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Kinetics of Folate Turnover in Pregnant Women (Second Trimester) and Nonpregnant Controls during Folic Acid Supplementation: Stable-Isotopic Labeling of Plasma Folate, Urinary Folate and Folate Catabolites Shows Subtle Effects of Pregnancy on Turnover of Folate Pools¹

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.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
To investigate the effects of pregnancy on folate metabolism, we conducted an 84-d study in second-trimester (gestational wk 14–25) pregnant women (n = 6) and nonpregnant controls (n = 6) with stable-isotopic tracer methods. All subjects were fed a diet containing 272 nmol/d (120 µg/d) folate from food, along with supplemental folic acid that contained 15% [3',5'-²H₂] folic acid ([²H₂]folic acid) during d 1–41 and that was unlabeled during d 42–84 to yield a constant total folate intake of 1.02 or 1.93 µmol/d (450 or 850 µg/d). Isotopic enrichment of plasma folate, urinary folate and the urinary folate catabolites para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) was determined at intervals throughout the study. The labeling of pABG and ApABG reflected that of tissue folate pools from which the catabolites originate. After the intake of labeled folic acid was terminated on d 41, labeling of urinary folate exhibited a biphasic exponential decline with distinct fast and slow components. In contrast, during d 42–84, the enrichment of urinary pABG and ApABG exhibited primarily monophasic exponential decline, and plasma folate underwent little decline of labeling during this period. Pregnant women and controls did not differ in estimates of body folate pool size and most aspects of the excretion of labeled urinary folate and catabolites, rates of decline of excretion, and areas under the curves for folate and catabolite excretion. Pregnant women, however, tended to have a slower rate of decline of pABG than ApABG and higher enrichment at d 42 of ApABG and pABG. These data support and extend our previous findings indicating that pregnancy (gestational wk 14–26) causes subtle changes in folate metabolism but does not elicit substantial increases in the rate or extent of folate turnover at these moderately high folate intakes.

KEY WORDS: • folate • kinetics • catabolism • stable isotopes • humans • pregnancy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The maintenance of adequate folate status during pregnancy requires a folate intake sufficient to support an adequate concentration and mass of maternal and fetal folate pools to allow proper folate-dependent metabolic processes to occur. Folate intake must at least balance the accretion of maternal and fetal folate pools and the overall elimination of folates and folate catabolites. The kinetics of folate metabolism in humans have been examined in many short-term and several long-term studies (1 , 2) ; however, little information exists regarding the influence of pregnancy on the rate and manner of folate turnover.

Although much recent investigation has been directed toward clarifying the relationships between periconceptional folate intakes and the risk of a neural tube defect–affected pregnancy (3) , it also is known that the folate intake required to maintain adequate nutritional status increases during pregnancy (4 , 5) . Maintaining adequate folate status throughout pregnancy is important to minimize the risk of adverse outcomes including habitual abortion, preeclampsia, placental abruption, and low birth weight and reduced duration of gestation, as reviewed by Scholl and Johnson (6) . Many questions remain unanswered regarding optimal intake and the physiologic or metabolic basis for such increased requirements (5) . The incidence of folate deficiency in pregnancy has been reported to increase toward the end of gestation (7) . Various researchers have proposed that increased folate requirements during pregnancy are caused by increases in maternal and fetal tissue mass and blood volume, increased metabolic demands for folate coenzymes to support growth and cell division, potentially increased urinary excretion of folate and possibly increased breakdown or catabolism of folates (4 ,5) .

Whether or to what extent folate catabolism increases during pregnancy has not been resolved. The catabolism of folates occurs by cleavage of the C9-N10 bond to yield one or more pterins and para-aminobenzoylglutamate (pABG)³ (8 ,9) . The extent of folate catabolism is most directly assessed by measurement of urinary excretion of pABG and its acetylated derivative, para-acetamidobenzoylglutamate (ApABG) (10) ; however, the mechanism is unclear. The possibility of increased folate catabolism during the second and third trimesters of human pregnancy was first reported in 1993 (11) and in a subsequent study conducted by the same research group (12) , but neither involved long-term control of folate intake. Caudill et al. (13) conducted a study of controlled folate intake in second-trimester women (wk 14–25) and nonpregnant controls consuming 450 or 850 µg/d total folate and found no increase in pABG or ApABG excretion by pregnant women at either intake. In addition to long-term control of folate intake, another difference between these studies is the period of pregnancy investigated. Caudill et al. (13) measured catabolite excretion from gestational wk 14 through wk 25. In contrast, Higgins et al. (12) examined excretion in nonpregnant controls and during gestational wk 12–16, 26–30 and 34. Although they did not examine the time period studied by Caudill et al., they did observe significantly increased catabolite excretion at wk 26–30 (relative to nonpregnant controls and pregnant women in wk 12–16) and still greater catabolite excretion after wk 34. The excretion of both catabolic products (pABG and ApABG) is related to long-term folate intake (13 14 15 16 17) , although to a lesser extent than several other measures of folate nutriture (17) .

A better understanding of the overall magnitude of folate turnover and the kinetics of its various components will aid in defining more precisely the nutritional requirement for this vitamin. We have shown that the long-term kinetics of folate metabolism can be examined in humans using protocols involving chronic administration of stable isotopically labeled folic acid (14 ,18) . In our 12-wk study of controlled folate intake in pregnant women and controls discussed above and reported previously (13 ,19) , all subjects received [3',5'-²H₂] folic acid ([²H₂]folic acid) chronically for the initial 6 wk followed by replacement of the tracer with nonlabeled folic acid to permit a direct examination of possible pregnancy-related differences in the in vivo kinetics of folate metabolism. We report here an evaluation of the kinetics of folate turnover in these second-trimester pregnant women and nonpregnant controls based on analysis of isotopic labeling of plasma folate, urinary folate and urinary folate catabolites.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects and protocol

This study was conducted with a 2 x 2 factorial design in which pregnant women (n = 12) and nonpregnant female control subjects (n = 12) were randomly assigned to consume folate at either 450 µg/d (1020 nmol/d; n = 6) or 850 µg/d (1926 nmol/d; n = 6) for 84 d (19) . Because of financial constraints, the kinetic analysis reported here was conducted on only half of the subjects (selected randomly) within each treatment group; i.e., nonpregnant control, 450 µg/d (n = 3), 850 µg/d (n = 3); pregnant, 450 µg/d (n = 3), 850 µg/d (n = 3).

The subjects consumed folate as a combination of dietary folate from a controlled, low folate diet and synthetic folic acid dissolved in apple juice taken with meals. This diet provided 120 ± 15 µg total folate/d as determined by analysis of composite diet samples (19) . Of the folate intake, 15% consisted of [²H₂]folic acid during d 1–41 of the study; the folic acid administered was unlabeled (¹H) during d 42–84. The total folate intake in the 450 and 850 µg/d groups corresponded to 681 and 1191 µg dietary folate equivalents (DFE), respectively, where DFE = µg dietary folate + 1.7 x µg synthetic folic acid (4) . Blood and urine were collected at baseline and thereafter on a weekly basis with more frequent sampling immediately after the switch to nonlabeled folic acid at d 42 (13 ,19) .

The pregnant subjects (18–35 y, initially 14 wk gestation) and nonpregnant controls (18–35 y) had normal blood chemistry profiles, normal blood folate concentrations and normal health status. Exclusion criteria included chronic drug (including oral contraceptives and folate antagonists), alcohol or tobacco use. The majority of pregnant subjects (n = 10) were consuming prenatal vitamins containing folic acid (0.4–1.0 mg) before starting the study. Gestational age in pregnant subjects was determined by sonogram in conjunction with calculation from d 1 of the last menstrual period. The study was approved by the Institutional Review Board of the University of Florida, and signed informed consent was obtained from each subject. Compliance with the study protocol was promoted by daily contact with the research team who observed consumption of the folic acid supplements. Nonpregnant women maintained their body weight within 5% of baseline throughout the study and pregnant women gained 0.45 kg/wk (18) .

Urine was collected into 2-L opaque brown plastic bottles containing 3 g sodium ascorbate and stored refrigerated during the collection periods. After pooling all urine collected by a subject during a 24-h collection and measuring the volume, 200-mL portions of the urine were stored in plastic containers at -20°C until analyzed. Samples of peripheral venous blood were collected into EDTA Vacutainer tubes in the morning of designated days after an overnight fast before consumption of breakfast. Portions of plasma also were stored at -20°C until analyzed.

Synthetic folates

Nonlabeled folic acid used for the preparation of folate supplements and analytical standards was obtained commercially (Sigma Chemical, St. Louis, MO); [²H₂]folic acid used as a stable isotopically labeled tracer was synthesized in this laboratory (20) . Each was analyzed before use by HPLC, proton nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (GC-MS) to verify purity and identity (20) . The concentration of stock solutions of labeled and unlabeled folic acid was determined spectrophotometrically using published molar absorptivities (21) .

Analytical methods

    Assessment of folate nutritional status. As described previously, serum, erythrocyte and urinary folate concentrations were determined by microbiological assay with Lactobacillus casei (22) .

    Isolation and preparation of urinary and plasma folate, pABG and ApABG for GC-MS determination of isotopic enrichment. Urinary folate concentration was determined by HPLC after affinity chromatography (23) . In this analysis, a 5-mL fraction containing urinary folate was collected from the folate-binding protein affinity chromatography column. A portion of the eluate (1 mL) was used for HPLC determination of folate (18 ,23) , and the remainder was prepared for GC-MS determination of isotopic enrichment (24) . Preparation for GC-MS analysis involves cleavage of the 9C-10N bond, isolation of the resulting pABG by HPLC and derivatization with combined trifluoroacetic anhydride and trifluoroethanol (23) to form N-trifluoroacetyl-p-aminobenzoylglutamate lactam -trifluoroethyl ester.

A modification of the procedure of McPartlin et al. (10) was used for the quantitative analysis of urinary pABG and ApABG, as reported previously (13) . An adaptation of this procedure was employed for the purification and isolation of urinary pABG and ApABG in preparation for determination of their isotopic labeling by GC-MS as the N-trifluoroacetyl-p-aminobenzoylglutamate lactam -trifluoroethyl ester derivative (14) .

Plasma samples (1–2 mL) were deproteinated by incubating in a boiling water bath, centrifuged at 10,000 x g for 20 min at 2°C and subjected to cleavage treatment exactly as done with affinity-purified urinary folate to induce conversion of plasma folate to pABG. The resulting pABG was purified by HPLC, then derivatized as described above to allow measurement of the isotopic labeling of plasma folate by GC-MS (23) .

    Gas chromatography-mass spectrometry analysis. GC-MS analysis of derivatized pABG (derived from urinary and plasma folate, pABG and ApABG) was performed in electron-capture negative ionization mode with selected-ion monitoring at mass-to-charge ratios (m/z) 426 and 428 (23) . 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 essentially as described by Storch et al. (25) . Isotopic enrichment values are expressed as mole percent excess (mol % excess) above the natural abundance of each isotopomer.

Kinetic analysis

All kinetic analysis was conducted by nonlinear regression using the numerical module of SAAM II, Simulation, Analysis, and Modeling Software, version 1.1 (SAAM Institute, University of Washington, Seattle, WA; 26 ). This analysis was conducted for isotopic enrichment (²H₂) of plasma and urinary folate, pABG and ApABG vs. time after withdrawal of [²H₂]folate (d 42–84) for each subject and then repeated for urinary excretion (nmol/d) of labeled urinary folate, pABG, ApABG and total urinary excretory products ([²H₂]folate, [²H₂]pABG, and [²H₂]ApABG) using nonlinear regression to fit the following equations:

For each data set analyzed (data for d 42–84 for each subject), the regression constants for each variable were manually altered in systematic fashion until reasonable fits to the data were obtained. In these calculations, we designated model parameters as A1, A2, a1 and a2 for urinary folate, B1 and b1 for urinary pABG; C1 and c1 for urinary ApABG, and D1 and d1 for plasma folate. The iterative "fit" function of SAAM II was then used to solve the equations and adjust these values until the "objective function" was minimized to indicate best fit.

Of particular interest in this analysis of the withdrawal phase (d 42–84) were the following: 1) initial isotopic enrichment (i.e., d 42) of plasma folate and urinary folate, pABG and ApABG; 2) rates of change in isotopic enrichment of plasma and urinary folate and urinary pABG and ApABG, and rates of change of urinary excretion of [²H₂]folate, [²H₂]pABG and [²H₂]ApABG; 3) the kinetic nature of the decay pattern of enrichment, whether mono- or biexponential; 4) relative rates of change of fast (if observed) and slow turnover processes of folate pools from which urinary [²H₂]folate, [²H₂]pABG and [²H₂]ApABG were derived; and 5) the area under the curve (AUC) and the fractional catabolic rate (FCR) for each data set. These values were calculated from the results of the nonlinear regression analysis using the following equations (27) :

The mean residence time (MRT) for body folates in pools from which urinary folate, pABG and ApABG were derived was calculated as MRT = 1/FCR.

We also estimated the total body folate pool size using the principle that, under conditions approximating steady state, folate input rate = folate turnover rate = total body pool size · system fractional catabolic rate [FCR(system)]. Assumptions necessary in this calculation were as follows: 1) 50% bioavailability of food folate and 85% bioavailability of folic acid consumed (4) ; 2) the first-order rate constant for the slow turnover of major tissue folate pools, as determined by measuring the rate constants for [²H₂]ApABG enrichment and excretion, approximates the FCR(system). For each subject, pool size was calculated as µmol total folate and as µmol total folate/kg body weight.

Statistical analysis

Differences among treatment groups for each kinetic term were evaluated by two-way ANOVA, with multiple comparisons by the Student-Newman-Keuls procedure, using SigmaStat Version 1.0 software (Jandel, San Rafael, CA; 28 ). In this analysis, the folate intake (450 or 850 µg/d) and pregnancy status (i.e., pregnant or control) served as the variables tested. Differences with P < 0.05 were considered significant.

	RESULTS

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Folate nutritional status, excretion of folate and pABG and ApABG

As reported previously, pregnant subjects and nonpregnant controls maintained normal folate status while consuming either 450 or 850 µg/d (Table 1 ; 19 ). Urinary folate consisted entirely of 5-methyl-tetrahydrofolate in all subjects except nonpregnant controls who consumed 850 µg folate/d. These findings indicate that essentially all of the ingested folic acid underwent reduction and incorporation into the reactions of one-carbon metabolism in pregnant subjects at each folate intake and in controls at the 450-µg/d level of folate intake. In contrast, the 850-µg/d nonpregnant control group excreted unchanged folic acid that constituted 19% of total urinary folate (19) .

    Isotopic enrichment of urinary folate, pABG and ApABG. The isotopic enrichment of urinary folate, pABG and ApABG increased through d 1–41, then declined through d 42–84. In contrast, the enrichment of plasma folate exhibited an initial small rapid rise, followed by a slow increase through d 1–41 with only a slight decline after cessation of tracer administration (d 42–84). To illustrate the overall time course of changes in enrichment, the mean values for the enrichment of urinary folate, pABG and ApABG over the entire 84-d study for pregnant and nonpregnant subjects at each folate intake are presented in Figure 1 . To illustrate specific relationships among treatments and to linearize the curves for variables that exhibit an exponential decline over time after tracer withdrawal, group means for d 42–84 (i.e., after tracer withdrawal) for each treatment group are presented on a logarithmic scale in Figure 2 . At the 450-µg/d intake, there was no difference between pregnant and nonpregnant subjects in overall shape of labeling disappearance curves. Nonpregnant subjects at the 850-µg/d folate intake reached a noticeably higher enrichment of total urinary folate than did pregnant subjects at this intake level at the time of tracer withdrawal (Fig. 2) .

View larger version (24K): [in this window] [in a new window]   Figure 1. Isotopic enrichment of urinary folate, para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) by nonpregnant and pregnant women consuming two levels of folate (upper panel, 450 µg/d; lower panel, 850 µg/d) during administration of [2H2]folic acid (d 1–41) and after its withdrawal and replacement with nonlabeled folic acid (d 42–84). Values are presented as means ± SEM of both pregnant and nonpregnant women for each level of total folate intake (450 and 850 µg/d; combined n = 6) to show general relationships in labeling patterns over the entire protocol.

View larger version (38K): [in this window] [in a new window]   Figure 2. Semilogarithmic plots of the isotopic enrichment (mole percent excess) of urinary folate, para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) in nonpregnant and pregnant women consuming two levels of folate (upper panels, 450 µg/d; lower panels, 850 µg/d) after withdrawal of [2H2]folic acid that had been administered for the previous 6 wk (d 1–41). Values are means for pregnant and nonpregnant subjects at each level of folate intake (n = 3/group). The lines are model predictions based on these mean values for illustration of overall relationships.

The kinetic values calculated from rates of change in isotopic enrichment of urinary folate, pABG and ApABG over d 42–84 are presented in Table 2 . The initial fast decline in enrichment of urinary folate occurred with a mean rate constant (a1) of 0.58 d^-1. This corresponded to a MRT of this rapid-turnover in vivo folate pool of origin of 1.7 d, for which there was no significant effect of pregnancy or folate intake. The long slow decline of urinary folate enrichment progressed from d 45 to 84 with a mean rate constant (a2) of 0.046 d^-1, with MRT of 21.7 d. Although there was no significant effect of either folate intake or pregnancy due to large variation among individuals, the mean slow-turnover rate constants (a2) for pregnant subjects (0.081 and 0.064 d^-1 for 450 and 850 µg/d intakes, respectively) appeared to exceed those for nonpregnant subjects (0.025 and 0.016 d^-1 for 450 and 850 µg/d intakes) (P = 0.242). In addition, the initial enrichment of fast and slow turnover pools (A1 and A2, respectively), Ya(0) and AUC values were significantly increased at 850 µg/d folate intakes. In the case of the isotopic enrichment kinetics of urinary pABG during d 42–84, the rate constant for decline of pABG enrichment (b1) showed an overall mean of 0.023 d^-1 with a trend toward a significant effect of pregnancy status (P = 0.087). The interaction between folate intake and pregancy status also approached significance (P = 0.065) for the initial enrichment of pABG (B1). The initial enrichment of ApABG (C1) was significantly affected by the level of folate intake (P = 0.003) and effects of pregnancy status exhibited a trend toward significance (P = 0.086). The rate constant for decline of ApABG enrichment (c1) exhibited a mean of 0.020 d^-1 with no significant effects of folate intake or pregnancy status observed. This corresponded to a MRT of 50 d.

View larger version (26K): [in this window] [in a new window]   Figure 3. Semilogarithmic plots of the isotopic enrichment (mole percent excess) of plasma folate in nonpregnant and pregnant women consuming two levels of folate after withdrawal of [2H2]folic acid that had been administered for the previous 6 wk (d 1–41). Values are means ± SEM for both pregnant and nonpregnant subjects at each level of folate intake because no discernible effect of pregnancy was observed (combined n = 6). The lines are model predictions based on these mean values for illustration of overall relationships.

Urinary excretion of [²H₂]folate, [²H₂]pABG and [²H₂]ApABG

The urinary excretion of deuterium-labeled forms of folate, pABG and ApABG after termination of [²H₂]folate administration is presented to illustrate the relationships among urinary excretion of folate, pABG and ApABG as routes of folate elimination as well as to show rates of decline (Fig. 4 ). The most notable difference in excretion curves among the experimental groups of this study was the markedly greater excretion of urinary folate at the 850-µg/d intake for both pregnant and nonpregnant subjects.

View larger version (38K): [in this window] [in a new window]   Figure 4. Excretion (nmol/d) of urinary [2H2]folate, [2H2]para-aminobenzoylglutamate (pABG) and [2H2]para-acetamidobenzoylglutamate ([2H2]ApABG) by nonpregnant and pregnant women consuming two levels of folate (upper panels, 450 µg/d; lower panels, 850 µg/d) after withdrawal of [2H2]folic acid that had been administered for the previous 6 wk (d 1–41). Values are means for pregnant and nonpregnant subjects at each level of folate intake (n = 3/group). The lines are model predictions based on these mean values for illustration of overall relationships.

Estimates of total body folate pool size, calculated on the basis of the rate of change of [²H₂]ApABG excretion after tracer withdrawal, are presented in Figure 5 . No significant differences were observed among treatment groups, and the effects of pregnancy, folate intake and their interaction were not significant by ANOVA (P > 0.05). Total pool size in nonpregnant women was 50 µmol (22 mg) and 70 µmol (31 mg) at the 450 and 850 µg/d folate intakes, respectively. In spite of the lack of difference due in part to the limited statistical power of this study, these results suggest somewhat higher folate pool size in second-trimester pregnant women than in nonpregnant controls. Although not significant under the conditions of this protocol, the apparent phenomenon of slightly greater total body folate pool size in pregnant subjects is also illustrated by least square means of 59.2 and 76.2 µmol for nonpregnant controls and pregnant subjects, respectively (pooled SEM = 7.6, n = 6 each). It should be noted that these values are similar to those determined kinetically in nonpregnant women (14) . Under the conditions of this study, the higher folate intake had only a slight and nonsignificant effect on folate pool size.

View larger version (29K): [in this window] [in a new window]   Figure 5. Kinetic estimates of total body folate pool size in nonpregnant controls and pregnant women consuming two levels of folate. Values are expressed as total pool size (µmol) and as µmol/kg body. Values are means and pooled SEM (n = 3/group); no significant differences existed among groups. P, pregnant; NP, nonpregnant control; 450 and 850 refer to total folate intake (µg/d).

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Folate kinetics

Our previous reports of folate status and urinary excretion of folate and folate catabolites in this study (13 ,19) , together with the present isotopic investigation conducted during the same study, constitute an in-depth investigation of folate metabolism in human pregnancy under conditions of controlled, ample folate intake. The kinetic and metabolic data extend the conclusions of Caudill et al. (13 ,19) that the effects of pregnancy on whole-body economy of folate constitute relatively small kinetic changes in metabolism, turnover and excretion. The influence of pregnancy on folate status might be greater under conditions of marginal folate intake, and subtle effects not observed in this study might be detectable in an investigation with greater statistical power. On the basis of earlier data gathered from this study, Caudill et al. (19) concluded that the intake of 450 µg/d (681 µg DFE) was adequate to support folate requirements of these women during the second trimester of pregnancy, which indicates that all subjects were in adequate folate status.

We previously described a compartmental model of whole-body folate turnover consisting of fast- and slow-turnover nonsaturable folate pools and a large saturable slow-turnover tissue folate pool (14) . This model had provisions for urinary folate excretion, catabolism and fecal excretion of endogenous folate and/or folate catabolites, with assumed enterohepatic circulation rates based on literature values for biliary folate concentration and bile flow. Although not all components of this model were kinetically identifiable using accessible isotopic enrichment data, with several assumptions involving previously measured rates of biliary folate secretion and the existence of a large tissue pool of protein-bound folates, this model did allow estimates of physiologically relevant turnover rates and pool sizes (14) . We initially attempted to use an extension of this compartmental model for integrated analysis of urinary folate, pABG and ApABG data from both tracer administration (d 1–41) and tracer disappearance (d 42–84) phases of this study. These attempts were not successful in precisely fitting the model to data collected during early time points of tracer administration, probably due to the differences between the folate intake during the study and intake before enrollment of many subjects. In addition, the physiologically nonsteady-state nature of pregnancy would complicate the use of a model that had been developed using assumptions of steady state. As stated above, the kinetic analysis reported here focused mainly on data obtained during the tracer withdrawal phase of the study (d 42–84). We believe that the constant and relatively high folate intakes during the initial 6-wk period of the study allowed the subjects to approach a steady state of folate nutritional status indicators (13 ,19) . Thus, the 6-wk period of tracer administration and controlled folate intake effectively served as a period for stabilization of the subjects as well as a period of labeling the various endogenous folate pools to facilitate their kinetic assessment when tracer administration was discontinued.

If isotopic equilibrium is reached during a period of chronic tracer intake, the enrichment of body pools would be equivalent to that of ingested folate (15% ²H₂). The kinetic terms indicative of the isotopic enrichment at the start of the withdrawal phase (d 42) of the various folate pools examined are A1 and A2 (fast- and slow-turnover pools, respectively, from which urinary folate originates), B1 and C1 (presumably tissue folate pools from which urinary pABG and ApABG originate, respectively), and D1 (plasma folate). The highest of these values was A1 (fast-turnover pool yielding urinary folate) for all subjects receiving the 850-µg/d dose, with A1 values of 6.5–8.0 mol % excess. Because [²H₂]folic acid constituted only 15% of ingested folate, we conclude that the in vivo pools had progressed at most only about halfway toward isotopic equilibrium during the 41-d period of tracer intake even at the 850 µg/d folate intake. The whole-body MRT for whole-body folate observed in our previous study of folate kinetics in nonpregnant women were 212 ± 8, 169 ± 12 and 124 ± 7 d for intakes of 200, 300 and 400 µg/d, respectively (14) , suggesting that the major tissue folate pools attain isotopic equilibrium slowly, and that the rate at which it is attained is strongly related to folate intake. We hypothesized that the enrichment of plasma folate would quickly come into equilibrium with ingested folate and that the isotopic enrichment of plasma folate would decline quickly during the tracer withdrawal phase. However, the observation that plasma folate enrichments [i.e., Yd(0) values] were substantially less than that of ingested folate is evidence that plasma folate reflects a balance between labeling of ingested folate and the labeling of tissue folates, much of which is protein bound, and which exhibit much slower rates of folate turnover. As noted previously, the fact that the MRT of folate in the various folate pools examined in this study decreased with increasing folate intake is evidence that newly absorbed folate molecules compete for binding sites in tissues and, thus, accelerate turnover.

This study is apparently the first in which the enrichment of fasting plasma folate and folate catabolites from a 24-h urine collection have been measured and compared after chronic intake of stable isotopically labeled folate. We observed similar enrichments of urinary and plasma folate at the lower level of folate intake (450 µg/d) but far greater enrichment of the 24-h urinary folate pool than seen for fasting plasma folate at the 850-µg/d folate intake. This may be indicative of large increases in the overall enrichment of plasma folate and, hence, urinary folate from which it is partially derived, in the short-term after consumption of the folic acid supplements. These results also suggest that plasma folate enrichment also returns to a more steady-state enrichment within 12–24 h of consuming a folate supplement that, in this protocol, contained labeled folic acid. Concurrently collected urine and blood sampling over the course of a 24-h period during a protocol such as this would be required to clarify this issue. The analytical method used in this study examined the aggregate enrichment of plasma folate by GC-MS and total folate concentration by microbiological assay reported previously (19) . Thus, we cannot determine the extent of appearance of unmetabolized folic acid in plasma, as was reported by Kelly et al. (29) . In all subjects of this study, the majority of urinary folate was 5-methyl-tetrahydrofolate. Unmetabolized folic acid was observed only in the urine of control subjects consuming 850 µg/d folate and constituted a mean of only 19% of urinary folate in those subjects (19) . Additional studies are required to clarify more completely the issue of rate and extent of reduction and further metabolism of folic acid at various doses in pregnant and nonpregnant women.

Folate catabolism

The results of this study provide new insight into the role of pABG and ApABG in folate turnover as well as the origin of these catabolic products. We previously reported the measurement of ApABG and pABG excretion in this study (13) , which showed the predominance of ApABG over pABG as an excreted catabolic product, confirming the observations of McPartlin et al. (10 ,11) . Another previous notable finding was that the excretion of ApABG exceeded that of urinary folate at the 450-µg/d folate intake, whereas urinary folate was markedly greater at the 850-µg/d intake (13 ,19) . In the present study, the urinary excretion of isotopically labeled forms of these catabolites and folate followed these same patterns.

The isotopic enrichment of urinary pABG and ApABG serves as a convenient indicator of the body folate pools from which they are formed. These catabolites appear to be formed by cleavage of tissue folates (8 ,9) , although the mechanism is unclear. An enzymatic process in which a ferritin-like protein catalyzes a folate cleavage reaction was reported (30) , but its physiologic role has not yet been demonstrated.

A comparison of the kinetic terms describing the d-42 enrichment of urinary folate, pABG and ApABG (A1 and A2, B1 and C1, respectively) shows clear differences in these values, regardless of the experimental group (Tables 2 and 3) . The fact that such differences in enrichment exist suggests that pABG and ApABG are produced by catabolism of tissue folate pools that differ in their rate of turnover and, hence, their extent of labeling in this study. The fact that both pABG and ApABG enrichments exhibited first-order kinetics and slow decline is evidence that each comes from a distinct, kinetically identifiable pool having a slow rate of turnover. These results also indicate that urinary pABG is not formed by simple oxidative breakdown of urinary folate because urinary pABG and folate generally differed in isotopic enrichment. It is possible that pABG and ApABG represent the main catabolic products of tissues that vary substantially in the activity of an arylamine acetyltransferase that was proposed to be responsible for the in vivo acetylation of pABG (31) .

A novel aspect of this study is the use of folate catabolites pABG and ApABG as indicators of isotopic labeling of the tissue folate pools from which they are derived. The results of this study suggest that each catabolite is formed mainly in a single, slow-turnover folate pool (Figs. 2 , 4) . Unlike the observations of this study in women consuming 450 or 850 µg/d total folate, we previously reported data that were consistent with substantial folate catabolism from both fast- and slow-turnover folate pools in nonpregnant women consuming controlled levels of 200–400 µg/d (14) . The differing initial enrichments of pABG and ApABG [Yb(0) and Yc(0)] observed here comprise the first evidence of different in vivo origins of pABG and ApABG.

We reported previously that there was no significant difference in the extent of excretion of folate catabolites between pregnant and nonpregnant women at either folate intake in this study during gestational wk 14–25 (13) . Obstetrical terminology conventionally divides pregnancy into three equivalent trimesters of 14 wk; thus wk 1–13 or 14 comprise the first trimester, wk 14 or 15–28, the second trimester, and wk 28 or 29–42 constitute the third trimester (32 33) . In the study of Higgins et al. (12) , the measurement of folate catabolites was conducted at gestational wk 12–16, 26–30 and 34, designated as the 1st, 2nd and 3rd trimesters, respectively. Thus, their "2nd trimester" observation actually described excretion at late 2nd and early 3rd trimesters, whereas that of the Caudill study investigated early and mid-2nd trimester. Assuming that the protocols of Caudill et al. (13 ,19) and Higgins et al. (12) were comparable (i.e., little effect of the differences in experimental design), these observations indicate that the rise in folate catabolism occurs near the end of the 2nd trimester of pregnancy and continues to escalate through the 3rd trimester. Further investigation of folate catabolite excretion in late pregnancy is clearly warranted.

The rates of excretion of labeled pABG and ApABG reported here are based on a calculation using the excretion data of Caudill et al. (13) ; thus, these data do not constitute fully independent observations. However, our examination of in vivo kinetics further supports this conclusion and provides new information regarding the rate of turnover of tissue folate pools from which pABG and ApABG are derived. For both pABG and ApABG, these rate constants (b1 and c1, respectively) were the same or lower in pregnant women compared with nonpregnant women with no difference in estimated body folate pool sizes (Table 2 and Fig. 5 ). This indicates similar, or possibly even slower turnover of the tissue folate pools from which these catabolites originate in the pregnant women of our study. Had pregnancy caused a large increase in folate catabolism, one would expect that the rate of turnover of these tissue folate pools would accelerate correspondingly. In addition, a substantially greater rate of turnover of tissue increase folate pools presumably would have led to depressed body folate pool size, which clearly was not the case (Fig. 5) . Thus, these results provide further evidence that there is little quantitatively important change in folate catabolism during pregnancy under the conditions of this study, i.e., controlled folate intakes of 450 and 850 µg/d with women of 14–25 wk gestation.

The results of this study extend our understanding of whole-body folate metabolism and the influence of folate intake and pregnancy; this study has illustrated a useful design for further investigation of folate metabolism. Of particular importance is the fact that this stable-isotopic approach allows the study of sensitive populations, such as pregnant women, in which radiolabeled folate tracers could not be ethically utilized regardless of dose.

	FOOTNOTES

 
¹ Supported by National Institutes of Health Grant # HD 29911, National Institutes of Health General Clinical Research Center Grant # RR00082, and funds from the Florida Agricultural Experiment Station. Florida Agricultural Experiment Station Journal Series No. R-08078.

³ Abbreviations used: ApABG, para-acetamidobenzoylglutamate (N-acetyl-para-aminobenzoylglutamate); AUC, area under the curve; DFE, dietary folate equivalents; FCR, fractional catabolic rate; GC-MS, gas chromatography-mass spectrometry; [²H₂]folic acid, [3',5'-²H₂]folic acid; MRT, mean residence time; pABG, para-aminobenzoylglutamate; SAAM, simulation, analysis, and modeling software.

Manuscript received October 31, 2000. Initial review completed January 3, 2001. Revision accepted April 11, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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