Background: Current thinking, which is based mainly on rodent studies, is that physiologic doses of folic acid (pterylmonoglutamic acid), such as dietary vitamin folates, are biotransformed in the intestinal mucosa and transferred to the portal vein as the natural circulating plasma folate, 5-methyltetrahydrofolic acid (5-MTHF) before entering the liver and the wider systemic blood supply.Objective: We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa.Design: We conducted a crossover study in which we sampled portal and peripheral veins for labeled folate concentrations after oral ingestion with physiologic doses of stable-isotope labeled folic acid or the reduced folate 5-formyltetrahydrofolic acid (5Formy1THF) in 6 subjects with a transjugular intrahepatic porto systemic shunt (TIPSS) in situ. The TIPSS allowed blood samples to be taken from the portal vein.Results: Fifteen minutes after a dose of folic acid, 80 12% of labeled folate in the hepatic portal vein was unmodified folic acid. In contrast, after a dose of labeled 5-Formy1THF, only 4 18% of labeled folate in the portal vein was unmodified 5-Formy1THF, and the rest had been converted to 5-MTHF after 15 min (postdose).Conclusions: The human gut appears to have a very efficient capacity to convert reduced dietary folates to 5-MTHF but limited ability to reduce folic acid. Therefore, large amounts of unmodified folic acid in the portal vein are probably attributable to an extremely limited mucosal cell dihydrofolate reductase (DHFR) capacity that is necessary to produce tetrahydrofolic acid before sequential methylation to 5-MTHF. This process would suggest that humans are reliant on the liver for folic acid reduction even though it has a low and highly variable DIAFR activity. Therefore, chronic liver exposure to folic acid in humans may induce saturation, which would possibly explain reports of systemic circulation of unmetabolized folic acid.

The fractional absorption of a stable isotope-labeled folate dose can be estimated from the subsequent short-term temporal changes in the concentration of labeled L-5-methyltetrahydrofolate (L-5-methyl-THF) in plasma using mathematical modeling. However, the model is dependent on the use of an accurate value for the apparent volume of distribution of L-5-methyl-THF. Previous studies that estimated the apparent volume of distribution of L-5-methyl-THF used large (nonphysiological) doses of unlabeled folates that are not found to any great extent in the circulatory system. The current study estimates the apparent volume of distribution at steady state in 16 healthy humans aged 18-65 y after an i.v. dose (440 nmol) of a stable isotope-labeled version of the naturally circulating plasma folate, L-5-methyl-THF. Blood was collected from 2 min to 2 h postinjection and plasma assayed by specific and sensitive liquid chromatography-tandem MS. The apparent volume of distribution for L-5-methyl-THF was 32.0 ± 11.6 L (mean ± SD; 392 ± 110 mL/kg bodyweight). There was a positive association with volunteer body weight (r = 0.64; P = 0.010), which allowed a simple linear equation to be developed relating apparent volume of distribution to body weight. This has important implications for predicting apparent absorption of labeled folates in future bioavailability studies.

Scope: The objective was to perform an inventory and critical evaluation of folate data in selected European and international databases. The ultimate aim was to establish guidelines for compiling standardized folate databases for international nutritional studies.Methods and results: An ad hoc questionnaire was prepared to critically compare and evaluate folate data completeness, quantification, terminologies, and documentation of 18 European and international databases, and national fortification regulations. Selected countries participated in the European Prospective Investigation into Nutrition and Cancer project and European Food Information Resource Network (EuroFIR). Folate completeness was generally high. "Total folate" was the most common terminology and microbiological assay was the most frequently reported quantification method. There is a lack of comparability within and between databases due to a lack of value documentation, the use of generic or non-appropriate terminologies, folate value conversions, and/or lack of identification of synthetic folic acid.Conclusion: Full value documentation and the use of EuroFIR component identifiers and/or INFOODS tagnames for total folate ("FOL") and synthetic folic acid ("FOLAC"), with the additional use of individual folates, will increase comparability between databases. For now, the standardized microbiological assay for total folate and HPLC for synthetic folic acid are the recommended quantification methods.

Folic acid (pteroylmonoglutamic acid) has historically been used as the reference folate in human intervention studies assessing the relative bioavailability of dietary folate. Recent studies using labelled folates indicated different plasma response kinetics to folic acid than to natural (food) folates, thus obviously precluding its use in single-dose experiments. Since differences in tissue distribution and site of biotransformation were hypothesised, the question is whether folic acid remains suitable as a reference folate for longer-term intervention studies, where the relative bioavailability of natural (food) folate is assessed based on changes in folate status. Healthy adults aged 18-65 years (n 163) completed a 16-week placebo-controlled intervention study in which the relative bioavailability of increased folate intake (453 nmol/d) from folate-rich foods was assessed by comparing changes in plasma and erythrocyte folate concentration with changes induced by an equal reference dose of supplemental (6S)-5-methyltetrahydrofolic acid or folic acid. The relative increase in plasma folate concentration in the food group was 31 % when compared with that induced by folic acid, but 39 % when compared with (6S)-5-methyltetrahydrofolic acid. The relative increase in erythrocyte folate concentration in the food group when compared with that induced by folic acid was 43 %, and 40 % when compared with (6S)-5-methyltetrahydrofolic acid. When recent published observations were additionally taken into account it was concluded that, in principle, folic acid should not be used as the reference folate when attempting to estimate relative natural (food) folate bioavailability in longer-term human intervention studies. Using (6S)-5-methyltetrahydrofolic acid as the reference folate would avoid future results' validity being questioned.

Objective: Hyperhomocysteinaemia is associated with peripheral arterial disease (PAD). There are inter-individual variations in the metabolism of homocysteine because of genetic polymorphisms. This study analyzed the role of one polymorphism that is associated with raised homocysteine, as a risk factor for PAD.Methods. This study considered the association of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with the incidence of PAD by performing a case-control study and a cross sectional study of homocysteine levels. We recruited 133 patients with PAD in Norfolk and compared the MTHFR allele distribution with 457 healthy individuals. We also carried out a meta-analysis to place our data within the context of other published studies. We searched Medline, Embase, and Cochrane databases up to March 2008 for any studies on the association between MTHFR C677T polymorphism and PAD.Results. The MTHFR C677T allele frequencies in the cases and controls were 0.37 and 0.33, and the odds ratios for the association of the 677 T allele or TT genotype with PAD were 1.18 (95% Confidence Interval [CI] 0.89, 1.58) and 1.99 (95% CI 1.09, 3.63). Homozygotes for the MTHFR C677T mutation had higher concentrations of plasma total homocysteine, odds ratio 2.82 (95% CI 1.03, 7.77) compared to homozygotes for the MTHFR 677 CC genotype.Twelve of 72 articles retrieved from the database search reported the prevalence of mutations in PAD patients. A meta-analysis of 9 appropriate studies, including our own, showed that being homozygous for the C677T allele was associated with an increased risk of PAD, pooled odds ratio 1.36 (95% CI 1.09, 1.68).Conclusion: We have found a strong association between raised homocysteine, the TT genotype, and PAD. (J Vasc Surg 2009;49:711-8.)

The association between novel atherosclerotic risk biomarkers and severity of peripheral arterial disease (PAD) was assessed. Patients (n = 133) with PAD were recruited. Established risk biomarkers including low- and high-density cholesterol, triglycerides, and blood pressure were measured. Novel risk biomarkers including plasma C-reactive protein, von Willebrand factor (vWF), interleukin 6, red cell folate (RCF), vitamin B12, total homocysteine (tHcy), and Hcy genotypes were also determined. The severity of PAD was evaluated, using ankle—brachial pressure index (ABPI), brachial—knee, and brachial—ankle pulse wave velocity (bk- and ba-PWV). Plasma tHcy and systolic blood pressure had a positive independent correlation with bk-PWV (β = +0.56, P = .02 and β = +0.38, P < .001, respectively). Red cell folate had an independent inverse correlation with bk-PWV (β = —0.01, P = .01). Systolic blood pressure showed an independent positive correlation with ba-PWV only after adjustment for other risk biomarkers (β = +0.1, P = .04). Novel markers, plasma tHcy, and RCF levels correlated with the severity of PAD.

Following an introduction of the importance of folates and the rationale for seeking to estimate fractional folate absorption from foods (especially for countries not having a mandatory folic acid fortification policy), scientific papers covering the mechanisms of folate absorption and initial biotransformation are discussed. There appears (post-1983) to be a consensus that physiological doses of folic acid undergo biotransformation in the absorptive cells of the upper small intestine to 5-methyltetrahydrofolic acid (as happens for all naturally-occurring reduced 1-carbon-substituted folates). This 'validates' short-term experimental protocols assessing 'relative' folate absorption in human subjects that use folic acid as the,reference' dose. The underlying scientific premise on which this consensus is based is challenged on three grounds: (i) the apparent absence of a 5-methyltetrahydrofolic acid response in the human hepatic portal vein following absorption of folic acid, (ii) the low dihydrofolate reductase activity peculiar to man and (iii) the implications derived from recent stable-isotope studies of folate absorption. It is concluded that the historically accepted case for folic acid being a suitable 'reference folate' for studies of the 'relative absorption' of reduced folates in human subjects is invalid. It is hypothesised that the liver, and not the absorptive cells of the upper small intestine, is the initial site of folic acid metabolism in man and that this may have important implications for its use as a supplement or fortificant since human liver's low capacity for reduction may eventually give rise to saturation, resulting in significant (and potentially deleterious) unmetabolised folic acid entering the systemic circulation.

We tested the hypothesis that some cereal-product vehicles may reduce fortificant bioavailability below 85% and examined the feasibility of using single dose, dual-label, non-saturation protocols for studying bioavailability based on urinary excretion ratios (UER; % oral 13C6 isotope dose excreted in intact folate/% IV 2H4 isotope dose excreted). Fifteen females received 225 μg oral folate (capsules, fortified bran flakes, and fortified white bread), mainly as 13C6-PteGlu, followed by IV injection of 100 μg 2H4-PteGlu. UERs were used as the primary index of absorption. Urinary folate was cleaved to p-aminobenzoylglutamic acid, dervivatized and determined by gas chromatography/mass spectroscopy (GC/MS). The UER mean (95%CI) for folic acid was 2.18 (1.2-3.8) at 48 hours and as these were greater than 1.0, it was concluded that oral and IV isotopes of folic acid are handled differently by the body. Compared to the 48 hour UER for folic acid, UERs for white bread and bran flakes were 0.71 and 0.37, respectively, thus indicating some matrix inhibition of absorption. Consideration should be given to the choice of cereal-based fortification vehicles in order to maximize bioavailability. Plasma enrichment of folate can be measured using LC/MSMS (liquid chromatography/mass spectroscopy-mass spectroscopy) but seems unfeasible with the GC/MS method.

Since its discovery in 1931 by Lucy Wills, and its first isolation in 1941 by Mitchell, Snell and Williams, our understanding of the fascinating world of folic acid and one-carbon metabolism, and its role in health and disease, has come a long way. However, there is still much to do in perfecting methods to measure folate bioavailability, and status, with a high degree of precision and accuracy. Future examination of the relationships of common gene polymorphims involved in folate bioavailability (folate polyglutamate deconjugation and carrier-mediated absorption) and one-carbon metabolism (methylation cycle, folate cycle and DNA synthesis/repair) to folate status, morbidity, mortality and longevity, need to be considered concurrently rather than as a series of individual associations, as has been the usual practice.

The bioavailability of folates from foods has become a popular area of interest. A common method of bioavailability measurement in human volunteers is using foods extrinsically and intrinsically labelled with stable isotopes. Chromatographic techniques such as high-performance liquid chromatography, gas chromatography or mass spectrometry are utilised for the separation and identification of folates from plasma and urine samples collected during dietary intervention, although most procedures at present are long winded and time consuming. This paper gives a short overview of the use of extrinsic and intrinsic labels for the use of studying folate absorption and metabolism. It also describes the production of an intrinsically labelled spinach for use in intervention studies, and discusses a simplified method for folate analysis and detection.

Using a simplified version of Johnson’s core model analysis of the plastic zone, one may determine the plastic zone size around a contact or, alternatively, determine the yield strength by measuring the plastic zone. The theoretical model contains three parameters: indentation load, yield strength and zone size so that knowing any two gives the third. This is experimentally demonstrated for a series of single crystals (Fe–3wt%Si, tungsten, zinc) and polycrystals (1100-0 Al, copper and 2024-T6 aluminum). In addition, two of these are evaluated in several work-hardened states. Plastic zone sizes are estimated principally by atomic force microscopy and Zygo interferometry imaging with some verification by transmission electron microscopy. On the theoretical side, verification of the relationship is obtained by elastic–plastic numerical analysis of a bi-material system based upon an Fe–3wt%Si single crystal with a thin oxide film. It is shown that in general predictions are reasonable down to nanometer level contacts except for two cases where an indentation size effect may dominate. The proposed relationship is suggested to be an alternative measure of yield strength compared to the often cited value of H/3, particularly at light contacts.

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.

The effects of sugar-beet fibre and wheat bran on iron and zinc absorption in rats The dietary habits and patterns of Third World countries imposed on them by economic and financial constraints have now been acclaimed as being universally beneficial (Cannon, 1990). Thus the World Health Organization (1990) stated that the health needs of the population are best met by a high carbohydrate low fat diet, rich in starchy foods (e.g. cereals, tubers and pulses) including a substantial intake of vegetables and fruits. A daily fibre intake of 30 g/d has also been recommended (World Health Organization, 1990). It is important that the negative aspects of this eating habit are considered along with the recommendations. This is particularly necessary because of the mineral imbalances characteristic of the people who have, over the years, subsisted on such diets (Hercberg et al. 1987). It was therefore pertinent to read the article entitled ‘The effects of sugar-beet fibre and wheat bran on iron and zinc absorption in rats’ by Fairweather-Tait & Wright (1990). The authors reported that the addition of 1 g sugar-beet fibre (Beta fibre) to 3 g semi-synthetic diet resulted in a 54 YO increase in Fe and a 39 O h increase in Zn absorption in rats. I quite agree with the feeding of the semi-synthetic diet to the rats for 2 weeks before the introduction of the test diet, as it has been demonstrated that the Fe contents of previous diets do influence Fe absorption from test diets (Fairweather-Tait & Wright, 1984). However, I feel strongly that the FeSO,. 7H,O component of the semi-synthetic diet should be excluded in the formulation of the test diets. Hence, the semi-synthetic diet contributed 34 and 42 YO of the total Fe in the Beta fibre and wheat bran diets respectively. These values are significant and therefore make the interpretation of the results difficult, particularly because the total non-haem Fe in the diet forms a common pool in the gastrointestinal tract in which there is complete isotopic exchange of Fe (Bjorn-Rasmussen et al. 1973). It is quite difficult to envisage the proportion of the test Fe that is absorbed. Apart from the low phytate level in Beta fibre, the various carbohydrate components could in fact be enhancing the FeSO, .7H,O fraction of the test diet. I presume the interaction in the gut of the food components will be more complex than when the test diet provides the only source of Fe. Since the Beta fibre has a naturally high level of Fe, i.e. 326 ,ug/g, would it not be appropriate to determine the availability of this Fe source alone? Perhaps it could even be fed as a component of a standard typical breakfast meal. What do the authors think about the practice of substituting test food items in semi-synthetic meals with reference to human diets? Couldn’t the test meals be administered to the rats, as is done in human 59Fe availability studies? I agree with the authors that the preliminary results should be interpreted with caution in the application to man. G. 0. LATUNDE-DADA Department oJ Biochemistry, Obafemi Aurolowo College of Health Sciences, Ogun State University, AgoZuroy e, Niger ia

Folic acid fortification, mandatory in the United States, is currently being considered by the UK. The hypothesis that the matrix of some cereal-product vehicles may result in low fortificant bioavailability was tested using a dual oral/intravenous (i.v.) isotopic-label approach, which was evaluated concurrently. Fifteen women received 225 g oral folate (capsules, fortified white bread and fortified branflakes), mainly as folic acid labeled with 13C on 6 car- bons of the benzoyl ring (13C6-PteGlu), followed by i.v. in- jection of 100 g folic acid labeled with 2H on 4 hydrogens of the glutamic acid group (2H4-PteGlu). The urinary excre- tion ratio (UER) in intact folate of the percentage of labeled oral dose excreted divided by the percentage of i.v. dose excreted was used as the primary index of absorption. The geometric mean (95% confidence interval) UER for folic acid capsules was 3.68 (1.90, 7.14) at 24 h and 2.18 (1.24, 3.83) at 48 h. Because these were significantly in excess of 1.0, indicative of 100% absorption of the oral dose, it was concluded that oral and i.v. labeled folic acid are handled differently by the body and that ''absolute'' absorption can- not be calculated. Compared with the 48-h UER for folic acid capsules, the ''relative'' 48-h UER for white bread and branflakes was 0.71 and 0.37, respectively, indicating that some cereal-based vehicles may inhibit absorption of for- tificant. However, even the validity of this "relative" ap- proach is questioned. J. Nutr. 132: 936 -939, 2002.