June E. Ayling

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.

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

BackgroundIreland 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.MethodsStudy 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 Womens and Infants University Hospital, Dublin. Whole blood samples were collected by venipuncture from mothers (n = 20), and from their infants 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.ResultsBlood 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,ConclusionWhile 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.

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 ascor‐bate 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, submicromo‐lar 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 photo‐degradation influenced the evolution of human skin color.–Offer, T., Ames, B. N., Bailey, S. W., Sabens, E. A., Nozawa, M., Ayling, J. E. 5‐Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA. FASEB J. 21, 2101–2107 (2007)

Hyperphenylalaninemia with high levels of 7-biopterin is associated with mutations in the PCBD gene encoding the bifunctional protein pterin-4a-carbinolamine dehydratase and transcriptional coactivator (DCoH)

Pterin-4a-carbinolamine dehydratase (PCD) is required for efficient tetrahydrobiopterin regeneration after phenylalanine hydroxylase activity. This catalytic function was proposed to be specifically defective in newborns with a mild form of hyperphenylalaninemia (HPA) and persistent high urinary levels of primapterin (7-biopterin). A second regulatory task of the same protein is DCoH, a coactivation of transcription by hepatocyte nuclear factor 1alpha (HNF-1alpha), a function that is apparently not impaired in these HPA individuals. It has been shown elsewhere that the human PCD/DCoH bifunctional protein is encoded by a single 4-exon-containing gene, PCBD, located on chromosome 10q22. We have now examined the PCBD gene for mutations at the genomic level in six such HPA patients from four different families. By the use of new intron-specific primers, we detected, in all six patients, single, homozygous nucleotide alterations, in exon 4, that were inherited from their parents. These homozygous alterations predicted mutant PCD/DCoH with a single amino acid exchange, in two cases (alleles T78I), or premature stop codons, in the other four patients (alleles E86X and Q97X). Recombinant expression in Escherichia coli revealed that the mutant proteins-T78I, E86X, and Q97X-are almost entirely in the insoluble fraction, in contrast to wild type, which is expressed as a soluble protein. These data support the proposal that HPA in combination with urinary primapterin may be due to autosomal recessive inheritance of mutations in the PCBD gene specifically affecting the dehydratase activity.

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.

The Mechanism of Cofactor Regeneration During Phenylalanine Hydroxylation

Although the ability of the liver to convert phenylalanine to tyrosine has been known since 19131, characterization of the components of the system did not begin until the mid 1950’s. Tetrahydrobiopterin was identified as the cofactor for the hydroxylase reaction2, which uses molecular oxygen as the source of the new hydroxyl group, Figure 1. It was first thought that only one other enzyme, dihydropteridine reductase (DHPR), was involved in the overall process. This converts the oxidized form of cofactor, a quinoid dihydropterin3, back to the starting tetrahydrobiopterin at the expense of NADH allowing cofactor to be used catalytically. The clinical symptoms of patients with a deficiency of DHPR have shown that cofactor regeneration is essential4.

Rearrangement and depletion of folate in human skin by ultraviolet radiation

DEAR EDITOR, Exposure to sunlight, especially its ultraviolet (UV) component, is a major risk factor for several forms of skin cancer. Both the more energetic UVB and the more abundant and deeply penetrating UVA (used in modern tanning beds) stimulate the progression of melanoma. Long duration or high frequency use of tanning beds and sunlamps has been associated with increased risk for basal and squamous cell carcinoma, while meta-analysis also suggests a link to melanoma. Skin colour has been proposed to have evolved to balance the need for vitamin D biosynthesis at higher latitudes with protecting not only DNA but also folate from the more intense radiation near the equator. It has been suggested that skin cancer could have been responsible for early development of protective pigmentation. By slowing photodegradation of the folate pool, dark skin may also help prevent birth defects. Blood folate levels have been reported in several studies to not decrease following exposure of humans to UVA or sunlight, except in subjects consuming folic acid supplements, folic acid itself being a photosensitizer. 5–Methyltetrahydrofolate (5–MTHF, the predominant folate in the epidermis and circulation) quenches photosensitizers and scavenges singlet oxygen (thus protecting DNA from strand breaks during UV irradiation). 5–MTHF may be lost specifically in the skin while blocking photo-oxidation reactions (Fig. 1). Damage by UV irradiation to DNA in skin is well documented. While folate loss in keratinocytes has also been studied, nothing is understood about the effect of UV on folate directly in skin, the largest of human organs. Therefore, to reveal the potential vulnerability of skin folate, after obtaining Institutional Review Board approval, we undertook a pilot experiment exposing fresh ex vivo whole human skin to UVA (Data S1 and S2; see Supporting Information). Relative to an unexposed section of skin subjected to the same conditions, total folate decreased by about 28% after 187 J cm 2 UVA (similar to the maximal daily UVA dose at sea level near the equator) (Fig. 2a; Data S3; see Supporting Information). The average loss of total folate from the epidermal samples (210 pmol g 1 tissue) signifies a decrease of about 120 nmol upon exposure of the total body skin area. This is a very small portion of the folate in the whole body (60–225 lmol). Therefore, after reaching homeostasis with the other organs, a single dose of UVA, as used in the current study, would not be expected to produce a major change in overall folate status. However, folate loss could be meaningful

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.

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 CVs 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.

Nicotinic Cholinergic Regulation of Tetrahydrobiopterin Levels in Bovine Adrenal Chromaffin Cells

Catecholamines (CA) in adrenergic neurons are maintained at a relatively constant level despite wide variations in neural activity1. The mechanism of this regulation is the modulation of the conversion of tyrosine to dopa, the rate limiting step in CA biosynthesis. The enzyme catalyzing this step, tyrosine hydroxylase2 (TH; EC 1.14.16.2; 1-tyrosine, tetrahydropteridine:oxygen oxidoreductase, 3-hydroxylating), is subject to a variety of regulatory mechanisms. Acutely, TH is regulated by negative feedback inhibition of TH by intracellular CA3 and activation through phosphorylation of TH by four second messenger-activated protein kinases4. At the chronic level the quantity of TH is regulated through trans-synaptic mediated increase in the synthesis of enzyme molecules5.