Identifying sources of measurement error in assessing dietary intakes - Results of a multi-country ring-trial.

BACKGROUND AND AIMS: Epidemiological investigations include dietary intakes as primary exposures or potential confounders. To reduce bias, data collection protocols include the administration of questionnaires together with measurements of biomarkers. Some error, however, remains and needs to be considered in the analysis and interpretation of results. The European Food Safety Authority supported a ring-trial to compare the precision and reproducibility of dietary assessment methods applied in Europe. METHODS AND RESULTS: Software applications used to collect 24-hour recalls and food records in six countries (Estonia, Italy, Latvia, Portugal, Spain, and Sweden) were assessed. The intake of 256 foods was identically reported to each method. Experienced interviewers participated and were instructed to repeat national protocols closely. The error in recording quantities, compared with reference values, was variable but in about 60% of recorded quantities was in the range of ±20%. Errors were however unsystematic and independent of the food type or quantification method used - although food pictures performed better. The reproducibility of some tools was limited. The methods generally captured additional ingredients (usually flavoring agents), but not sweetening agents or fortification and failed to record packaging information in about 60% of the cases. CONCLUSION: In a design that eliminated respondent bias, this study indicates that softwares, supporting databases and interviewers generally introduce random error in dietary assessments. The inclusion of large sample sizes and food pictures to quantify portions, together with enhanced attention on interviewers' training, standardisation of procedures and regular tool upgrades are essential in assuring a study's quality and comparability.

ePlantLIBRA: A composition and biological activity database for bioactive compounds in plant food supplements.

The newly developed ePlantLIBRA database is a comprehensive and searchable database, with up-to-date coherent and validated scientific information on plant food supplement (PFS) bioactive compounds, with putative health benefits as well as adverse effects, and contaminants and residues. It is the only web-based database available compiling peer reviewed publications and case studies on PFS. A user-friendly, efficient and flexible interface has been developed for searching, extracting, and exporting the data, including links to the original references. Data from over 570 publications have been quality evaluated and entered covering 70 PFS or their botanical ingredients.

EuroFIR eBASIS: application for health claims submissions and evaluations.

BACKGROUND: The European Food Information Resource (EuroFIR) network has established the eBASIS (Bioactive Substances in Food Information System) online food composition and biological effects database for plant-derived bioactive compounds (phytochemicals). On the basis of submitted evidence, the European Food Safety Authority (EFSA) expert panel on Dietetic Products, Nutrition and Allergies assesses whether claims made under articles 13.1, 13.5 or 14 of the Regulation (EC) 1924/2006, which governs the use of nutrition and health claims on foods, are scientifically justified. This report evaluates the eBASIS biological effects database in the preparation and evaluation of health claims dossiers. METHODS: The eBASIS biological effects database is a compilation of expert-evaluated data extracted from the literature, prioritizing human intervention studies to investigate health effects of phytochemicals. Currently included are >750 records from 445 studies providing data on 56 validated biomarkers, mainly relating to cardio-metabolic and bone health outcomes. The data cover 144 bioactive compounds from 17 compound classes. Using the EFSA Register of Questions and the database of general function health claims, we identified claims relating to phytochemicals made under articles 13.1, 13.5 and 14 and compared them with the eBASIS database to identify overlap between them. RESULTS: The EFSA online health claims database contains 4240 submissions under article 13.1, of which 2157 pertain to plants or plant-based bioactive compounds; 496 of these relate to plants or bioactive compounds included in the eBASIS biological effects database. Out of the 18 current 13.5 'new function' claims on EFSA's register of questions, 7 are for plants or plant-based bioactive compounds, of which 6 are included in eBASIS. Of the 222 defined article 14 claims, 21 pertain to plants or plant-based bioactive compounds, of which 19 are in eBASIS. CONCLUSIONS: There is extensive overlap between eBASIS and the submitted health claims that relate to plant-based bioactive compounds. EuroFIR eBASIS is a useful tool for regulators to independently check completeness of health claims applications relating to phytochemicals and is a potentially valuable resource to assist claimants in the compilation of dossiers on functional foods and health claims.

New nutritional data on traditional foods for European food composition databases.

BACKGROUND/OBJECTIVES: There are many different cultures within Europe, each with its own distinct dietary habits. Traditional foods are the key elements that differentiate the dietary patterns of each country. Unfortunately, in most countries, there is little information on the nutritional composition of such foods. Therefore, there is a need to study traditional foods to preserve these elements of European culture and, if possible, enrich and improve dietary habits across the continent. The Traditional Foods work package within the European Food Information Resource (EuroFIR) project aimed to provide new nutritional data on traditional foods for use in national food composition tables. SUBJECTS/METHODS: A EuroFIR consensus-based method with standardised procedures was applied for the systematic study of traditional foods and recipes in selected European countries. Traditional foods were selected on the basis of the EuroFIR definition of the term 'traditional food' and prioritized according to specific criteria. From the prioritized list, the five traditional foods per country to be investigated were selected to represent a full course meal. Protocols with guidelines for the recording of traditional recipes, the collection, preparation and distribution of laboratory samples, as well as quality requirements for laboratory selection, were developed to establish a common approach for use by all countries for the acquisition of reliable data. RESULTS: The traditional character of the selected foods has been documented and traditional recipes have been recorded. Chemical analyses to determine the nutritional composition of 55 traditional foods were performed and the data were evaluated and fully documented according to EuroFIR standards. Information on food description, the recipe, component identification, sampling plan, sample handling, analytical method and performance was collected for each of the 55 investigated traditional foods. CONCLUSIONS: This common methodology for the systematic study of traditional foods will enable countries to further investigate their traditional foods and to continue to update their national food composition databases and EuroFIR's food databank system.

New food composition data on selected ethnic foods consumed in Europe.

BACKGROUND: Reliable data on the composition of foods is needed to better understand individual diets, measure nutrient intakes and provide nutritional guidance for improving the health of the populations. Ethnic foods are becoming increasingly popular among all European consumers, and are the main source of nutrients in the diets of ethnic groups. However, there is limited information on the nutrient composition of ethnic foods in Europe. The objective of this study therefore was to generate new and reliable data on ethnic foods using harmonised methods for chemical analyses. METHODS: New data on 128 ethnic foods were generated for inclusion in the national databases within the European Food Information Resource Network of Excellence through participants from France, Israel, Spain, Denmark, Italy, The Netherlands, Belgium and the United Kingdom. In each selected country, the list of prioritized foods and key nutrients, methods of analyses and quality assurance procedure were harmonised. RESULTS: This paper presents the nutrient composition of 40 ethnic foods consumed in Europe. The nutrient composition of the foods varied widely because of the nature and variety of foods analysed, with energy content (kcal) ranging between 24 (biteku-teku, Blegium) and 495 (nachos, Italy) per 100 g of edible food. Polyunsaturated and monounsaturated fatty acids were generally higher in most ethnic foods consumed in Italy and Spain compared with ethnic foods of other countries. CONCLUSIONS: The new data were scrutinised and fully documented for inclusion in the national food composition databases. The data will aid effective diet and disease interventions, and enhance the provision of dietary advice, in all European consumers.

[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.

Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography.

A trienzyme treatment (conjugase, alpha-amylase, protease) followed by affinity chromatography and reversed-phase HPLC with UV and fluorescence detection was performed for the quantification of folate vitamers in legumes (chickpea and beans), processed meats (salami Milano and Parma ham) and in an Italian reference diet. This method allowed a good separation of six folate vitamers: 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, folic acid, 10-formylfolic acid, 10-formyldihydrofolate and tetrahydrofolate within 30 min. Recovery, reproducibility and limits of detection of the method are reported. HPLC results were 24-52% lower than the microbiological assay findings.

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.