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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:619-623, March 2005

Nutritional Models

Differential Kinetic Behavior and Distribution for Pteroylglutamic Acid and Reduced Folates: a Revised Hypothesis of the Primary Site of PteGlu Metabolism in Humans1

Anthony J. A. Wright2, Paul M. Finglas, Jack R. Dainty, Caroline A. Wolfe, David J. Hart, Dawn M. Wright and Jesse F. Gregory*

Nutrition Department, Institute of Food Research, Norwich NR4 7UA, United Kingdom; and * Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370

2To whom correspondence should be addressed. E-mail: tony.wright{at}bbsrc.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Single 13C6-labeled doses of pteroylmonoglutamic acid (PteGlu: 634 nmol; n = 14), (6S-)5-formyltetrahydrofolic acid (431–569 nmol; n = 16), or [15N1–7]-intrinsically labeled spinach (mainly 5-methyltetrahydrofolate) (588 nmol; n = 14) were fed to fasting adult volunteers. Plasma-labeled 5-methyltetrahydrofolic acid responses were monitored for 8 h. There was a slower rate of increase in plasma-labeled 5-methyltetrahydrofolic acid and longer time to peak (171 ± 9 min; mean ± SEM) following an oral dose of [13C6]PteGlu than either [13C6]5-formyltetrahydrofolic acid (54 ± 10 min) or [15N1–7]spinach folate (60 ± 13 min) suggesting saturated metabolic capacity for the biotransformation of PteGlu. Mathematical modeling generated a significantly higher mean "apparent absorption" for 5-formyltetrahydrofolic acid (38%) and spinach folate (44%) than for PteGlu (24%). The high "relative absorption" of reduced folates to PteGlu was unexpected given that PteGlu itself, from 14C-tracer mass balance experiments, is almost completely absorbed. Although it is ubiquitously accepted that a physiological dose of PteGlu is reduced and methylated in the epithelial cells of the small intestine, and that essentially only 5-methyltetrahydrofolic acid is exported into the hepatic portal vein (HPV), as is the case for absorbed reduced 1-carbon-substituted folates, modeling indicated greater liver sequestration when PteGlu was used as the test dose, suggesting that PteGlu enters the HPV unaltered and that the liver is the primary site of initial metabolism. Because of the observed differential plasma response and the hypothesized difference in the site of initial metabolism, the historical use of PteGlu as a "reference folate" in studies of folate bioavailability is seriously questioned.

KEY WORDS: • pteroylmonoglutamic acid • folate • absorption • isotopes • modeling

Because there is an extensive hepatic uptake (liver "first-pass") of newly absorbed folate (1–4), which prevents direct estimation of the degree of folate absorption from any plasma response, the vast majority of work over the past 35 or more years attempting to assess folate absorption in humans has centered on methods comparing the serum/plasma response to a single unlabeled oral test-dose relative to that of an equal "reference" dose of pteroylmonoglutamic acid (PteGlu)3 (4,5) and the subsequent computation of "relative absorption." This may entail either measurement of the rate of increase, or the maximum increase, in plasma folate concentration over 2 to 3 h (6–11) or measurement of the dose-normalized rise in plasma folate concentration area under the curve (AUC) over 6 h or more (12–19).

Comparison of AUC between test and reference folate has been accepted as a valuable indicator of absorption, provided that the postdosing plasma measurement test period is long enough to capture ≥80% of the whole AUC (20). This approach also requires experimental conditions that satisfy the following 3 principles: Assumption 1 states that PteGlu is absorbed by the same mechanism as reduced folates and in a similar manner. PteGlu and reduced folates are absorbed mainly in the proximal small intestine by a saturable, carrier-mediated, pH- and energy-dependent transport mechanism, which, unlike other epithelial tissues, appears to be unique in its lack of hierarchy of transport, having a similar affinity for both PteGlu and reduced folate forms (21,22). Assumption 2 states that physiological doses of PteGlu are initially reduced and then methylated in the epithelial cells of the small intestine and that only 5-methyltetrahydrofolic acid is exported from the mucosa to the hepatic portal vein. Without exception, review articles from 1983 onward agree that the small intestine efficiently reduces and methylates physiological doses of PteGlu and, as with absorbed naturally occurring reduced and 1-carbon-substituted folates, subsequently transfers only 5-methyltetrahydrofolic acid (5-CH3H4PteGlu) to the hepatic portal vein (e.g., 2,22,23). Thus, logically, one would expect no differential in hepatic uptake in response to oral doses of PteGlu compared to other test folates, and the calculation of relative absorption from the comparative plasma AUC response of a test-dose to that for PteGlu is a valid "pragmatic" maneuver. Assumption 3 states that the kinetics of the plasma response to PteGlu is the same as that for reduced 1-carbon-substituted naturally occurring folates. Until recently, no systematic difference has been reported between the plasma 5-methyltetrahydrofolic acid response to test folates and PteGlu.

Controversy has arisen recently because it has been shown that much of the plasma AUC response to oral folate doses is induced by, but is not actually derived from, the dose itself (18), a phenomenon recently reported to also affect vitamin C (24). We interpreted our reported large, variable, and unpredictable displacement of tissue 5-methyltetrahydrofolic acid into the plasma pool as making a comparison of the relative absorption of folate from an unlabeled test dose (food or supplement) versus an unlabeled PteGlu reference dose untenable. Also, concurrent examination of the plasma 13C-labeled 5-methyltetrahydrofolic acid response to 13C-labeled [6S]5-formyltetrahydrofolic acid (5-HCOH4PteGlu) and a "reference dose" of 13C-labeled PteGlu unmasked an underlying serious discrepancy in plasma response that completely violated Assumption 3, rendering direct estimates of relative absorption using even labeled AUCs potentially invalid.

Fortunately, the violation of Assumption 3 need not necessarily be fatal to the overall methodologic approach. The application of suitable mathematical modeling (25), which makes allowance for any differences in the kinetics of plasma-labeled 5-methyltetrahydrofolic acid response, can be used to estimate important parameters. We have applied such an approach to the current study where, in fasting human volunteers, plasma response profiles for labeled 5-CH3H4PteGlu are followed over a period of 8 h after ingestion of single oral doses of [13C6] PteGlu, (6S-) [13C6]5-HCOH4PteGlu, or intrinsically labeled [15N1–7]spinach folate. Folate binding affinity columns were used to isolate extracted plasma folate and, following HPLC analysis of folate concentrations, a recently developed LC-MS analytical method (26) was used to determine the proportions of labeled and unlabeled 5-CH3H4PteGlu in the plasma samples.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. With the exception of intrinsically labeled spinach folate, a description of materials was given previously (18). A hydroponics system was used for the production of [15N]-intrinsically labeled spinach, which leads to intrinsic labeling of some or all of the 7 nitrogen atoms in the folate molecule. In brief, spinach seeds were grown under glass and, once seed leaves opened, were watered daily with a growing medium containing 50% of its nitrogen as [15N]-nitrates. Plants were harvested after 8 wk. Woody stems and outer leaves were removed and the remaining leaves were washed in lightly salted cold water, rinsed in cold water, shaken dry, and shredded and a composite was made. Portions (200 g) were then weighed into bowls and blanched in a microwave oven (750 W) for 3 min. After the bowls were cooled in cold water, the contents (leaf and liquid) were transferred to sealable bags, frozen over solid CO2, and stored at –20°C. Total spinach folate, 2.94 (± 0.38) µmol/kg, predominantly 5-CH3H4PteGlu but with some 5-HCOH4PteGlu, was determined using liquid chromatography-microbiologic assay (27) after polyglutamates were deconjugated to the monoglutamate form with hog kidney deconjugase (28). Isotopic enrichment of spinach [15N]5-CH3H4PteGlu isolated on folate binding affinity columns, compared to natural abundance 5-CH3H4PteGlu, was measured using negative ion [M-H]– electrospray LC-MS (m/z 458–465) (26). Isotopic spectra revealed that spinach [15N]5-CH3H4PteGlu had measurable ions at m/z 458–465 (i.e., M to M + 7), with 18% of all the naturally occurring 14N atoms being replaced by 15N atoms.

    Human study design. The study was approved by the Norwich Local Research Ethics Committee (Norfolk & Norwich University Hospital NHS Trust). After written consent was obtained, a blood sample was taken from healthy adult volunteers who had fasted for 12 h and analyzed at the hematology department of the Norfolk & Norwich University Hospital for full blood count, blood glucose, erythrocyte folate, serum vitamin B-12, urea and electrolytes, and liver function tests. If all results were within normal assay ranges, volunteers were then invited to attend 3 test days. Following an overnight fast and the measurement of blood pressure, a baseline blood sample (10 mL) was taken via cannula. Volunteers were then given a single oral dose of [13C6]PteGlu (634 nmol), (6S-)[13C6]5-HCOH4PteGlu (431–569 nmol), or a portion of [15N1–7]-labeled spinach (588 nmol folate). The dose of [13C6]PteGlu or (6S-)[13C6]5-HCOH4PteGlu was administered as previously described (18). Spinach portions were thawed, homogenized, reheated by microwave, and then served and consumed within 10 min. A timer was started only after the spinach had been completely consumed. Volunteers were always allowed access to water, and were given a light lunch (a sandwich with a only slice of either processed chicken breast or processed cream cheese spread and cucumber) only after the 4-h postdosing venous blood sample had been obtained. Few volunteers completed all 3 test days.

    Blood sampling and storage. Venous blood samples (10 mL) were taken by cannula: a baseline "time zero" prior to test dose and 11 time points over an 8-h period following each test dose; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 h. Blood samples were transferred immediately into tubes containing K2-EDTA and mixed gently. Samples were centrifuged (1500 x g, 10 min), and plasma was removed and frozen immediately over solid CO2 and then stored at –30°C until analysis.

    Sample preparation, folate extraction, and analysis. The extraction of folate from plasma samples, purification on affinity columns using folate binding protein, and subsequent analysis by HPLC (total plasma 5-CH3H4PteGlu) or LC-MS (labeled and unlabeled plasma 5-CH3H4PteGlu) was conducted as described previously (18) with the exception that selected ion monitoring was conducted additionally on the [M-H]– ion over the range of m/z 458–465 to determine 5-CH3H4PteGlu derived from the [15N1–7]-labeled spinach.

    Mathematical modeling of the labeled plasma response. We started with the application of a simple one-compartment model (Fig. 1), which is designed specifically to elicit an understanding of the kinetics of initial absorption, metabolism, and transport of absorbed folates. Mathematical modeling was derived from pharmacokinetic principles (29); all formulas were executable within cells of standard operating Microsoft Excel spreadsheet software. Following an oral dose of labeled folate, the plasma appearance of labeled 5-CH3H4PteGlu is assumed to approximate that of an infusion of rate R over an absorption time period. The absorption time period (T) is defined as the time to peak plasma labeled 5-CH3H4PteGlu concentration (tmax) minus the time during which the plasma-labeled enrichment initially remains at baseline (tlag).



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  		FIGURE 1 Simple 1-compartment model for folate absorption.

 
The definition of R is

(1)

If t is time after ingestion of the test dose (doseoral), k is the rate constant of elimination from the plasma compartment to body tissues and/or excretion, and V is the apparent volume of distribution for folate in the sampled plasma compartment, then plasma concentration (C) of labeled 5-CH3H4PteGlu can be approximated as

(2)


(3)

The time during which the plasma enrichment remains at baseline (tlag), the time to peak plasma concentration (tmax), the value of T (tmax – tlag), and the rate constant of elimination (k) are established from the plasma-labeled 5-CH3H4PteGlu enrichment curve. The volume (V) of distribution in the sampled compartment is large and estimated to be 387 mL/kg body wt in humans (30).

By fitting the pair of simultaneous equations (Eqs. [2]and [3]) to the plasma concentration of labeled 5-CH3H4PteGlu over time (t), M can be calculated. The apparent absorption can then be calculated according to

(4)

A liver first-pass effect occurs when folate is absorbed from a meal (12–15). By assuming that the absorption, at least of PteGlu, in the present study is ~90% (31–34), the first pass effect can be estimated:

(5)

    Statistics. Originally, our study had a crossover within-subject design. However, because of the intrusive nature of 8-h cannulations, most subjects did not complete all 3 test days and data are treated as independent observations. Because distributions were not significantly different from normal (Kolmogorov-Shapiro test), data (means ± SEM) were compared using ANOVA. When the ANOVA was significant (P ≤ 0.05), means for reduced-folate test doses were compared to that for the PteGlu reference folate test dose by t test. Linear regression analysis was used to assess the correlation (r) of mathematically modeled "apparent absorption," "first-pass effect," and "plasma 5-CH3H4PteGlu elimination rate constant" (k) to fasting baseline plasma or erythrocyte folate concentration.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Age, BMI, erythrocyte, and plasma (at t = 0 h) folate concentrations of volunteers did not differ in each of the 3 folate test groups (Table 1).


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  		TABLE 1 [13C6]- or [15N1–7]-labeled plasma 5-methyltetrahydrofolic acid-response in fasting healthy adult volunteers following oral test doses of [13C6]PteGlu (634 nmol), (6S)[13C6]5-HCOH4PteGlu (431–569 nmol), and [15N1–7]spinach folate (588 nmol)1

 
Selected ion monitoring indicated that neither PteGlu nor 5-formyltetrahydrofolic acid from the respective test doses appeared in the plasma in its unmetabolized form.

Plasma-labeled 5-methyltetrahydrofolic acid response to the 3 test folates is depicted in Figure 2.



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  		FIGURE 2 Labeled plasma 5-methyltetrahydrofolic acid (5-MTHF) response to labeled folate test doses. PteGlu ({blacksquare}), 5-HCOH4PteGlu ({diamondsuit}), and spinach folate ({blacktriangleup}). Values are means ± SEM, n = 14 (PteGlu or spinach) or 16 (5-HCOH4PteGlu). Neither labeled PteGlu nor 5-HCOH4PteGlu is detected in plasma, only their labeled 5-methyltetrahydrofolic acid metabolite. To convert 5-MTFH to nmol/L, multiply ng/mL by 2.17.

 
The labeled plasma response to PteGlu was slower and peaked much later (tmax = 171 ± 9 min) than the response to either of the reduced-folate test doses; tmax = 54 ± 10 min in response to 5-formyltetrahydrofolic acid test dose and tmax = 60 ± 13 min in response to spinach-folate test dose (Table 1).

The mean modeled apparent absorption (an estimate of folate absorption that takes no account of the fact that a fraction of newly absorbed folate is sequestered by the liver) for reduced folates (38% for 5-formyltetrahydrofolic acid and 44% for spinach-folate) was significantly higher than for the PteGlu (24%) reference dose (Table 1). Calculation of relative absorptions (i.e., response for test dose divided by response for PteGlu reference dose) yields 158 and 183% for 5-formyltetrahydrofolic acid and spinach-folate, respectively.

By assuming that the actual absorption of the test folate dose is 90%, the mean calculated first-pass-effect, where a percentage of absorbed labeled folate is sequestered (presumably to the liver), was significantly greater for PteGlu (73%) than for either 5-formyltetrahydrofolic acid (58%) or spinach-folate (52%) (Table 1).

The rate constant of elimination (k) of plasma-labeled 5-CH3H4PteGlu response was not associated with fasting baseline plasma or erythrocyte folate concentration (Table 2).


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  		TABLE 2 Correlation among apparent absorption, first-pass effect, or labeled plasma 5-CH3H4PteGlu clearance rate in fasting healthy adult volunteers following oral labeled-folate test doses and fasting baseline plasma or erythrocyte folate concentration1

 
Apparent absorption and the first-pass effect were not correlated to fasting erythrocyte folate concentration, but they did significantly correlate with fasting baseline plasma folate concentration (Table 2).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Most previous work over the past 4 decades attempting to assess relative folate absorption in humans in studies of folate bioavailability has centered on methods comparing the serum/plasma AUC response toward a single oral test dose (food or supplement) to that of an equal reference dose of PteGlu. This approach has been undertaken universally using unlabeled test and reference folates. We have already demonstrated that a substantial portion of any total plasma response is elicited by, but does not originate directly from, an oral test dose itself (18). Arguably, in all previous studies utilizing unlabeled test doses, calculations of relative absorption to PteGlu have been ambiguous and potentially erroneous.

When restricting our current analysis to plasma-labeled 5-methyltetrahydrofolic acid response, we note that the time to maximum response to a dose of PteGlu is ~3 h in comparison to 1 h for reduced folates. This large difference in the kinetics of plasma response violates Assumption 3 and thus disqualifies the idea of comparing the plasma AUC response to an oral test dose of folate with that of an equal reference dose of PteGlu and the subsequent computation of relative absorption. We interpret this delay to indicate that the metabolic capacity for the biotransformation of PteGlu was saturated when using our dose of 634 nmol, possibly due to a limitation in the rate of initial reduction of PteGlu to H2-PteGlu (35). A study published recently (36), using accelerator mass spectrometry to quantify the plasma [14C]5-methyltetrahydrofolic acid response to a small 80 nmol dose of [14C]PteGlu, reports a similar delayed peak plasma response of 3 h. This evidence suggests that the metabolic capacity for the biotransformation of PteGlu in humans may be rate limiting; a conclusion that is consistent with the lower dihydrofolate reductase activity detected in human tissue compared to corresponding animal tissue (37–39) and a report that concludes that low levels of dihydrofolate reductase activity are a feature peculiar to humans (40).

Fortunately, the violation of Assumption 3 is not fatal because mathematical modeling of plasma response circumvents the issue. Modeling indicates an apparent absorption of reduced folates that is significantly higher than for the reference dose of PteGlu. These generate relative absorptions significantly in excess of 100%. This is unexpected and biologically impossible, because the "true absorption" of doses of [14C]PteGlu in 2 human volunteers by mass balance has been reported to approximate 90% or more (33,34), and tritiated forms of PteGlu and reduced folates are almost fully absorbed in rats (31). It is only when the first-pass effect is estimated from the current data (Table 1) that it becomes apparent that more absorbed folate may be sequestered by the liver when PteGlu is the test dose than when reduced-folates are the test dose. However, such a differential in distribution to body tissues should not be so if, as generally assumed, absorbed folate forms undergo mucosal biotransformation and successive transfer to the hepatic portal vein primarily as 5-methyltetrahydrofolic acid. Therefore, contrary to current theory, it appears that a substantial fraction of absorbed [13C6]PteGlu may be entering the hepatic portal vein unchanged, to be more effectively removed by the liver than would 5-methyltetrahydrofolic acid, prior to subsequent biotransformation and partial enterohepatic recirculation. Although we agree that physiologic doses of PteGlu are indeed extensively reduced and methylated in the mucosal epithelial cells of the small intestine in rats (the historical experimental animal model used in formulating much of our current understanding of folate absorption and metabolism), we are not the first to suggest that this may not take place, or not so extensively, in humans. High concentrations of untransformed PteGlu were reported to appear in the hepatic portal vein of humans following oral administration of both 1.0 mg (2264 nmol) PteGlu (41) and 0.5 mg (1132 nmol) PteGlu (42). Despite criticisms of these studies, e.g., the use of relatively high doses of PteGlu and the analysis of folate concentrations by possibly problematic differential microbiological assay, we think that reports of the almost complete absence of assayable 5-methyltetrahydrofolic acid in the hepatic portal vein are remarkable—particularly when a dose of 0.5 mg (1132 nmol) was used (42). This is only twice the dose of 260 µg (589 nmol) reported much later (11) to be the threshold at which PteGlu may start to appear in the plasma.

It is noteworthy that apparent absorption correlates positively with fasting baseline plasma folate concentration (Table 2). Because a large fraction of newly absorbed labeled folate undergoes immediate removal to the liver, irrespective of the form appearing in the hepatic portal vein, much of any subsequent plasma-labeled response may actually be derived from enterohepatic recirculation. Greater enterohepatic folate recirculation would not only result in a higher fasting baseline plasma folate concentration but also then automatically assist in generating a greater labeled plasma response to any test folate by recycling a larger proportion of newly sequestered folate, mainly as 5-methyltetrahydrofolic acid.

We have serious doubts as to the ubiquitous use of PteGlu as the reference folate in most nutritional studies conducted in humans and conclude that currently held views on the absorption, metabolism, and subsequent tissue distribution of folates need careful re-evaluation, as does the methodology in current use for estimating the relative absorption of single folate test doses. We think it is essential, using labeled folates and sensitive HPLC/MS techniques, to establish whether a substantial fraction of absorbed PteGlu from even small oral doses enters the human hepatic portal vein unchanged. If it does, then the initial primary site of PteGlu metabolism would be the liver, Assumption 2 would be violated, and the use of PteGlu as a reference folate in most human folate bioavailability studies would be fatally undermined.


   FOOTNOTES
 
1 Supported by UK Food Standards Agency, the UK Biotechnology & Biological Sciences Research Council, and EU project "Folate: From Food to Functionality and Optimal Health," QLK1-CT-1999–00576. Back

3 Abbreviations used: 5-CH3H4PteGlu, 5-methyltetrahydrofolic acid; 5-HCOH4PteGlu, 5-formyltetrahydrofolic acid; AUC, area under the curve; PteGlu, pteroylmonoglutamic acid; m/z, mass-to-charge ratio; spinach folate, mainly 5-methyltetrahydrofolate polyglutamate. Back

Manuscript received 21 October 2004. Initial review completed 4 December 2004. Revision accepted 17 December 2004.


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