The pharmacokinetic advantage of 5-methyltetrahydrofolate for minimization of the risk for birth defects.

Despite efforts to increase folic acid (FA) intake, even within countries mandating FA fortification, there remain pregnant women with folate levels inadequate to minimize congenital disorders (e.g., of the neural tube, heart, and lip/palate). The pharmacokinetics of FA and [6S]-5-methyltetrahydrofolate (5-MTHF) were examined to find a reliable and minimal dose for rapidly rescuing folate status prior to critical periods of embryonic development. Serum total folate increased much more rapidly over the first four days in insufficient women given 7.5 mg doses of 5-MTHF than the same regimen of FA (P for trend <0.0001). Nearly all women given 7.5 mg 5-MTHF (every 12 hours, five doses total) almost immediately reached 50 nM serum total folate. Moreover, this level could be maintained by subsequent administration of 0.4 mg/d of folic acid. Thus, 5-MTHF enables repletion of folate stores more quickly and uniformly than FA and without exposure to unmetabolized FA.

Differential coulometric oxidation following post column-switching high pressure liquid chromatography for fluorescence measurement of unmetabolized folic acid in human plasma.

Although many countries have fortified their grain supplies with folic acid (FA) to decrease the incidence of neural tube defects, others have not due to concerns that this synthetic folate might have some adverse effects. Persistent unmetabolized FA has been found even in plasma from fasted subjects. To facilitate measurement of low levels of folic acid in human plasma, post-column coulometric oxidative cleavage was used to convert poorly fluorescent FA into a highly fluorescent compound determined to be 6-formyl-pterin. To minimize sample work-up and maximize recovery, column-switching HPLC transferred a window of eluate containing the FA from the first column (C8) onto a second column (phenyl-hexyl). The pH of two mobile phases were adjusted to be above and then below a pK of the FA α-carboxyl group, thus promoting separation from compounds coeluting from the C8-column. This permitted sample preparation using only a simple high recovery protein precipitation. Definitive identification of FA in human plasma was accomplished by duplicate injections of sample with the electrochemical voltage set above and below its half-potential. The LOD (S/N=3) was 0.10 nM. The intra- and inter-assay CV's were 2.3% and 5%, respectively. Comparison of these results with those obtained by HPLC/MS/MS with stable isotope internal standard showed a slope of 1.00 ± 0.019. This simple, sensitive, and repeatable assay facilitates a more thorough investigation of the response of various human populations to folic acid intake. Post-column differential coulometric electrochemistry can expand the variety of compounds amenable to fluorescence detection.

Effect of serum folate status on total folate and 5-methyltetrahydrofolate in human skin.

BACKGROUND: It has been suggested that human skin color adapts to balance the need for vitamin D synthesis in comparison with the protection of DNA and folate from photodegradation. However, the folate content of human skin is unknown and may affect the effectiveness of the antifolate methotrexate for the treatment of psoriasis. OBJECTIVES: We examined whether total folate and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) in human skin can be predicted by serum concentrations and whether there are differences in the proportion of 5-MTHF in dermis compared with epidermis. DESIGN: Total folate (by using a microbiological assay) and 5-MTHF (by using high-pressure liquid chromatography) were measured in fasting serum and fresh skin obtained at surgery by using a recovery validated extraction method. RESULTS: Total folate in human epidermis was shown to be low compared with in many other tissues, and dermal folate was an order-of-magnitude even lower. These concentrations were directly and linearly linked to serum folate status. Although the percentage of 5-MTHF of the total in the dermis was similar to that in other organs, it was especially high in the epidermis and increased to >65% as serum folate decreased. CONCLUSIONS: The high proportion of 5-MTHF in the epidermis, which is further emphasized in subjects with a lower (10-20-nmol/L) serum folate status, points to a special role for this form of folate in skin, perhaps as a protectant from ultraviolet-induced photosensitization reactions. 5-MTHF may also maintain methylation reactions that influence the proliferative activity. These results may help to individualize the treatment of psoriasis patients with methotrexate and folate.

Unmetabolized folic acid prevalence is widespread in the older Irish population despite the lack of a mandatory fortification program.

BACKGROUND: In 2006 the Food Safety Authority of Ireland recommended mandatory folic acid fortification of flour for the prevention of neural tube defects in addition to the existing extensive voluntary folic acid fortification culture in place there. This recommendation is now suspended until further scientific evidence surrounding safety becomes available. The safety issues include concerns about the masking of vitamin B-12 deficiency and potential cancer acceleration, both of which may be of concern for the elderly population. OBJECTIVE: The aim of this study was to measure the basal (fasted) concentrations of unmetabolized folic acid in the plasma of an elderly population group exposed to this liberal voluntary fortification of foodstuffs in Ireland. DESIGN: We invited participants aged 60-86 y from the Lifeways Cross-Generation Cohort Study to participate in this project. After providing informed consent, the participants were invited to provide fasting blood samples and to complete a standard food-frequency questionnaire and a questionnaire on recent and habitual intakes of folic acid. Samples were assayed for total plasma folate, red blood cell folate, homocysteine, and unmetabolized folic acid. RESULTS: A total of 137 subjects with a mean age of 67.4 y were studied. Unmetabolized folic acid was detected in 94.1% of the cohort with a mean concentration of 0.39 nmol/L (range: 0.07-1.59 nmol/L), accounting for 1.3% of total plasma folate. CONCLUSION: These results indicate unmetabolized folic acid in plasma in most of this elderly Irish cohort, even after an overnight fast. These results should be considered carefully by those legislating in this area.

Persistent circulating unmetabolised folic acid in a setting of liberal voluntary folic acid fortification. Implications for further mandatory fortification?

BACKGROUND: Ireland is an example of a country that has extensive voluntary fortification with folic acid. After a public consultation process, in 2006, the Food Safety Authority in Ireland FSAI 1 recommended mandatory fortification. However due to safety considerations this decision is now on hold. Before mandatory fortification goes ahead, existing levels of unmetabolised folic acid and their anticipated increase after fortification needs investigation because of the potential of folic acid to mask pernicious anaemia and possibly accelerate the growth of existing cancers. The aim of this study was to examine the levels of circulatory unmetabolised folic acid in Irish adults (both fasted and un-fasted) and new-born infants (fasted) before the proposed implementation of mandatory folic acid fortification. A secondary aim was to predict the increase in circulatory unmetabolised folic acid levels after fortification. METHODS: Study 1. SETTING: Irish Blood Transfusion Service (IBTS). Whole blood samples were collected from blood donors (n=50) attending for routine blood donation sessions (representing the general population). Subjects were not fasted prior to sampling. Study 2. SETTING: Coombe Women's and Infant's University Hospital, Dublin. Whole blood samples were collected by venipuncture from mothers (n=20), and from their infant's umbilical-cords (n=20) immediately after caesarean section. All women had been fasted for at least 8 hours prior to the surgery. A questionnaire on habitual and recent dietary intakes of folic acid was administered by an interviewer to all subjects. The data collection period was February to April 2006. Serum samples were analysed for plasma folate, plasma folic acid and red cell folate. RESULTS: Blood Donor Group: Circulatory unmetabolised folic acid was present in 18 out of 20 mothers (fasted) (CI: 68.3%-99.8%) comprising 1.31% of total plasma folate, 17 out of 20 babies (fasted) (CI: 62.1%-96.8%), and 49 out of 50 blood donors (unfasted) (CI: 88.0%-99.9%), comprising 2.25% of total plasma folate, CONCLUSION: While the levels of circulatory unmetabolised folic acid reported are low, it is persistently present in women immediately after caesarean section who were fasting indicating that there would be a constant/habitual exposure of existing tumours to folic acid, with the potential for accelerated growth. Mandatory fortification might exacerbate this. This has implications for those with responsibility for drafting legislating in this area.

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake.

Numerous clinical trials using folic acid for prevention of cardiovascular disease, stroke, cognitive decline, and neural tube defects have been completed or are underway. Yet, all functions of folate are performed by tetrahydrofolate and its one-carbon derivatives. Folic acid is a synthetic oxidized form not significantly found in fresh natural foods; to be used it must be converted to tetrahydrofolate by dihydrofolate reductase (DHFR). Increasing evidence suggests that this process may be slow in humans. Here we show, using a sensitive assay we developed, that the reduction of folic acid by DHFR per gram of human liver (n = 6) obtained from organ donors or directly from surgery is, on average, less than 2% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a 5-fold variation of DHFR activity among the human samples. This limited ability to activate the synthetic vitamer raises issues about clinical trials using high levels of folic acid. The extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. These results are also consistent with the reports of unmetabolized folic acid in plasma and urine.

Determinants of oligomerization of the bifunctional protein DCoHalpha and the effect on its enzymatic and transcriptional coactivator activities.

DCoH and DCoHalpha are bifunctional proteins that function as 4a-hydroxytetrahydrobiopterin dehydratases and as coactivators of HNF1alpha-dependent transcription. Although these isoforms share sequence and structural similarity and equivalent enzyme activities, DCoH is a hyperstable tetramer whereas DCoHalpha readily forms dimers. Differences in quaternary structure affect the formation of the DCoH(alpha):HNF1alpha complex. Because the interface used to bind HNF1alpha is masked in tetrameric DCoH, the DCoH:HNF1alpha complex is only formed in vivo, presumably by co-translational folding. Conversely, the DCoHalpha:HNF1alpha complex readily forms in vitro. We identified residues in DCoHalpha that differed from those in the dimer-dimer interface of tetrameric DCoH. Mutating these residues altered the quaternary state and concomitantly the ability of the mutated proteins to affect HNF1alpha-dependent DNA binding. Our results indicate that three residues, Asn61, Gln45, and Lys98 in DCoHalpha play a role in oligomeric flexibility, which enables DCoHalpha to more readily interact with HNF1alpha and increase DNA binding.

5-Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA.

The known functions of folate are to support one-carbon metabolism and to serve as photoreceptors for cryptochromes and photolyases. We demonstrate that 5-methyltetrahydrofolate (5-MTHF, the predominant folate in plasma) is also a potent, near diffusion limited, scavenger of singlet oxygen and quencher of excited photosensitizers. Both pathways result in decomposition of 5-MTHF, although ascorbate can protect against this loss. In the absence of photosensitizers, 5-MTHF is directly decomposed only very slowly by UVA or UVB. Although synthetic folic acid can promote DNA damage by UVA, submicromolar 5-MTHF inhibits photosensitization-induced strand breaks. These observations suggest a new role for reduced folate in protection from ultraviolet damage and have bearing on the hypothesis that folate photodegradation influenced the evolution of human skin color.

Can the DCoHalpha isozyme compensate in patients with 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH deficiency?

4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1alpha (HNF1). Patients with a deficiency of dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, dehydratase/DCoHalpha, has been detected which shares 60% identity with dehydratase/DCoH. This investigation was undertaken to ascertain if dehydratase/DCoHalpha has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHalpha demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHalpha, demonstrates that DCoHalpha does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The dehydratase activity of each isoform was measured by a direct spectrophotometric assay. Km and Vmax for DCoHalpha were both 2-3 times higher than for DCoH, thus leaving the catalytic efficiency (Vmax/Km) the same for both enzymes. In conclusion, the properties of dehydratase/DCoHalpha are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in dehydratase/DCoH.