Trueness evaluation and verification of inter-assay agreement of serum folate measuring systems.

Background Definitive data to establish if the use of the WHO International Standard (IS) 03/178 as a common calibrator of commercial measuring systems (MSs) has improved the harmonization of serum total folate (tFOL) measurements to a clinically suitable level are lacking. Here, we report the results of an intercomparison study aimed to verify if the current inter-assay variability is acceptable for clinical application of tFOL testing. Methods After confirming their commutability, the IS 03/178 and National Institute for Standards and Technology SRM 3949 L1 were used for evaluating the correctness of traceability implementation by manufacturers and the MSs trueness, respectively. The inter-assay agreement was verified using 20 patient pools. The measurement uncertainty (U) of tFOL measurements on clinical samples was also estimated. An outcome-based model for defining desirable performance specifications for bias and imprecision for serum tFOL measurements was applied. Results The majority of evaluated MSs overestimated the WHO IS value of +5% or more with the risk to produce an unacceptably high number of false-negative results in clinical practice. The mean inter-assay CV on all pools and on those with tFOL values >3.0 µg/L (n = 15) was 12.5% and 7.1%, respectively. In neither case the goal of 3.0% was fulfilled. The residual bias resulted in an excessive U of tFOL measurement on clinical samples. Conclusions The implementation of traceability of tFOL MSs to the WHO IS 03/178 is currently inadequate, resulting in an inter-assay variability that does not permit the use of a common threshold for detecting folate deficiency.

Determination of fortified and endogenous folates in food-based Standard Reference Materials by liquid chromatography-tandem mass spectrometry.

The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33 ± 0.06) µg/g and (16.0 ± 0.7) µg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839 ± 0.0071) µg/g, (0.211 ± 0.014) µg/g, and (0.838 ± 0.044) µg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.

The development and role of international biological reference materials in the diagnosis of anaemia.

Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B12), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B12 and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.

Comparison of Nutrient Reference Values for Food Labeling in Japan with CODEX Recommendations, Based on DRIs and Nutrient Intake in Japan.

To clarify the degree of consistency between the international recommendations and the national Japanese system, the nutrient reference values (NRVs) adopted by the CODEX were compared with current Japanese NRVs 2015, the dietary reference intakes for Japanese (DRIs-J) 2015, and actual nutrient intake levels by the Japanese population. The Japanese NRV for protein was high relative to CODEX NRV-R (i.e., NRV-Requirement). The Japanese NRVs for folate and calcium were low, and vitamin K was high, relative to each CODEX NRV-R. However, it was similar to the DRI-J values, and current intake levels for the Japanese population. For iron, calculation methods were different between the CODEX and Japan. Japanese iron NRV was calculated based on the RDA without menstruatating women, whereas CODEX NRV-R was calculated based on the INL98 of all adult men and women. Actual intake levels of iron for the Japanese population were similarly low. The Japanese NRV for sodium was high and potassium was low based on DRI-J values, relative to the CODEX NRV-NCD. For nutrients that show large discrepancies between the CODEX and Japanese NRVs, the values should be discussed further.

The Protective Effect of Maternal Folic Acid Supplementation on Childhood Cancer: A Systematic Review and Meta-analysis of Case-control Studies.

OBJECTIVES: Maternal folic acid supplementation is considered mandatory in almost every country in the world to prevent congenital malformations. However, little is known about the association of maternal folic acid intake with the occurrence of childhood cancer. Hence, this study aimed to determine the effects of maternal folic acid consumption on the risk of childhood cancer. METHODS: A total of 158 related articles were obtained from PubMed, Google Scholar, Scopus, and ProQuest using standardized keywords, of which 17 were included in the final review. RESULTS: Eleven of the 17 articles showed a significant protective association between maternal folic acid supplementation and childhood cancer. Using a random-effects model, pooled odds ratios (ORs) showed a protective association between maternal folic acid supplementation and childhood acute lymphoblastic leukaemia (OR, 0.75; 95% confidence interval [CI], 0.66 to 0.86). However, there was no significant association between maternal folic acid supplementation and acute myeloid leukaemia (OR, 0.70; 95% CI, 0.46 to 1.06) or childhood brain tumours (OR, 1.02; 95% CI, 0.88 to 1.19). CONCLUSIONS: Maternal folic acid supplementation was found to have a protective effect against childhood acute lymphoblastic leukaemia. Thus, healthcare professionals are recommended to provide regular health education and health promotion to the community on the benefits of folic acid supplementation during pregnancy.

[Folic acid supplementation--voluntary enrichment of different foodstuffs or mandatory fortification of a single staple food?]. / Folsäure--Freiwilliger Zusatz bei diversen Lebensmitteln oder obligate Anreicherung eines Grundnahrungsmittels?

Regular, customary nutrition is not sufficient to cover the recommended daily intake of 400 microg of folic acid as it has been defined by several scientific committees. The provision of various foodstuffs enriched with folic acid would therefore seem to be justified. Whether such products would have a prophylactic effect against different homocysteine-associated diseases of the second part of life is not (yet) proven but certainly a well founded hypothesis. It has been shown that for the prevention of neural tube defects and other embryonic malformations, as well as malignancies of early childhood a pharmaceutical supply of 0,4-0,8 mg before and during the first 12 weeks of pregnancy is required. Mandatory general fortification of all grain products would be an attractive alternative to meet this goal. This measure has been successful in many countries, mainly on the North American continent. The controversy surrounding possible mandatory folic acid fortification of flour in our country is discussed.

Using folic acid-fortified flour should be mandatory policy.

Research published last month states that pregnancy multivitamins do not contribute anything towards boosting the health of mothers and babies.

A review of European guidelines on periconceptional folic acid supplementation.

Strong evidence that folic acid (FA) prevents the majority of cases of neural tube defects (NTDs) has led to national organisations developing guidelines for women concerning periconceptional supplementation. In Europe, there is evidence of national variations in the incidence of NTDs, with a recent Irish study reporting an increase in the rate. This review compares the periconceptional FA supplementation guidelines between the different countries in Europe. An online search of country-specific guidelines produced before 2015 concerning periconceptional FA supplementation was conducted. If an English version was not available directly, the EUROCAT register was searched for the English version of the recommendations. We identified national guidelines from 20 European countries. Over half recommended that FA supplements be taken by women planning a pregnancy, but three recommended that they should be taken by all women of child-bearing age. Four guidelines recommended starting FA at least 4 weeks preconceptionally, but no country recommended starting FA at least 12 weeks preconceptionally as suggested by recently published studies. There is a need for further consideration of the duration of preconceptional FA supplementation specifically. The latest scientific evidence in this area should inform the development of European guidelines on FA, as there is wide variation in current recommendations. Overall, the wide variation in national guidelines concerning periconceptional FA supplementation may in part explain the differences in national rates of NTDs reported by EUROCAT. National guidelines on FA supplementation should be standardised across European countries.

What is the safe upper intake level of folic acid for the nervous system? Implications for folic acid fortification policies.

Between 1945 and 1959 it was convincingly documented that folic acid can precipitate or aggravate the neurological and haematological consequences of vitamin B12 deficiency by increasing the demand for vitamin B12. Since then there has been much misunderstanding of the issues, mainly by advocates of folic acid fortification who have been inclined to minimise or even dismiss the risks by misinterpreting the evidence as only a 'masking' of the anaemia of pernicious anaemia. Recent studies in the era of fortification are rediscovering the risks to the nervous system, especially cognitive function, of excess folate in the presence of vitamin B12 deficiency. I have reviewed the Reports of four Expert Advisory Committees in Europe and the USA, which suggest that the safe upper tolerable limit (UL) for folic acid is 1 mg in adults. These reports are unsound and there is already evidence of neurological harm from long-term exposure to doses of folic acid between 0.5 and 1 mg in the presence of vitamin B12 deficiency. There is an urgent need to review the safe UL for folic acid and to consider the addition of vitamin B12 to folic acid fortification policies.

Applying inappropriate cutoffs leads to misinterpretation of folate status in the US population.

BACKGROUND: Folate cutoffs for risk of deficiency compared with possible deficiency were originally derived differently (experimental compared with epidemiologic data), and their interpretations are different. The matching of cutoffs derived from one assay with population-based data derived from another assay requires caution. OBJECTIVE: We assessed the extent of folate-status misinterpretation with the use of inappropriate cutoffs. DESIGN: In the cross-sectional NHANES, serum and red blood cell (RBC) folate were first measured with the use of a radioprotein-binding assay (RPBA) (1988-2006) and, afterwards, with the use of a microbiologic assay (2007-2010). We compared prevalence estimates for assay-matched cutoffs (e.g., with the use of an RPBA cutoff with RPBA data) and assay-mismatched cutoffs (e.g., with the use of microbiologic assay cutoff with RPBA data) for risk of deficiency on the basis of megaloblastic anemia as a hematologic indicator in persons ≥4 y of age (e.g., serum folate concentration <7 nmol/L and RBC folate concentration <305 nmol/L derived with the use of a microbiologic assay), possible deficiency on the basis of rising homocysteine as a metabolic indicator in persons ≥4 y of age (e.g., serum folate concentration <10 nmol/L and RBC folate concentration <340 nmol/L derived with the use of an RPBA), and insufficiency on the basis of elevated risk of neural tube defects in women 12-49 y old (e.g., RBC folate concentration <906 nmol/L derived with the use of a microbiologic assay). RESULTS: Pre-folic acid fortification (1988-1994), risks of deficiency for assay-matched compared with assay-mismatched cutoffs were 5.6% compared with 16% (serum folate), respectively, and 7.4% compared with 28% (RBC folate), respectively; risks declined postfortification (1999-2006) to <1% compared with <1% (serum folate), respectively, and to <1% compared with 2.5% (RBC folate), respectively. Prefortification (1988-1994), risks of possible deficiency for assay-matched compared with assay-mismatched cutoffs were 35% compared with 56% (serum folate), respectively, and 37% compared with 84% (RBC folate), respectively; risks declined postfortification (1999-2006) to 1.9% compared with 7.0% (serum folate), respectively, and to 4.8% compared with 53% (RBC folate), respectively. Postfortification (2007-2010), risks of insufficiency were 3% (assay matched) compared with 39% (assay mismatched), respectively. CONCLUSIONS: The application of assay-mismatched cutoffs leads to a misinterpretation of folate status. This confusion likely applies to clinical assays because no comparability data are available, to our knowledge.

Are the Recommended Dietary Allowances for Vitamins Appropriate for Elderly People?

BACKGROUND: An adequate vitamin intake is essential for a good nutritional status, especially in older women, who are more sensitive to nutritional deficiencies. The American, European and Italian Recommended Dietary Allowances (RDAs) derive mainly from studies on adults, and it is not clear whether they also apply to elderly people. Comparing the RDAs with the actual vitamin intake of a group of healthy older women could help to clarify the real needs of elderly people. OBJECTIVE: Our aim was to compare the American, European, and Italian RDAs with the actual vitamin intake of a group of healthy older women. DESIGN: This was a cross-sectional study. PARTICIPANTS: The study included 286 healthy women aged older than 65 years. MAIN OUTCOME MEASURES: For each micronutrient, the 50th percentile of the distribution of its intake was considered as the average requirement, and the corresponding calculated RDA for our sample was the average requirement×1.2, as recommended by the US Food and Nutrition Board. This calculated RDA was then compared with the American, European, and Italian RDAs. STATISTICAL ANALYSES PERFORMED: Student's t test or the Mann-Whitney test (after checking the normal distribution of the micronutrient) for continuous variables; the χ(2) test for categorical variables. RESULTS: The calculated RDA were 2,230 µg retinol equivalents for vitamin A, 2.8 µg for vitamin B-12, 0.9 mg for thiamin, 1.4 mg for riboflavin, 3.6 mg for pantothenic acid, 1.4 mg for vitamin B-6, 320 µg for folic acid, and 115 mg for vitamin C. CONCLUSIONS: Our findings suggest that the current RDAs are adequate for older women's intake of riboflavin, vitamin B-6, and folic acid, but should be raised for vitamin B-12 and for vitamin C.

Will an increased dietary folate intake reduce the incidence of cardiovascular disease?

Based on a meta-analysis of published studies, it has been estimated that approximately 10% of coronary artery disease cases are attributable to hyperhomocyst(e)inemia. It has also been calculated that food fortification with folate might reduce the number of cases of coronary artery disease in the United States by 50,000 per year. However, the use of statistical concepts to estimate the expected benefits of this intervention strategy may be misleading.

Folic acid fortification.

The relationship of folic acid to neural tube defects has provided us with one of the richest case studies in nutrition science policy of this half century. The concepts of nutritional adequacy in the periconceptual period, the power of well-designed and controlled intervention trials, the challenges of altering the intake of a critical nutrient, the risks and benefits of food fortification, proper targeting with supplementation, and the effectiveness of achieving dietary change are all elements of this case study that we will continue to explore throughout the pages of this journal in the months ahead.

Purity profiles of pteroylglutamate reference substances by high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) in the reversed-phase mode was used for the purity analysis of three pteroylglutamic acid-type reference substances (folic acid, leucovorin calcium, and methotrexate). The influence of the pH of the mobile phase on the separation of an artificial mixture of six pteroylglutamic acid derivatives and three potential impurities was studied. Results of purity analysis of current lots of USP reference standards are reported. A better separation of methotrexate from its major impurities was achieved by using a standard buffer, rather than an ion-pairing mobile phase. A separation of methotrexate and its biologically inactive 7-isomer is reported.

Non-fasting reference intervals for the Abbott IMx homocysteine and AxSYM plasma folate assays: influence of the methylenetetrahydrofolate reductase 677 C-->T mutation on homocysteine.

Plasma homocysteine comes under both genetic and nutritional control. B vitamins and particularly folate are important factors in homocysteine metabolism. We have obtained reference intervals for total plasma homocysteine and plasma folate. We have also determined the influence of methylenetetrahydrofolate reductase (MTHFR) genotype on plasma homocysteine concentrations in healthy individuals. Reference intervals for Abbott IMx homocysteine and AxSYM plasma folate assays were established using 116 volunteers recruited from hospital staff. Exclusion criteria included cardiac, hepatic or renal disorders, and use of over-the-counter prescription medications. An exception was the inclusion of three women using oral contraceptives and one woman receiving post-menopausal oestrogen supplementation. Methylenetetrahydrofolate reductase 677C-->T genotyping was performed on 101 of the volunteers to determine whether the MTHFR 677T allele influences homocysteine concentrations in healthy individuals. Reference intervals for homocysteine and folate were determined using the mean+/-2 standard deviations of the data. Folate/homocysteine ratios were sorted by MTHFR C677T genotype. Homocysteine correlated negatively with plasma folate. Mean male homocysteine concentrations were significantly higher (9.0 micromol/L; P<0.05) than the mean value (7.1 micromol/L) obtained for females. Mean homocysteine values were significantly higher in subjects who were homozygous for the MTHFR 677T allele when compared with the 677CC genotype (P<0.05). Ratios of folate/homocysteine were 20% and 7.4% lower in the male and female 677TT group than in the 677CC group, respectively. The mean homocysteine value of 43 volunteers who were taking multivitamins was not significantly different from that of 73 who were not vitamin supplemented. Conversely, the mean folate value was slightly greater, and statistically significant, in the group taking vitamin supplements. The mean folate values and reference intervals were not significantly different when grouped by sex or age. MTHFR 677C-->T mutations influenced homocysteine values observed in our study of healthy volunteers, even though we did not observe outright folate-deficient individuals. Our random homocysteine values were similar to the fasting homocysteine values obtained in other studies.

Recommended dietary allowances (RDAs) for genomic stability.

Diet as a key factor in determining genomic stability is more important than previously imagined because we now know it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis. Current recommended dietary allowances for vitamins and minerals are based largely on the prevention of diseases of deficiency such as scurvy in the case of Vitamin C. Because diseases of development, degenerative disease and ageing itself are partly caused by damage to DNA, it seems logical that we should focus better our attention on defining optimal requirements of key minerals and vitamins for preventing damage to both nuclear and mitochondrial DNA. To date our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganised. Appropriately designed placebo, controlled trials are required to define recommended dietary allowances for genomic stability. Recently, it has been shown that above RDA intakes of folic acid and Vitamin B12 are required to reduce the micronucleus index in humans by 25%. In the future, clinical trials with a defined wider array of complementary DNA damage end-points would be necessary. That there is a need for an international collaborative group to establish RDAs for genomic stability is self-evident and this paper is a call for such a process to begin.

Folic acid and total parenteral nutrition.

The stability of folic acid in a variety of solutions used for parenteral nutrition has been determined over a 2-wk period. Provided that the acidity of the solution remains above pH 5.0 the folate, in the concentrations usually used for parenteral nutrition, will remain stable in solution, and all of the folate added to the solution will be delivered to the patient. The applicability of this in vitro work to a group of patients requiring parenteral nutrition was assessed, in order to determine a suitable dose. A dose of 0.2 mg of folic acid daily was inadequate to meet the requirements of these patients. In contrast a dose of 1.2 mg daily for 7 days was sufficient to cause an increase in the serum folate concentration.

Micronutrients and genomic stability: a new paradigm for recommended dietary allowances (RDAs).

Diet as a key factor in determining genomic stability is more important than previously imagined because we now know that it impacts on all relevant pathways, namely exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis. Current recommended dietary allowances for vitamins and minerals are based largely on the prevention of diseases of deficiency such as scurvy in the case of vitamin C. Because diseases of development, degenerative disease and aging itself are partly caused by damage to DNA it seems logical that we should focus better our attention on defining optimal requirements of key minerals and vitamins for preventing damage to both nuclear and mitochondrial DNA. To date, our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganised. However, there is already sufficient evidence to suggest that marginal deficiencies in folate, vitamin B12, niacin and zinc impact significantly on spontaneous chromosome damage rate. The recent data for folate and vitamin B12 in humans with respect to micronucleus formation in blood and epithelial cells provide compelling evidence of the important role of these micronutrients in maintenance of genome integrity and the need to revise current RDAs for these micronutrients based on minimisation of DNA damage. Appropriately designed in vitro studies and in vivo placebo controlled trials with dose responses using a complementary array of DNA damage biomarkers are required to define recommended dietary allowances for genomic stability. Furthermore these studies would have to be targeted to individuals with common genetic polymorphisms that alter the bioavailability of specific micronutrients and the affinity of specific key enzymes involved in DNA metabolism for their micronutrient co-factor. That there is a need for an international collaborative effort to establish RDAs for genomic stability is self-evident.