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Background: Evidence is conflicting as to whether the bioavailability of food folates is influenced by the extent of their conjugation., Objective: The objective was to compare the bioavailability of 3 representative food folate sources with various degrees of glutamylation-ie, egg yolk, spinach, and yeast, whose polyglutamyl folate content measured 0%, 50%, and 100%, respectively., Design: In a randomized crossover trial, 13 male subjects, after a prestudy folate saturation procedure, received in random order either placebo or 500 mug total folate, which was provided as concentrated freeze-dried extract removed from the normal food matrix of egg yolk, spinach, or yeast. Blood samples (n = 10) were collected before and up to 10 h after treatments, which were administered at weekly intervals., Results: A significant increase from baseline plasma folate concentrations was observed by 0.5 h after treatment with egg yolk folate or spinach folate and by 1 h after treatment with yeast folate, and the concentrations remained significantly elevated for 3-5 h; no plasma folate response was observed after placebo treatment. The overall responses, calculated as plasma folate area under the curve (AUC) for egg yolk, spinach, and yeast folate, were 122.6 +/- 23.6, 136.2 +/- 21.4, and 102.5 +/- 21.1 nmol . h/L, respectively. No significant differences in AUC were seen between monoglutamyl (egg yolk) folate and either of the polyglutamate-containing folates examined., Conclusion: These results suggest that the ratio of monoglutamate to polyglutamate in natural folates is not a factor that limits the extent of intestinal absorption of food folate.

The natural folate derivative, 5-methyltetrahydrofolate ([6S]-5-MTHF), could be an option for supplementation and fortification but its bioavailability remains unclear. This study compared the bioavailability of [6S]-5-MTHF with that of folic acid (FA) by measuring plasma folate responses after a single ingestion of equivalent doses of the two folate forms. In a double-blind, crossover study, 13 men (presaturated with FA) received in random order each of the following treatments administered orally at 1-wk intervals: 1) placebo capsule; 2) 500 micro g FA capsule; and 3) 500 micro g [6S]-5-MTHF capsule. Plasma total folate concentrations were measured before and up to 10 h after each treatment (n = 10 samples per treatment). Plasma folate concentrations increased significantly (compared with baseline) from 0.5 to 5 h after both folate treatments. The maximum plasma folate response did not differ between the two treatments (mean +/- SEM, 33.4 +/- 3.9 vs. 31.8 +/- 3.9 nmol/L, P = 0.7, for FA and [6S]-5-MTHF, respectively) and typically occurred in individuals between 0.5 and 3 h postprandially. The area under the plasma folate response curve was significantly greater after both folate treatments compared with placebo, and the response did not differ between the treatments. These results indicate that the short-term bioavailabilities of [6S]-5-MTHF and FA are equivalent. Supplementation with the natural folate derivative could have all the beneficial effects associated with FA, but without the potential disadvantage of masking the anemia of vitamin B-12 deficiency.

Folate intake is strongly influenced by various methods of cooking that can degrade the natural forms of the vitamin in foods. The aim of the present study was to determine the effect of different cooking methods on folate retention in various foods that contribute to folate intake in the UK diet. Typical purchasing and cooking practices of representative food folate sources were determined from a questionnaire survey of local shoppers (n 100). Total folate was determined by microbiological assay (Lactobacillus casei NCIMB 10463) following thermal extraction and tri-enzyme (alpha-amylase, protease and conjugase) treatment in raw foods and after typical methods of cooking. Boiling for typical time periods resulted in only 49 % retention of folate in spinach (191.8 and 94.4 microg/100 g for raw and boiled spinach respectively; P<0.005), and only 44 % in broccoli (177.1 and 77.0 microg/100 g for raw and boiled broccoli respectively, P<0.0001). Steaming of spinach or broccoli, in contrast, resulted in no significant decrease in folate content, even for the maximum steaming periods of 4.5 min (spinach) and 15.0 min (broccoli). Prolonged grilling of beef for the maximum period of 16.0 min did not result in a significant decrease in folate content (54.3 and 51.5 microg/100 g for raw and grilled beef respectively). Compared with raw values, boiling of whole potatoes (skin and flesh) for 60.0 min did not result in a significant change in folate content (125.1 and 102.8 microg/100 g for raw and boiled potato respectively), nor was there any effect on folate retention whether or not skin was retained during boiling. These current results show that the retention of folate in various foods is highly dependent both on the food in question and the method of cooking. Thus, public health efforts to increase folate intake in order to improve folate status should incorporate practical advice on cooking.

Background: Fasting plasma total homocysteine (tHcy) decreases during pregnancy. Previous reports suggested that this is due to folic acid supplementation, hemodilution, or a decrease in albumin. However, these hypotheses have not been tested in a longitudinal study., Objective: We investigated the relation between pregnancy-related physiologic changes and tHcy in a group of healthy women who were either unsupplemented or supplemented with folic acid., Design: In a longitudinal study from preconception throughout pregnancy, we studied 54 unsupplemented women and 39 women who were supplemented with folic acid during the second or third trimester of pregnancy. tHcy, hematocrit, and serum albumin were determined preconceptionally and at 8, 20, and 32 wk of pregnancy., Results: For the entire group, geometric mean tHcy concentrations at preconception (8.2 micro mol/L) were significantly greater (P < 0.001) than those at 8 wk of pregnancy (6.4 micro mol/L). When the unsupplemented and supplemented groups were regarded separately, geometric mean tHcy concentrations at preconception were significantly greater than those at 20 (5.22 and 4.18 micro mol/L, respectively) and 32 (5.16 and 4.42 micro mol/L, respectively) wk of pregnancy (P < 0.001 for both). Mean reductions from preconception concentrations at 8, 20, and 32 wk of pregnancy were significantly greater (P < 0.001) for tHcy (-11.5%, -25.5%, and -24.5%, respectively) than for hematocrit (-1.9%, -4.2%, and -4.3%, respectively) or serum albumin (-1.1%, -9.8%, and -13.4%, respectively). There was no correlation between changes in either hematocrit or serum albumin and changes in tHcy., Conclusions: This study refutes the previous explanations for the reduction in plasma tHcy known to occur in pregnancy, namely, folic acid supplementation, hemodilution, and a decrease in serum albumin. We suggest that the changes may be endocrine-based.

Objective: To determine the plasma concentration of total homocysteine (tHcy), a recognized risk factor for vascular disease, in patients with type 1 diabetes and to examine the relationships with age, sex, duration of diabetes, microvascular complications and neuropathy, and folic acid concentration., Research Design and Methods: Plasma tHcy and folic acid concentrations were measured in a randomly selected cohort of type 1 diabetic patients (n = 119), well characterized as regards microvascular complications, and in a matched control group (n = 51)., Results: Plasma tHcy was higher in male than in female control subjects (geometric mean [95% CI]: 9.3 [8.0-10.9] vs. 6.1 [5.2-7.2] micromol/l, P < 0.001), as previously described, but there was no sex difference in diabetic patients. Plasma tHcy significantly correlated with age in patients (r = 0.348, P < 0.01) but not in control subjects (r = 0.007, P = 0.96). Male patients without microvascular complications had lower plasma tHcy concentrations than did male control subjects (6.2 [5.1-7.5] vs. 9.3 [8.0-10.9] micromol/l, P < 0.001), but values in female patients without complications were similar to those of female control subjects. Plasma folic acid concentration was higher in diabetic patients than in control subjects. The expected negative association between plasma tHcy and folic acid was stronger in control subjects than in patients., Conclusions: Subnormal tHcy concentrations in male patients, the absence of a sex difference, and the positive association with age indicate that homocysteine metabolism differs between type 1 diabetic patients and control subjects. Homocysteine is unlikely to be of pathogenic significance in patients, particularly male subjects, with early microvascular disease and/or neuropathy.

Thermolabile 5,10-methylenetetrahydrofolate reductase (MTHFR) is the first folate-related variant to be associated with an increased risk of neural tube defects (NTDs). The variant causes high plasma homocysteine levels and reduced red cell folate (RCF) levels, both of which have also been linked to an increased risk of NTDs. We examined the relationship between folate status and presence of the common mutation MTHFR C677T in 82 NTD-affected and 260 control mothers. Homozygosity for the TT genotype was associated with very low folate status among both the cases (n = 13) and the controls (n = 21). However, after exclusion of TT homozygotes, only 10% of the remaining 240 controls had RCF levels less than 200 microg/L compared with 29% of the 69 cases (odds ratio, 3.67; 95% confidence interval, 1.88-7.18; P < 0.001), and those with RCF less than 150 microg/L had eight times higher risk of NTD than subjects with levels over 400 microg/L. Plasma homocysteine levels of non-TT cases were also higher than those of controls (P = 0.047). This study shows that homozygosity for the C677T MTHFR variant cannot account for reduced blood folate levels in many NTD-affected mothers. Thus, a strategy of genetic screening of all childbearing women for this variant would be ineffective as a method of primary prevention of NTDs. The data suggest that low maternal folate status is itself the major determinant of NTD risk, or else that other folate-dependent genetic variants confer risk through the reduction of folate levels. These results emphasize the importance of a food-fortification program as a population strategy for reducing the occurrence of NTDs.

It is now well established that folic acid, when taken periconceptionally, can prevent many neural tube defects. It is also becoming clear that folic acid does not work by correcting a nutritional deficiency in pregnant women. Rather, it appears that a metabolic defect is responsible for these neural tube defects and that this defect or defects can be corrected by a sufficiently large dose of folic acid. Our recent work demonstrates that homocysteine metabolism is likely to be the critical pathway affected by folic acid. We have demonstrated significantly higher homocysteine levels in women carrying affected fetuses than in control women. These findings indicate that one of the enzymes responsible for homocysteine metabolism is likely to be abnormal in affected pregnancies. Animal studies suggest that the conversion of homocysteine to methionine could be the critical step. Rat embryos in culture require methionine for neural tube closure. Methionine synthase, cystathionine synthase, and 5,10 methylene tetrahydrofolate reductase are all important in the metabolism of homocysteine in humans. If methionine synthase is the critical enzyme, it would raise the interesting public health issue that vitamin B-12 might be able to stimulate the abnormal enzyme as folic acid does. Adding vitamin B-12 might make it possible to reduce the dose of folic acid required in fortified food, thus allaying concerns about overexposure to folic acid.

The amount of biologically active folate excreted in the urine corresponds to a small fraction of the recommended dietary allowance, suggesting that a large amount of folate is excreted as its catabolites. A strategy of assessing folate requirement by measuring the daily urinary levels of products of folate catabolism depends on the demonstration of an exclusive mechanism of breakdown as well as a suitable marker of the catabolic process. Rats were given [3H] and [14C]folic acid by gastric intubation daily for 10 d to simulate normal dietary intake of the vitamin. Total urine was collected throughout this period as well as for the following 10 d. Reverse-phase HPLC of the radiolabeled urinary products revealed the presence of a variety of intact folates as well as products of C9-N10 scission of the folate molecule, pteridines, para-aminobenzoylglutamate and para-acetamidobenzoylglutamate. We detected no other N10-containing catabolites, nor did we find the oxidized folate derivative '4 alpha-hydroxy-5-methyltetrahydrofolate'. Of all the urinary folate metabolites, only para-acetamidobenzoylglutmate persisted at high levels up to 10 d after radiolabel treatment was withdrawn. We conclude that folate catabolism occurs exclusively through C9-N10 cleavage and that measurement of urinary para-acetamidobenzoylglutmate provides a suitable indicator of daily folate turnover.

Folic acid taken around the time of conception can prevent many neural-tube defects. Women with low-normal vitamin B12 values may also be at increased risk. We considered whether homocysteine metabolism via the enzyme methionine synthase, which requires both folate and B12, could be the critical defect in folate-related neural tube defects. Blood was obtained during pregnancies that produced 81 infants with neural-tube defects and 323 normal children. Samples were assayed for homocysteine, methylmalonic acid, plasma folate, red-cell folate, and B12. Mothers of children with neural-tube defects had significantly higher homocysteine values (8.62 [SD 2.8] mumol/L) than did B12-matched controls (7.96 [2.5] mumol/L, p = 0.03). The difference was significant (p = 0.004) in the lower half of the B12 distribution after adjusting for plasma folate. Our study shows that an abnormality in homocysteine metabolism, apparently related to methionine synthase, is present in many women who give birth to children with neural-tube defects. Overcoming this abnormality is likely to be the mechanism by which folic acid prevents neural-tube defects. These findings suggest that the most effective periconceptional prophylaxis to prevent neural-tube defects may require B12 as well as folic acid.

The relationship between growth rate and folate catabolism was determined using a catch-up growth model in weanling rats. A recently developed HPLC method was employed to compare the urinary excretion of the major end product of folate catabolism, acetamidobenzoylglutamate (apABGlu), in normally growing (n = 8) and age-matched growth-restricted (n = 8) weanling animals, during and after food restriction. During the 12-d period of food restriction in which restricted rats were fed (per gram of body weight) at a level of 50% of the required dietary intake for normal growth, apABGlu excretion was significantly higher in normally growing rats compared with growth-restricted rats. In the latter group after restriction, apABGlu concentrations increased markedly, tending to exceed those of normally growing rats of comparable age. Thus growth retardation resulted in a decreased rate of folate catabolism, whereas accelerated growth following dietary restriction was associated with an increase in catabolism. Because early growth in rats is predominantly hyperplastic, these results suggest that the rate of folate catabolism is related to folate utilization in cell division.

The hypothesis examined in the present study is that pregnancy results in a greater rate of folate catabolism. Mammalian folate catabolism proceeds by cleavage of the vitamin at the C9-N10 position to yield p-aminobenzoylglutamate, which is quantitatively excreted in the urine after its acetylation. A recently developed HPLC method for the determination of endogenous levels of this catabolite, acetamidobenzoylglutamate, in rat urine was used to investigate folate catabolism in three groups of female rats (n = 6/group), one nonpregnant and two pregnant groups. One of the pregnant groups was allowed to feed freely whereas the other was pair-fed to the nonpregnant control group. The daily excretion of acetamidobenzoylglutamate was stable in the nonpregnant group over the experimental period but increased significantly with progression of gestation in both pregnant groups, in which values peaked at d 18 to concentrations of up to three times those of the nonpregnant animals. In both pregnant groups the values fell significantly before parturition, demonstrating that increased catabolism of folate is a feature of pregnancy per se and not simply due to increased weight. These results suggest that increased catabolism may make an important contribution to the folate deficiency associated with pregnancy.

All official recommendations for folate tend to be far in excess of current estimated intakes. Several recent studies have highlighted this, showing satisfactory folate status in healthy populations consuming considerably less than official RDAs. This study takes a critical look at current RDAs for folate. We have recently developed an assay to quantify the end product of folate catabolism in human urine, thus affording the possibility of a more rational and scientific approach to setting the RDA for folate. Results of this study (now confirmed by further work) suggest that, under normal dietary conditions, the body appears to catabolize only about 100 micrograms total folate per day. Furthermore, this rate of catabolism remained relatively unchanged in response to saturating doses of a 5 mg folic acid supplement over several days. It is therefore most unlikely that our diet is as seriously deficient in folate as current official RDAs seem to imply.