women consuming 450 and 850 µg/d total folate, mean urinary excretion of [13C5]total was 16.0 and 19.0 nmol, respectively, which corresponded to 4.3 and 5.0% of the tracer dose.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Moreover, folate degradation within the gastrointestinal tract during digestion also appears to be likely (23
). The present study is the first to examine the contribution of an oral dose of pABG to urinary pABG and apABG excretion and the influence of pregnancy and folate intake on this process. Of the 376 nmol of [13C5]pABG administered, a mean of 17.5 nmol (range: 4.7–33.2 nmol) was detected in the urine over the 4-d collection period. This represents 4.6% of the tracer dose and demonstrates that the contributions of dietary pABG and of pABG formed from any preabsorptive cleavage of folate (23
) to urinary pABG and apABG excretion are small.
The low recovery of labeled pABG as urinary pABG and apABG was unexpected. Studies conducted in rodents have reported rapid excretion (65% in urine/48 h) of free and acetamido-pABG after administration of an oral dose of labeled pABG (30
). In humans, it is possible that pABG, an unusual dipeptide, is not absorbed. More likely, however, it undergoes hydrolysis by peptidases and/or proteases in the gastrointestinal tract producing glutamate and para-aminobenzoic acid (pABA) which are subsequently absorbed. The ability of the intestine to metabolize pABG to pABA during the absorption process has been demonstrated in rodents (30
). After absorption, pABA would be rapidly excreted in urine as free and acetylated pABA as demonstrated by Drucker and colleagues in humans (31
). Alternatively, pABG may be absorbed intact, deglutamylated in vivo and excreted in urine as free and acetylated pABA. The present study was unable to provide definitive data in this regard because the glutamic acid portion of pABG was labeled, not the pABA portion, and no attempt was made to measure urinary pABA. It is also possible that pABG may have been absorbed and retained by the body; this is unlikely, however, because the majority of the tracer (84%) that was detected in urine was eliminated between 25 and 48 h and < 0.05% was eliminated between 73 to 96 h. The use of a tracer with 13C labeling on the benzoyl moiety would help resolve these issues, especially if attempts were made also to determine fecal excretion of 13C.
Of the proportion of the tracer dose excreted, 86% was acetylated. This strongly suggests that the absorbed pABG was rapidly and extensively acetylated by various tissues in which arylamine N-acetyltransferases exists. As indicated by the absence of urinary [13C5]folate, there was no conversion of [13C5]pABG to [13C5]folate. This is consistent with the observation that pABG is a very poor substrate in bacterial folate synthesis (32
).
Upon examining the influence of pregnancy on urinary excretion of [13C5]pABG, [13C5]apABG and [13C5]total, it was noted that nonpregnant women excreted 2 times more [13C5]apABG than pregnant women and 4 times more [13C5]total. The practical importance of this finding is questionable, however, because the urinary excretion of total labeled pABG and apABG was minimal in both pregnant and nonpregnant women. No consistent effect of folate intake on urinary [13C5]pABG excretion derived from oral [13C5]pABG was observed, which suggests that exogenous folate/folic acid does not affect the absorption or metabolic handling of dietary pABG.
In summary, these findings demonstrate that dietary pABG contributes minimally to urinary pABG and apABG excretion. Thus, studies using measurement of folate catabolite excretion to assess folate status, turnover and/or minimal requirements can assume that, in humans, the majority of urinary catabolite excretion is derived from an endogenous source.
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FOOTNOTES |
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3 Abbreviations used: apABG, para-acetamidobenzoylglutamate (N-acetyl-para-aminobenzoylglutamate); DFE, dietary folate equivalents; GC-MS, gas chromatography-mass spectrometry; pABA, para-aminobenzoic acid, pABG, para-aminobenzoylglutamate.
Manuscript received 9 May 2002. Initial review completed 5 June 2002. Revision accepted 13 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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