Primed, constant infusion with [2H3]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism.

BACKGROUND: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved. OBJECTIVE: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one-carbon units in homocysteine remethylation in hepatic and whole-body metabolism. DESIGN: A healthy man aged 23 y was administered [2,3,3-(2)H(3)]serine and [5,5,5-(2)H(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling. RESULTS: [(2)H(1)]Methionine and [(2)H(2)]methionine were labeled through homocysteine remethylation. We propose that [(2)H(2)]methionine occurs by remethylation with [(2)H(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [(2)H(3)]serine, whereas [(2)H(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [(2)H(1)]formate to yield cytosolic [(2)H(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis. CONCLUSIONS: The appearance of both [(2)H(1)]- and [(2)H(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Chemical characteristics and relative bioavailability of supplemental organic zinc sources for poultry and ruminants.

Eight commercially available organic Zn products and reagent-grade ZnSO4 x 7H2O (Zn Sulf) were evaluated by polarographic analysis, and solubility in .1 M K2HPO4-KH2PO4 buffer (pH 5), .2 M HCl-KCl buffer (pH 2), and deionized water. Fractions from these solubility tests were evaluated by gel filtration chromatography for structural integrity. Degree of chelation was generally positively related to chelation effectiveness determined by polarography. The organic sources were Zn methionine complex A (Zn MetA), Zn methionine complex B (Zn MetB), Zn polysaccharide complex (Zn Poly), Zn lysine complex (Zn Lys), Zn amino acid chelate (Zn AA), Zn proteinate A (Zn ProA), Zn proteinate B (Zn ProB), and Zn proteinate C (Zn ProC). Three experiments were conducted to estimate the relative bioavailability of Zn from the organic Zn supplements for chicks and lambs when added at high dietary levels to practical diets. Bone Zn concentration increased (P < .001) as dietary Zn increased in both experiments. When Zn Sulf was assigned a value of 100% as the standard, multiple linear regression slope ratios of bone Zn from chicks fed 3 wk regressed on dietary Zn intake gave estimated relative bioavailability values of 83 +/- 14.6 and 139 +/- 16.9 for Zn AA and Zn ProA, respectively, in Exp. 1 and 94 +/- 11.6, 99 +/- 8.8, and 108 +/- 11.4 for Zn Poly, Zn ProB, and Zn ProC, respectively, in Exp. 2. In Exp. 3, 42 lambs were fed diets containing Zn Sulf, Zn ProA, Zn AA, or Zn MetB for 21 d. Based on multiple linear regression slope ratios of liver, kidney, and pancreas Zn and liver metallothionein concentrations on added dietary Zn, bioavailability estimates relative to 100% for Zn Sulf were 130, 110, and 113 for Zn ProA, Zn AA, and Zn MetB, respectively. Except for Zn ProA, which was greater, the organic Zn supplements had bioavailability values similar to that of Zn Sulf for chicks and lambs. Bioavailability of organic Zn products was inversely related to solubility of Zn in pH 5 buffer in chicks (r2 = .91) and pH 2 buffer in lambs (r2 = .91), but not to an estimate of degree of chelation.

Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration.

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [(2)H(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats [7 and 0.1 mg pyridoxine (PN)/kg diet]. [(2)H(3)]Leucine and [1-(13)C]methionine were also used to examine turnover of protein and methionine pools, respectively. All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [(2)H(3)]leucine. Hepatic [(2)H(2)]methionine production from [(2)H(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [(2)H(3)]cysteine from the [(2)H(3)]serine increased over twofold. In contrast, plasma appearance of [(2)H(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-(13)C]homocysteine/[1-(13)C]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

Effect of temperature and humidity on the formation of dibutylurea in benlate fungicide.

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate] is the active ingredient in DuPont Benlate fungicides. The formation of N, N'-dibutylurea (DBU), a phytotoxic degradation product of benomyl, in Benlate formulations was evaluated by analyzing Benlate samples maintained under simulated storage conditions and assessing the effects of temperature and humidity on sample moisture content, benomyl degradation, and the rate of DBU formation. Benomyl degraded during storage by the elimination of n-butylisocyanate (BIC) to form methyl 2-benzimidazole carbamate (MBC; carbendazim). Liberated BIC could then proceed to react with water to form DBU (first-order rate constant of 8.4 x 10(-)(4) s (-)(1)). The degradation of benomyl and subsequent formation of DBU were dependent on the temperature and highly dependent on the humidity of the storage environment. At the lower humidity storage conditions the rates of DBU formation were significantly higher in the dry flowable (DF) formulation than in the wettable powder (WP) formulation. The initial moisture content of Benlate DF samples was higher than those of Benlate WP samples, although the Benlate WP samples absorbed more moisture upon incubation. These results may yield insight on the appearance of high levels of DBU found in some boxes and bags of Benlate DF and Benlate WP formulations.

Dissociation of attentional processes in patients with focal frontal and posterior lesions.

A location-based ('select-what, respond-where') priming task was used to examine three measures of selective attention (interference (INT), negative priming (NP), and inhibition of return (IOR)) as a function of focal brain pathology and the complexity of target selection. Control subjects showed different patterns of performance for the three attentional measures as a function of complexity, suggesting some independence among INT, NP, and IOR. Brain-damaged subjects showed significant response slowing, as well as a number of lesion-specific attentional abnormalities. Right frontal (including bifrontal) damage resulted in proportionally increased interference related to task complexity. Left posterior damage increased IOR in the most complex task, while left frontal damage reversed the control pattern of IOR as a function of complexity. Right hemisphere (right posterior and right frontal damage) pathology resulted in a virtual loss of negative priming at all levels of task complexity; left and bifrontal damage resulted in diminished NP only related to increases in the complexity of selection. INT, NP, and IOR are mediated by different brain regions and their expression can be modulated by the complexity of the selection task.

Urinary excretion of [2H4]folate by nonpregnant women following a single oral dose of [2H4]folic acid is a functional index of folate nutritional status.

In a 10-wk study with nonpregnant women (21-27 y, n = 5-6 per group), subjects were fed a diet containing approximately 68 nmol/d (30 microg/d) folate from food that was supplemented with folic acid in apple juice to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 microg/d) to evaluate folate status and long-term in vivo kinetics. Reported here is an additional phase of this protocol conducted to determine the relationship between short-term urinary excretion after a single isotopically labeled dose and various measures of folate nutritional status. It was hypothesized that urinary excretion from a single [glutamate-2H4]folic acid ([2H4]folic acid) dose would increase in proportion to folate nutritional status due to saturable cellular uptake and retention processes along with saturation of renal reabsorption. Each subject was given 1.13 micromol (500 microg) of [2H4]folic acid orally on the morning of d 70 of the study, followed by a complete 24-h urine collection. Urine was analyzed to determine the isotopic enrichment of urinary folate by gas chromatography-mass spectrometry and the concentration of urinary folate by HPLC. Urinary excretion of [2H4]folate was greatest at the 907 nmol/d intake and was positively correlated with serum folate concentration but was not correlated with erythrocyte folate. Excretion of [2H4]folate tended to be greatest when plasma homocysteine concentrations were low (<8 micromol/L), although this relation was not significant. These results suggest that 24-h urinary excretion after a single oral dose of isotopically labeled folate is a functional indicator of folate nutritional status that complements other measures of folate nutriture.

Kinetic model of folate metabolism in nonpregnant women consuming [2H2]folic acid: isotopic labeling of urinary folate and the catabolite para-acetamidobenzoylglutamate indicates slow, intake-dependent, turnover of folate pools.

In a 10-wk study of folate metabolism in nonpregnant women (21-27 y, n -6 per group), subjects were fed a diet containing approximately 68 nmol/d (30 microg/d) folate from food. The remainder of the ingested folate was provided as folic acid in apple juice (as nonlabeled during wk 1-2, as [2H2]folic acid during wk 3-10) to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 microg/d). Isotopic enrichment of total urinary folate and the primary catabolite para-acetamidobenzoylglutamate (ApABG) was determined. Isotopic enrichment of ApABG served as an indicator of labeling of tissue folates. A kinetic model consisting of fast- and slow-turnover nonsaturable pools and a saturable slow-turnover pool, with provisions for urinary and fecal excretion, catabolism and enterohepatic circulation, yielded a close fit to the data. Mean residence times for total body folate were 212, 169 and 124 d for folate intakes of 454, 680, and 907 nmol/d, respectively. The model predicted that variation in folate intake over this range had little effect on the mass of the large saturable folate pool; however, the fast-turnover nonsaturable pools increased in proportion to folate intake, whereas the slow nonsaturable pool also tended to increase. This model will aid in evaluation of folate turnover and in predicting kinetic consequences of physiologic conditions associated with altered folate requirements.

Episodic memory and the self in a case of isolated retrograde amnesia.

Isolated retrograde amnesia is defined as impaired recollection of experiences pre-dating brain injury with relatively preserved anterograde learning and memory. We present findings from a patient (M.L.) with isolated retrograde amnesia following severe traumatic brain injury (TBI) that address hypotheses of the interrelationships of focal neuropathology, episodic memory and the self. M.L. is densely amnesic for experiences predating his injury, but shows normal anterograde memory performance on a variety of standard tests of recall and recognition. The cognitive processes underlying this performance were examined with the remember/know technique, which permits separation of episodic from non-episodic contributions to memory tests by quantifying subjects' reports of re-experiencing aspects of the encoding episode. The results demonstrated that M.L. does not episodically re-experience post-injury events to the same extent as control subjects, although he can use familiarity or other non-episodic processes to distinguish events he has experienced from those he has not experienced. M.L.'s MRI showed damage to the right ventral frontal cortex and underlying white matter, including the uncinate fasciculus, a frontotemporal band of fibres previously hypothesized to mediate retrieval of specific events from one's personal past. Recent functional neuroimaging evidence of an association between right frontal lobe functioning and episodic retrieval demands suggest that M.L.'s memory deficits are related to this focal injury. This hypothesis was supported by right frontal polar hypoactivation in M.L. in response to episodic retrieval demands when he was examined with a cognitive activation H2(15)O PET paradigm that reliably activated this frontal region in both healthy controls and patients with TBI carefully matched to M.L. (but without isolated retrograde amnesia). He also showed increased left inferomedial temporal activation relative to control subjects, suggesting that his spared anterograde memory is mediated through increased reliance on medial temporal lobe structures. Re-experiencing events as part of one's past is based on autonoetic awareness, i.e. awareness of oneself as a continuous entity across time. This form of awareness also supports the formulation of future goals and the implementation of a behavioural guidance system to achieve them. The findings from this study converge to suggest that M.L. has impaired autonoetic awareness attributable to right ventral frontal lobe injury, including right frontal-temporal disconnection. Reorganized brain systems mediate certain preserved cognitive operations in M.L., but without the normal complement of information concerning the self with respect to both past and future events.

Normative data for equivalent, parallel forms of the Judgment of Line Orientation Test.

The Judgment of Line Orientation Test (JLO; Benton, Hamsher, Varney, & Spreen, 1983) permits assessment of visuospatial processing without making demands on motor skills. However, its administration can be time-intensive and frustrating for patients, particularly when used in a geriatric population. We present a single set of normative data to be used for both the odd-item and even-item forms derived from Form V of the JLO based on responses from a healthy geriatric sample. Mean scores and distributions of odd-item and even-item forms were nearly identical, and both forms showed significant correlations with the Developmental Test of Visual- Motor Integration. Cross-validation using the odd form of the JLO on an independent sample from a different geographic location suggested good generalizability of the normative data. We conclude that these JLO short-form normative data may be used in clinical screening situations or when serial assessments are needed.

Controlled and automatic forms of memory and attention: process purity and the uniqueness of age-related influences.

Estimates of controlled and automatic processes hypothesized to underlie performance in a memory task and in an attention task were derived for 115 participants from 18 to 78 years of age using the process-dissociation procedure. Participants also performed speed and neuropsychological tests that were suspected to be negatively related to age. Process estimates showed good reliability (from .76 to .98), and the qualitative distinction between processes was supported by the overall pattern of correlations among measures. However, only estimated automatic processes exhibited unique variance, as they were either weakly related or unrelated both to performance on the other tests and to each other. Estimates of the control processes, in contrast, shared considerable variance with measures from other tests, and there were no unique, or independent, age-related effects on these measures. The results highlight the need to distinguish between process purity and the uniqueness of age-related influences in accounting for age differences in cognition.

Kinetic modeling of folate metabolism through use of chronic administration of deuterium-labeled folic acid in men.

This study was conducted as an initial investigation of in vivo folate kinetics in healthy men (n = 4) and made use of a chronic-administration protocol with stable-isotope labeling. Subjects were given 0.453 mumol (200 micrograms) total folic acid in aqueous solution daily throughout the 18-wk study while they consumed self-selected folate-adequate diets. After a 2-wk pretrial period with unlabeled folic acid, subjects were given 0.227 mumol (100 micrograms) pteroyl-L-[2H4]glutamic acid/d ([2H4]folic acid) combined with 0.227 mumol nonlabeled folic acid or [2H2]pteroylhexaglutamic acid/d for the next 8 wk; then for the next 8 wk the [2H4]folic acid was withdrawn and the subjects received only nonlabeled folic acid. Little unmetabolized folic acid was excreted in urine. Isotopic enrichment of urinary folate during [2H4]folic acid administration and withdrawal was consistent with a kinetic model having a rapid turnover pool and a slow turnover pool. In contrast with previous two-pool models, provisions were made for folate turnover by urinary folate excretion (as measured here) and by fecal excretion and catabolic processes. The precision of modeling will be improved in future studies by measurement of enrichment of additional pools. However, this study shows clearly the slow turnover of the whole-body folate pool (< or = 1% per day) and the feasibility of further long-term kinetic analysis.

Bioavailability for humans of deuterium-labeled monoglutamyl and polyglutamyl folates is affected by selected foods.

Dietary folate exists mainly as polyglutamyl forms that require deconjugation by Zn-dependent pteroylpolyglutamate hydrolase prior to intestinal absorption. Because deconjugation by pteroylpolyglutamate hydrolase is an essential step in the absorption of dietary polyglutamyl folates, factors influencing the deconjugation process may affect folate bioavailability. This study was conducted to evaluate in vivo the bioavailability of [2H4]folic acid (d4-PteGlu1) and [2H2]-pteroylhexaglutamate (d2-PteGlu6) administered in solution in water or citrate buffer or added to selected foods using a single-dose, dual-label protocol. In each of six trials, healthy men (n = 7) were given a single oral dose of d2-PteGlu6 and d4-PteGlu1 (677 nmol of each form) blended into orange juice, tomatoes, lima beans, 52 mmol/L citrate (pH 4.1), or water as the control. Urine was collected for 48 h and the isotopic labeling of urinary folates used as criteria of the relative bioavailability of administered PteGlu1 and PteGlu6. Urinary excretion of d4-folates and d2-folates derived from the respective oral doses did not differ from the control in any treatment within the statistical power of this protocol. High relative bioavailability of the polyglutamyl folate was reflected by ratios of urinary d2/d4 folates of approximately 1.0 for control, tomato, lima bean and citrate buffer trials, whereas the ratio of urinary d2/d4 folates when subjects consumed orange juice was approximately 33% less than the control ratio (P < 0.05). These findings suggest that the bioavailability of polyglutamyl folates in orange juice would be partially incomplete. However, this would be compensated by the high total folate concentration of orange juice. The relation of these findings to endogenous dietary folates requires further investigation.

Conceptual automaticity in recognition memory: levels-of-processing effects on familiarity.

Recognition memory can reflect both conscious recollection and automatically generated feelings of familiarity. Previous research has suggested that perceptual factors mediate familiarity. Three experiments show that familiarity can also arise from prior conceptual (meaning-based) processing. Each experiment manipulated level of processing (LoP) and tested recognition memory using two response-signal delays (500 and 1500 ms). In Experiment 1, a modality effect was found for fast, but not slow, responses, thus supporting dual-process theories; the LoP effect was reliable at both points in time. In Experiment 2, recollection was set in opposition to familiarity by telling subjects to accept only test items from a to-be-remembered list which followed the incidental (LoP) study list; fast responses were associated with significantly more "false-alarms" to words encoded semantically than those encoded nonsemantically. Experiment 3 used the process dissociation procedure (Jacoby, 1991) to obtain quantitative estimates of recollection and familiarity. Both estimates were elevated by prior conceptual processing. Moreover, estimates of recollection, but not familiarity, were affected by response-signal delay, suggesting functional independence between the two processes. Relations to implicit memory are discussed.

Dissociation of processes underlying spatial s-r compatibility: evidence for the independent influence of what and where.

The process-dissociation procedure was used to estimate the influence of spatial and form-based processing in the Simon task. Subjects made manual (left/right) responses to the direction of arrows (> or <) presented to the left or right of fixation. Manipulating the proportion of incongruent trials (e.g., a right-pointing arrow presented to the left of fixation) affected both the size and direction of the Simon effect. To account for this pattern of data, we compared process estimates based on three possible relationships between spatial and form-based processing: independence, redundancy, and exclusivity. The independence model provided the best account of the data. Most telling was that independent form-based estimates were superior at predicting observed performance on arrows presented at fixation and did so consistently across conditions (r's > .80). The results provide evidence that the form ("what") and spatial location ("where") of a single stimulus can have functionally independent effects on performance. They also indicate the existence of two kinds of automaticity--an associative ("implicit learning") component that reflects prior S-R mappings and a nonassociative component that reflects the correspondence between stimulus and response codes.

Folic acid absorption in women with a history of pregnancy with neural tube defect.

Folic acid absorption was compared in nonpregnant women with a history of pregnancy with a neural tube defect (cases)(n = 10) with that of control women (n = 10) with a normal pregnancy history. [2H4]folic acid was administered in an oral dose (400 micrograms) to fasting case and control subjects after a 30-d saturation protocol involving daily ingestion of two 1-mg folic acid supplements. Serum and red blood cell folate concentrations were not different for case and control subjects before or during the saturation protocol (P > 0.05). The percentage (x +/- SD) of the oral dose of [2H4]folic acid excreted in 24-h urine collections postdose was not different (P > 0.05) for case compared with control subjects (9.05 +/- 2.25% and 11.10 +/- 3.41%, respectively). These data suggest that the absorption of folic acid routinely consumed in supplements and fortified food products is not impaired in women with a history of a pregnancy with a neural tube defect. Further case-controlled studies are needed to compare the absorption of the predominant dietary form of the vitamin.

Toward a redefinition of implicit memory: process dissociations following elaborative processing and self-generation.

Does conceptual processing affect unconscious uses of memory? We used a process-dissociation procedure to separate automatic (unconscious) and consciously controlled uses of memory in a stem-completion task. Contrary to results from indirect test conditions, estimates derived from our procedure showed no effect of self-generation and no differential effect of semantic and nonsemantic study conditions on automatic uses of memory. These results provide evidence that (a) indirect tests are often contaminated by conscious uses of memory and (b) stem completion is highly dependent on prior perceptual (and perhaps lexical) processing. The experiments demonstrate the advantages of using process-dissociation procedures over comparisons between direct and indirect tests.

Caribbean fruit fly,Anastrepha suspensa (Loew), attraction to host fruit and host kairomones.

Extracts of 22 fruits were tested for their attractancy toAnastrepha suspensa (Loew), the Caribbean fruit fly. Box-orange, calamondin, carambola, cattley guava, loquat, and Surinam-cherry were about equal in attractiveness to males and females. Nine synthetic chemicals, including four found in box-orange ripe seed, were attractive to females. Five synthetic chemicals, including two in box-orange ripe seed, were attractive to males. Farnesol,α-phellandrene, and 3-carene were highest in attractiveness to both males and females. Females were more attracted than males to 12 synthetic chemicals. These data suggest that host chemicals serve as attractants and that female and male specific attractants and traps could be developed from host kairomone data. These data also suggest that the volatilization of chemicals from water may play an important role in kairomone biology.

Urinary excretion of deuterium-labeled folate and the metabolite p-aminobenzoylglutamate in humans.

Stable isotopic methods were employed to determine the proportion of ingested folate excreted as the metabolite p-aminobenzoylglutamate (pABG, free and N-acetyl) in urine. Adult male subjects (n = 7) were maintained on a 4.54 mumol/d (2 mg/d) folate saturation regimen. After 7 d, subjects were given a mixed oral dose containing 0.67 mumol (300 micrograms) each of bideuterofolic acid (d2) and tetradeutero-folic acid (d4). Urine was collected for the following 48 h and analyzed for folate and pABG. The extent of deuterium labeling of urinary folate and pABG was determined by gas chromatography-mass spectrometry. Urinary total pABG excretion increased less than twofold as a result of the folate saturation, whereas urinary folate increased over 10-fold. Urinary d2 and d4 folates each contained 14-15% of the respective oral doses of labeled compounds, whereas urinary d2 and d4 pABG comprised only 0.98-1.15% of the labeled doses. Molar ratios (d2/d4) of excreted folate and pABG indicated that there was no in vivo isotopic discrimination between the labeled folates. Urinary pABG accounted for 5.1 +/- 0.6% of total ingested folate, whereas labeled pABG was about 6.7-7.6% of the excretion of labeled compounds (i.e., labeled folate + pABG). This study indicated that pABG is not a major excretory product during folate supplementation, but its relative importance may increase in conditions of reduced folate nutriture.

In vivo folate kinetics during chronic supplementation of human subjects with deuterium-labeled folic acid.

Six healthy men (22-31 y) were supplemented for 4 wk with folic acid labeled with deuterium [3',5'-2H2; 3.6 mumol/d (1.6 mg/d)] to permit evaluation of in vivo kinetics of this vitamin. Total folate in urine, serum and erythrocytes was determined by microbiological assay, and isotopic labeling of urinary and erythrocyte folate was determined by gas chromatography-mass spectrometry. During supplementation, serum folate reached maximal concentration in approximately 18 d, whereas excretion of total and deuterium-labeled folates increased rapidly and reached isotopic steady state in 1-2 wk. Isotopic labeling of erythrocyte folate increased continually over the entire supplementation period. Upon cessation of supplementation, red blood cell folate and urinary folate excretion (total and labeled) decreased linearly. The decline in total serum folate could be described with a biexponential model that yielded a slow-phase half-life of 18.7 +/- 2.3 d. This model also indicated a turnover of 4.5% of the total body folate pool per day. Pool sizes of total body folate before and after supplementation (at steady state) were calculated to be 10 mumol (4.4 mg) and 98.9 mumol (43.7 mg), respectively. These kinetic data and stable isotope methodology may be used to address a wide range of experimental questions related to folate metabolism.

Relative bioavailability of deuterium-labeled monoglutamyl tetrahydrofolates and folic acid in human subjects.

The bioavailability of orally administered monoglutamyl folic acid and various (6S)-tetrahydrofolates was examined in humans with stable-isotope methods. Folic acid (PteGlu), tetrahydrofolate (H4folate), 5-formyl-H4folate, 10-formyl-H4folate, and 5-methyl-H4folate were prepared for oral administration in 3',5'-2H2 labeled (d2) form, and [glu-2H4]folic acid (d4-PteGlu) was prepared for intravenous injection. In each of five trials, fasting adult males (n = 7) on a folate saturation regimen (2 mg/d) were given a single oral dose of one of the d2-folates in apple juice, as well as an intravenous injection of d4-PteGlu as a control. Urine was collected for 48 h and the isotope labeling of urinary folates determined by mass spectrometry. Isotope excretion ratios of urinary folates were used as criteria of bioavailability (pooled SE = 0.10): PteGlu (1.53, least squares mean), 10-formyl-H4folate (1.02), 5-methyl-H4folate (0.99), 5-formyl-H4folate (0.1.13), and H4folate (0.71). These results indicate that differences exist in the bioavailability of monoglutamyl folates under these experimental conditions. This variation, whether due to differences in absorption or postabsorptive events, must be considered in quantitative studies of folate utilization with this type of protocol.