[6S]-5-methyltetrahydrofolate increases plasma folate more effectively than folic acid in women with the homozygous or wild-type 677C-->T polymorphism of methylenetetrahydrofolate reductase.

BACKGROUND AND PURPOSE: 5,10-Methylenetetrahydrofolate reductase (MTHFR) is responsible for the synthesis of 5-methyltetrahydrofolate (5-MTHF). The 677C-->T mutation of MTHFR reduces the activity of this enzyme. The aim of this study was, first, to compare pharmacokinetic parameters of [6S]-5-MTHF and folic acid (FA) in women with the homozygous (TT) and wild-type (CC) 677C-->T mutation, and second, to explore genotype differences. The metabolism of [6S]-5-MTHF and FA was evaluated by measuring plasma folate derivatives. EXPERIMENTAL APPROACH: Healthy females (TT, n= 16; CC, n= 8) received a single oral dose of FA (400 microg) and [6S]-5-MTHF (416 microg) in a randomized crossover design. Plasma folate was measured up to 8 h after supplementation. Concentration-time-profile [area under the curve of the plasma folate concentration vs. time (AUC)], maximum concentration (C(max)) and time-to-reach-maximum (t(max)) were calculated. KEY RESULTS: AUC and C(max) were significantly higher, and t(max) significantly shorter for [6S]-5-MTHF compared with FA in both genotypes. A significant difference between the genotypes was observed for t(max) after FA only (P < 0.05). Plasma folate consisted essentially of 5-MTHF irrespective of the folate form given. Unmetabolized FA in plasma occurs regularly following FA supplementation, but rarely with [6S]-5-MTHF. CONCLUSIONS AND IMPLICATIONS: These data suggest that [6S]-5-MTHF increases plasma folate more effectively than FA irrespective of the 677C-->T mutation of the MTHFR. This natural form of folate could be an alternative to FA supplementation or fortification.

Randomized clinical trial of folate supplementation in patients with peripheral arterial disease.

BACKGROUND: The aim was to determine whether folate supplementation improved arterial function in patients with peripheral arterial disease (PAD). METHODS: Individuals with PAD were randomly assigned to receive 400 microg folic acid (45 patients) or 5-methyltetrahydrofolate (5-MTHF) (48) daily, or placebo (40) for 16 weeks. Primary endpoints were changes in plasma total homocysteine (tHcy), ankle : brachial pressure index (ABPI) and pulse wave velocity (PWV). Secondary outcomes were changes in plasma inflammatory markers. RESULTS: Plasma tHcy was significantly reduced in folic acid and 5-MTHF groups compared with controls: median difference: - 2.12 (95 per cent confidence interval - 3.70 to - 0.75) micromol/l (P = 0.002) and - 2.07 (-3.48 to - 0.54) micromol/l (P = 0.007) respectively. ABPI improved significantly: median difference 0.07 (0.04 to 0.11) (P < 0.001) and 0.05 (0.01 to 0.10) (P = 0.009) respectively. Brachial-knee PWV (bk-PWV) decreased significantly in individuals receiving 5-MTHF and tended to be reduced in those taking folic acid compared with controls: median difference: - 1.10 (-2.20 to - 0.20) m/s (P = 0.011) and - 0.90 (-2.10 to 0.00) m/s (P = 0.051) respectively. Plasma levels of inflammatory markers were not affected. CONCLUSION: Folate administration reduced plasma homocysteine, and slightly improved ABPI and bk-PWV.

Research goals for folate and related B vitamin in Europe.

In the past decade, the understanding of folate bioavailability, metabolism and related health issues has increased, but several problems remain, including the difficulty of delivering the available knowledge to the populations at risk. Owing to the low compliance of taking folic acid supplements, for example, among women of child-bearing age who could lower the risk of having a baby with a neural tube defect, food-based strategies aimed at increasing the intake of folate and other B-group vitamins should be a priority for future research. These should include the development of a combined strategy of supplemental folate (possibly with vitamin B(12)), biofortification using engineered plant-derived foods and micro-organisms and food fortification for increasing folate intakes in the general population. Currently, the most effective population-based strategy to reduce NTDs remains folic acid fortification. However, the possible adverse effect of high intakes of folic acid on neurologic functioning among elderly persons with vitamin B(12) deficiency needs urgent investigation. The results of ongoing randomized controlled studies aimed at reducing the prevalence of hyperhomocysteinemia and related morbidity must be available before food-based total population approaches for treatment of hyperhomocysteinemia can be recommended. Further research is required on quantitative assessment of folate intake and bioavailability, along with a more thorough understanding of physiological, biochemical and genetic processes involved in folate absorption and metabolism.

[6S]5-methyltetrahydrofolate or folic acid supplementation and absorption and initial elimination of folate in young and middle-aged adults.

OBJECTIVES: To assess the effects of supplementation with the diastereoisomer of 5-methyltetrahydrofolate ([6S]5-methylTHF), as an alternative supplement for folic acid, on folate absorption and elimination, in two age groups. DESIGN: A randomized, double-blind intervention study. SUBJECTS: A total of 12 young (<30 y) and 12 middle-aged (> or =50 y) healthy volunteers were recruited. METHODS: Volunteers were randomized to receive daily supplementation with 400 mug folic acid or equimolar amounts of [6S]5-methylTHF during 5 weeks. Before and after supplementation, absorption and initial elimination were calculated following oral [(2)H(2)]folic acid test doses using isotope kinetics in plasma. RESULTS: Folic acid absorption was lower in the middle-aged as compared to the young adults, both before (P = 0.03) and after (P = 0.05) supplementation. In the young adults, absorption decreased by 22% after [6S]5-methylTHF and increased by 21% after folic acid (P = 0.02). In the other age group, no such changes were found. The folate rate constant of elimination increased after folic acid supplementation in the young (+50%; P = 0.05) but not in the middle-aged (+18%; P = 0.5) adults. CONCLUSIONS: Young adults show increased folate turnover after folic acid supplementation relative to the effect of [6S]5-methylTHF supplementation. Similar differences are not observed in middle-aged adults, in whom folic acid absorption was found to be lower as compared to the young adults. SPONSORSHIP: Financial support was received from the European Union 5th Framework Programme (Grant QLRT-1999-00576).

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism.

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.

Single oral doses of 13C forms of pteroylmonoglutamic acid and 5-formyltetrahydrofolic acid elicit differences in short-term kinetics of labelled and unlabelled folates in plasma: potential problems in interpretation of folate bioavailability studies.

Single (13)C6-labelled doses of pteroylmonoglutamic acid (PteGlu; 634 nmol) or 5-formyltetrahydrofolic acid (431-569 nmol) were given to fasted adult volunteers, and the rise in total and (13)C-labelled plasma 5-methyltetrahydrofolic acid metabolite monitored over 8 h by HPLC and liquid chromatography-MS. The dose-adjusted area under the curve (AUC) for total (labelled plus unlabelled) plasma 5-methyltetrahydrofolic acid following a 5-formyltetrahydrofolic acid test dose was 155 % that obtained following a PteGlu test dose. Surprisingly, an average 60 and 40 % of the total plasma 5-methyltetrahydrofolic acid response to [(13)C6]PteGlu and [(13)C6]5-formyltetrahydrofolic acid, respectively, was unlabelled; an observation never before reported. Short-term kinetics of plasma [(13)C6]5-methyltetrahydrofolic acid showed a slower initial rate of increase in plasma concentration and longer time to peak following an oral dose of [(13)C6]PteGlu compared with that for an oral dose of [(13)C6]5-formyltetrahydrofolic acid, while the [(13)C6]5-methyltetrahydrofolic acid AUC for [(13)C6]5-formyltetrahydrofolic acid was 221 % that for [(13)C6]PteGlu. These data indicate that PteGlu and 5-formyltetrahydrofolic acid, which are thought to be well absorbed (about 90 %) at physiological doses, exhibit dramatically different rates and patterns of plasma response. A limitation in the rate of reduction of PteGlu before methylation could result in slower mucosal transfer of [(13)C6]5-methyltetrahydrofolic acid derived from [(13)C6]PteGlu into the plasma. This, when coupled with an observed similar plasma clearance rate for [(13)C6]5-methyltetrahydrofolic acid metabolite derived from either folate test dose, would yield a comparatively smaller AUC. These findings suggest potential problems in interpretation of absorption studies using unlabelled or labelled folates where the rate of increase, the maximum increase, or the AUC, of plasma folate is employed for test foods (mainly reduced folates) v. a 'reference dose' of PteGlu.

Certification of B-group vitamins (B1, B2, B6, and B12) in four food reference materials.

In 1989, the Community Bureau of Reference started a research program to improve the quality of vitamin analysis in food. To achieve this task, vitamin methodology was evaluated and tested by interlaboratory studies and the preparation of certified reference materials, which will be used for quality control of vitamin measurements. The main improvements in methodology were achieved by testing and standardizing the extraction condition and enzymatic hydrolysis procedures. Results for each individual material are derived from five replicate determinations using at least two independent methods: liquid chromatography (HPLC) and microbiological assay for vitamins B1, B2, and B6; and radioprotein binding and microbiological assays for vitamin B12. The certificate of analysis for four reference materials gives mass fraction values for water-soluble vitamins. These certified values were based on the acceptable statistical agreement of results from collaborating laboratories. Certified values with uncertainties (mg/kg dry matter) for each CRM are as follows: 4.63 (0.20) and 4.10 (0.51) for vitamins B1 and B6, respectively, in CRM 121 (wholemeal flour); 6.51 (0.24), 14.54 (0.3), 6.66 (0.43), and 0.034 (0.003) for vitamins B1, B2, B6, and B12, respectively, in CRM 421 (milk powder); 3.07 (0.17) and 4.80 (0.40) for vitamins B1 and B6, respectively, in CRM 485 (lyophilized mixed vegetables), and 8.58 (0.55), 106.8 (2.8), 19.3 1.5), and 1.12 (0.044) for vitamins B1. B2, B6, and B12, respectively, in CRM 487 (lyophilized pig liver).

Erythrocyte folate analysis: saponin added during lysis of whole blood can increase apparent folate concentrations, depending on hemolysate pH.

BACKGROUND: The analysis of red cell folate (RCF) depends on complete hemolysis of erythrocytes, and it is assumed that complete hemolysis is achieved by 10-fold dilution of whole blood with hypotonic solutions of 10 g/L ascorbic acid/ascorbate. This report challenges this assumption. METHODS: The conventional method of erythrocyte lysis was modified to include saponin, a known effective hemolyzing agent. The influence of saponin was determined at various lysate pHs, using the microbiological (Lactobacillus rhamnosus) folate assay. The effect of saponin during lysate preparation was subsequently compared with either the effect of 30 s of sonication or a single 1-h freeze-thaw cycle. RESULTS: Saponin addition was found to increase assayable RCF up to ninefold, depending on lysate pH. Sonication of lysates had no effect, and freezing-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) without pH adjustment produced assayable folate concentrations significantly lower than optimal. CONCLUSIONS: A lysing agent should be incorporated into RCF assays to guarantee complete hemolysis. Ten-fold dilution of blood with 10 g/L ascorbic acid, without pH adjustment, produces lysates with pHs (pH 4.0) below the point (pH 4.7) at which hemoglobin can denature irreversibly. The optimum pH for hemolysates is approximately 5.0.

Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay.

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.

Effects of lycopene and lutein supplementation on the expression of functionally associated surface molecules on blood monocytes from healthy male nonsmokers.

It has been suggested that dietary carotenoids can enhance immune function. Supplementation with beta-carotene (15 mg daily) was previously shown to enhance human monocyte function. To examine the effect of other dietary carotenoids, two similar independent studies were done. Healthy adult male nonsmokers were randomly assigned to receive lycopene (study 1), lutein (study 2), or placebo for 26 days, followed by the alternative treatment for another 26 days. The expression of functionally related monocyte surface molecules was quantified by laser flow cytometry before and after each treatment period. There was a significant increase in plasma levels of each carotenoid following dietary supplementation, but the effects on monocyte surface molecule expression were not as striking as those observed after beta-carotene supplementation. These findings emphasize that it cannot be assumed that the effect of one carotenoid will be the same as another, even at the same level of intake.

Erythrocyte folate analysis: a cause for concern?

Neural tube defects can be prevented by adequate intake of periconceptional folate, and inverse associations between folate status and cardiovascular disease and various cancers have been noted. Thus, there is renewed interest in the analysis of red cell folate (RCF) as an indicator of folate deficiency risk. Assessment of the assumptions that underpin RCF assays indicates that many are false. Published literature suggests that increased deoxy-hemoglobin (which can bind RCF electrostatically) yields more assayable folate, and increased oxy-hemoglobin (which cannot bind RCF) yields less assayable folate. It is argued that as deoxy-hemoglobin picks up oxygen and switches quaternary structure, any bound folate must, on purely theoretical grounds, become physically "trapped". Venous blood taken for analysis is 65% to 75% saturated with oxygen, and pro-rata "trapping" will lead to serious underestimation of RCF. Hence, doubt is cast over the validity of all previous RCF values. Some strategies for accurately assessing RCF are suggested.

The effect of beta-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers.

Although there is strong epidemiologic evidence that diets rich in carotenoids such as beta-carotene are associated with a reduced incidence of cancer, the cellular mechanisms underlying this phenomenon remain unknown. This article describes the effect of dietary beta-carotene supplementation on both the expression of functionally associated surface molecules on human monocytes and on the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha) by monocytes, all of which are involved in the initiation and regulation of immune responses involved in tumor surveillance. A double-blind, placebo-controlled, crossover study was undertaken in which 25 healthy, adult male nonsmokers were randomly assigned to receive beta-carotene (15 mg daily) or placebo for 26 days, followed by the alternative treatment for a further 26 days. The expression of functionally related monocyte surface molecules was quantified by flow cytometry, and ex vivo secretion of TNF-alpha was quantified by an enzyme-linked immunosorbent assay, before and after each treatment period. After dietary supplementation there were significant increases in plasma levels of beta-carotene and in the percentages of monocytes expressing the major histocompatibility complex class II molecule HLA-DR and the adhesion molecules intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. In addition, the ex vivo TNF-alpha secretion by blood monocytes was significantly increased after supplementation. These findings suggest that moderate increases in the dietary intake of beta-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anticarcinogenic properties attributed to beta-carotene.

Relationships between micronutrient intake and biochemical indicators of nutrient adequacy in a "free-living' elderly UK population.

Nutritional assessments are frequently based on amounts of nutrients consumed. In the present paper the usefulness of nutrient intake data for assessing nutrient adequacy is examined in an elderly British population. Subjects were "free-living' elderly aged 68-90 years (sixty men, eighty-five women) in Norwich. Forty-two of forty-nine surviving males and sixty-seven of seventy-nine surviving females were reassessed after 2 years. With few exceptions, estimated micronutrient intake was not statistically predictive of biochemical measures of nutrient adequacy. Initial biochemical measures of nutritional adequacy were compared with those found 2 years later in an attempt to assess whether initial biochemical assessment was predictive of the "longer term' situation. Biochemical measurements at the start of the study were correlated to the same measurements made 2 years later for: serum ferritin, haemoglobin and erythrocyte count, whole-blood Se-glutathione peroxidase (EC 1.11.1.9; males only), plasma Cu, alkaline phosphatase (EC 3.1.3.1), ascorbic acid, vitamin B6 (pyridoxal-5-phosphate), folate and vitamin B12, total erythrocyte thiamin (males only), riboflavin (erythrocyte glutathione reductase (EC 1.6.4.1) activation coefficient): but not for: erythrocyte Cu-superoxide dismutase (EC 1.15.1.1) or plasma Zn. Either only small changes, or no changes, in mean values were seen over the 2 years for most of the biochemical measures. One exception was a large increase in plasma folate. The only important "negative' features seen at 2-year follow up were a large fall in serum ferritin concentration and a large increase in the activity of two antioxidant defence enzymes, superoxide dismutase and glutathione peroxidase. As judged by currently accepted biochemical deficiency threshold values, a small proportion of subjects were possibly at risk of Fe (3% men; 1% women), folate (7%, 3%), thiamin (12%; 3%) and vitamin C (15%; 17%) deficiency. Many more appeared to be at risk of vitamin B6 (42%; 47%) and riboflavin (77%; 79%) deficiency. It was concluded that the requirements of the elderly for vitamins B1, B2 and C, and the biochemical deficiency threshold values used to indicate vitamin B6 deficiency, need review.

Nutrient intake and biochemical status of non-instutionalized elderly subjects in Norwich: comparison with younger adults and adolescents from the same general community.

The Department of Health (1992) has recently stated that 'Nutritional reviews concerning elderly people are especially constrained by lack of data', and that much of the emphasis in the nutritional literature has been placed on the study of institutionalized, and often chronically ill, elderly subjects rather than the non-institutionalized elderly who form the majority of this population. The present study presents information on the dietary intake and biochemical status of non-institutionalized elderly subjects (68-73 and 74-90 years) and compares such data with those obtained for adult (20-64 years) and adolescent (13-14 years) populations living within the same community. Nutrient intakes and appropriate biochemical measurements of nutrient status, performed on fasting blood samples, were statistically examined and have been discussed in relation to potential age-related influences. The nutrient intake of elderly subjects was on a par with adolescents of corresponding sex but generally lower than that of adult counterparts. There were several significant differences in biochemical measurements of nutrient status between age groups. In general these did not suggest progressive age-related trends. However, there were significant suggestions of age-related increases in whole-blood glutathione peroxidase (EC 1.11.1.9) activity, serum ferritin, plasma cholesterol, LDL and triacylglycerol concentrations and decreases in plasma HDL and ascorbic acid concentrations. The significance of these differences is discussed. An age-related difference (suggestive of a decline) in vitamin C status together with a difference (suggestive of an increase) in glutathione peroxidase activity may indicate an imbalance in the regulation of O2-derived free-radicals with ageing. These observations are worthy of a further study in the light of current thinking which relates the induction of a number of diseases to oxidative damage.

Thiamin intake, erythrocyte transketolase (EC 2.2.1.1) activity and total erythrocyte thiamin in adolescents.

The relationships between thiamin intake, erythrocyte transketolase (EC 2.2.1.1) activity coefficient (ETK-AC) and total erythrocyte thiamin were investigated in a group of adolescents (13 to 14 years old; nineteen boys, thirty-five girls). Thiamin intakes were calculated from 7 d weighed records, using food composition tables, and compared with those obtained by direct analysis of duplicate diets. Average 7 d calculated thiamin intakes were significantly lower than analysed intakes for both sexes. On an individual basis, calculated intakes ranged from 30 to 143% of corresponding analysed values. Analysed and calculated intakes were significantly correlated when expressed as mg/d; however, when expressed in terms of energy intake, the correlation was significant for males only. Thiamin intake appeared largely adequate when compared with current UK dietary recommendations (Department of Health, 1991), but the limitations of such comparisons are considered. The major food groups contributing to thiamin intake were examined and showed breakfast cereals to contribute more than 25% of dietary thiamin. A proportion of the subjects had ETK-AC values in ranges usually associated with marginal or severe thiamin deficiency. There was, however, no statistically significant relationship between erythrocyte thiamin and basal or stimulated transketolase activity, or between thiamin intake and either of the methods used to assess status. The need to re-evaluate indices of thiamin status is discussed.

Dietary intake and micronutrient status of adolescents: effect of vitamin and trace element supplementation on indices of status and performance in tests of verbal and non-verbal intelligence.

Relationships between micronutrient intake and status, and micronutrient status and performance in tests of intelligence were investigated in a group of adolescents (13-14 years old). Dietary intakes were assessed using a 7 d weighed dietary record method, coupled with the collection of duplicate diets. Vitamin and trace mineral intakes calculated using food composition tables were compared with those obtained by direct analysis of duplicate diets. Micronutrient status was judged via a range of biochemical indices measured in blood samples taken after a 12-15 h fast. Blood samples were taken both before and after a 16-week period of vitamin and trace mineral supplementation. Individual tests of verbal and nonverbal intelligence were also performed pre- and post-supplementation. The results of this study indicate that the use of food table data may lead to substantial over- or underestimation of the intake of several micronutrients. In general, the total calculated or analysed amount of a specific micronutrient consumed did not adequately predict status, as judged by a range of biochemical indices. There were significant changes in status measurements over the 16-week study period, irrespective of supplementation, and these changes were markedly influenced by the initial status of the subject. There was no effect of supplementation on performance in tests of intelligence. However, there was a significant association between plasma ascorbic acid and initial non-verbal intelligence quotient (IQ) in the boys, and between whole blood glutathione peroxidase (EC 1.11.1.9) activity and non-verbal and verbal IQ in both sexes. These findings are discussed in relation to other recent studies of the influence of micronutrient supplementation on the psychological performance of children.