Cornelia M. Witthöft

Folates and Dairy Products: A Critical Update

In recent years, folates have come into focus due to their protective role against child birth defects, for example, neural tube defects. In addition, folates may have a protective role to play against coronary heart disease and certain forms of cancer. During the last few years most countries have established increased recommended intakes of folates, for example, between 300–400μg per day for adults. This review of folates in milk and dairy products compares some recent data based on high pressure liquid chromatography (HPLC) analyses and radioprotein-binding assays, with previous data based on microbiological assays. All three methods show similar ranges for folates in cow’s milk, 5–10μg per 100g, the variation being due to seasonal variations. Data on folates in fermented milk (buttermilk and yogurt) are also similar for these methods. Different starter cultures, however, might explain some of the variations in folate content and folate forms. Most cheese varieties contain between 10μg and 40μg folate per kg, with slightly higher values for whey cheese. Ripened soft cheeses may contain up to 100μg folate per 100g. Most previous and recent studies using HPLC indicate that 5-methyl-tetrahydrofolate (5-methyl-THF) is the major folate form in milk, but more studies are needed concerning folate forms in other, especially fermented dairy products. Relatively new data on actual concentrations in different dairy products show folate-binding proteins (FBP) to occur in unprocessed milk, but also in pasteurised milk, spray-dried skim milk powder and whey. In contrast, UHT milk, fermented milk and most cheeses only contain low levels or trace amounts.

HPLC determination of folates in raw and processed beetroots

Abstract A sensitive HPLC method with fluorescence detection and gradient elution has been developed for the determination of folates in vegetables. The method involved extraction of folates from food matrix by heat treatment, deconjugation of folate polyglutamates to monoglutamates by incubation with hog kidney conjugase and purification of food extracts by solid-phase extraction with strong-anion exchange cartridges. The chromatographic separation of folates was achieved on Zorbax SB C 8 column, which was found to be superior over conventional C 18 column in terms of selectivity and sensitivity. Validation of the method included linearity tests, the addition of standard folates for the determination of recovery, repeatability and stability tests. The method developed was applied to analysis of raw and processed beetroots; 5-methyltetrahydrofolate was found to be the main folate form in beetroots. Cultivar differences and growing conditions were found to have a pronounced effect on the folate content in beetroots. Processing resulted in considerable losses of folates, whereas losses during storage appeared to be moderate.

Folate, Vitamin B12, and Risk of Ischemic and Hemorrhagic Stroke A Prospective, Nested Case-Referent Study of Plasma Concentrations and Dietary Intake

Background and Purpose— Folate metabolism has been implicated in stroke. However, the possibility of a role for folate and vitamin B12, independent of their effects on homocysteine status, remains to be explored. The aim of this prospective, nested case-referent study was to relate plasma and dietary intake levels of folate and vitamin B12 to risk of stroke, taking into consideration plasma homocysteine concentrations and methylenetetrahydrofolate reductase polymorphisms. Methods— Subjects were 334 ischemic and 62 hemorrhagic stroke cases and matched double referents from the population-based Northern Sweden Health and Disease Cohort. Results— Plasma folate was statistically significantly associated with risk of hemorrhagic stroke in an inverse linear manner, both in univariate analysis and after adjustment for conventional risk factors including hypertension (odds ratio [OR] for highest versus lowest quartile 0.21 (95% confidence interval [CI], 0.06 to 0.71; P for trend=0.008)). Risk estimates were attenuated by inclusion of homocysteine in the model (OR, 0.34; 95% CI, 0.08 to 1.40; P for trend=0.088). A similar pattern was observed for increasing folate intake (multivariate OR, 0.07; 95% CI, 0.01 to 0.55; P for trend=0.031 without homocysteine, and OR, 0.16, 95% CI, 0.02 to 1.23; P for trend=0.118 with homocysteine in the analysis). We found little evidence of an association between plasma or dietary folate and risk of ischemic stroke. Neither plasma nor dietary vitamin B12 was associated with risk of either stroke subtype. Conclusions— The results of this study suggest a protective role for folate, possibly in addition to its effects on homocysteine status, in hemorrhagic but not ischemic stroke.

Standardisation of HPLC techniques for the determination of naturally-occurring folates in food

The aim of this work was to evaluate current in-house HPLC procedures for the determination of naturally-occurring folates in food, and to identify problem areas for further improvement. Five intercomparison studies were completed over the period 1990–1997 in which nine participants from six countries took part. Through careful validations and detailed discussions held at evaluation meetings, possible biases and sources of systematic error have been identified and reduced. The use of ascorbic acid and nitrogen flushing during extraction, sample clean-up using strong anion exchange columns, spectrophometrically calibrated standards and fluorescence detection are all recommended. Both in-house hog kidney and human plasma deconjugase enzymes gave similar results to the circulated common hog kidney enzyme which was prepared from fresh pig’s kidneys. The most consistently reported values were for 5-CH3H4-PteGlu, and to a lesser extent, for H4PteGlu. Four candidate reference materials (CRM 121, wholemeal flour; CRM 421, milk powder; CRM 485, lyophilised mixed vegetables, and CRM 487, lyophilised pig’s liver) have been proposed with both indicative values (mean ± uncertainty) for 5-CH3H4-PteGlu in CRM 421 (0.25; ± 0.02 mg/kg) and CRM 485 (2.14; ±0.42 mg/kg), and information values (mean; range) for 5-CH3H4-PteGlu in CRM 121 (0.04; 0.03–0.08 mg/kg) and CRM 487 (2.6; 1.9–3.8 mg/kg). Certified values are also given for total folate by microbiological assay: CRM 121 (0.50; ±0.07 mg/kg), CRM 421 (1.42; ±0.14 mg/kg), CRM 485 (3.15; 0.28 mg/kg), and CRM 487 (13.4; 1.3 mg/kg). Average recovery of 5-CH3H4-PteGlu, added prior to extraction and deconjugation, was 91% (84–95%) for the four CRMs. The average within- and between-laboratory variations were 6 and 15% for the determination of 5-CH3H4-PteGlu by HPLC, and 9 and 18% for the determination of total folate by microbiological assay. These CRMs will be used for quality control of folate measurements for nutritional labelling, and validation of new techniques. Further methodology work is required for the HPLC analyses of folate forms other than 5-CH3H4-PteGlu.1

Human Folate Bioavailability

The vitamin folate is recognized as beneficial health-wise in the prevention of neural tube defects, anemia, cardiovascular diseases, poor cognitive performance, and some forms of cancer. However, suboptimal dietary folate intake has been reported in a number of countries. Several national health authorities have therefore introduced mandatory food fortification with synthetic folic acid, which is considered a convenient fortificant, being cost-efficient in production, more stable than natural food folate, and superior in terms of bioavailability and bioefficacy. Other countries have decided against fortification due to the ambiguous role of synthetic folic acid regarding promotion of subclinical cancers and other adverse health effects. This paper reviews recent studies on folate bioavailability after intervention with folate from food. Our conclusions were that limited folate bioavailability data are available for vegetables, fruits, cereal products, and fortified foods, and that it is difficult to evaluate the bioavailability of food folate or whether intervention with food folate improves folate status. We recommend revising the classical approach of using folic acid as a reference dose for estimating the plasma kinetics and relative bioavailability of food folate.

Plasma folate and total homocysteine levels are associated with the risk of myocardial infarction, independently of each other and of renal function

Objectives.  To investigate the relationship between plasma folate, vitamin B12 and total homocysteine concentrations, dietary intake of folate and vitamins B12, B6 and B2, and the risk of first acute myocardial infarction (MI).

Evaluation of a radioprotein-binding assay (RPBA) for folate analysis in berries and milk

This study aimed to optimise a commercial radioprotein-binding assay (RPBA), routinely used for clinical samples, for folate quantification in foods containing mainly 5-CH3-H4folate. The assay was modified using external calibration with 5-CH3-H4folate in a lower concentration range diluted in food extraction buffer, rather than the buffer with human serum albumin (HSA) provided by the kit. We evaluated the modified RBPA on some selected food products; milk, whey powder, rose hips, strawberries and European certified reference materials (CRM 421, 485) and the adjustments did not affect the assay negatively. Performance parameters included control of selectivity, absence of matrix effects, recoveries of 94–113%, precision of 1–6 CV% (intra-assay) and 5–9 CV% (inter-assay). Folate concentrations in berries and milk, obtained by the modified RPBA were also compared with other quantification methods such as HPLC and MA. The optimised RPBA was found to be a quick and inexpensive complement to HPLC methods, reliable for folate quantification in foods such as milk and berries that contain mainly 5-CH3-H4folate. # 2002 Elsevier Science Ltd. All rights reserved.

Third EU MAT intercomparison study on food folate analysis using HPLC procedures

Three samples (milk powder, lyophilized pigs liver and wholemeal flour), a 5-methyltetrahydrofolic acid (5-MTHF) calibrant and two deconjugase enzymes (purified hog kidney and human plasma) were circulated to three laboratories taking part in the study. The objectives were to optimize the deconjugation step in these foods and to improve the between-laboratory agreement in HPLC results for folates. The predominant natural folate form in milk powder was 5-MTHF, together with appreciable amounts of folic acid. In pigs liver 5-MTHF was found to represent about one-third of the total folate content found. For these two foods, results from one laboratory of the sum of the folate vitamers agreed favourably with the microbiological data. 5-MTHF was most successfully determined by all three laboratories. There was little or no agreement found for the other folate vitamers detected.

Folate absorption from folate-fortified and processed foods using a human ileostomy model.

Data on folate absorption from food from validated human studies using physiological folate doses are still needed to estimate dietary requirements and to formulate recommendations. The aim of the present work was to study the effects from fortified and processed foods on folate absorption in ileostomy volunteers (n 9) using the area under the plasma concentration curve (AUC) and kinetic modelling. Using a standardized single-dose protocol, dairy products fortified with a candidate fortificant (6S)-5-methyltetrahydrofolate ((6S)-5-CH3-H4folate), folic acid-fortified bread and a dessert crème containing natural yeast folate polyglutamates were compared with folate supplements. Absorbed folate was estimated by AUC and a kinetic model, and non-absorbed folate by ileostomal folate excretion. Median apparent absorption from test foods ranged from 55 to 86 %. Added folate-binding proteins (FBP) significantly reduced folate absorption from dairy products, as in the absence of FBP, AUC-dose-corrected ratios were increased and ileal folate excretion decreased. After in vivo gastrointestinal passage of dairy products containing FBP, up to 43 % of the ingested FBP was found in ileostomal effluent. Folate absorption was similar for (6S)-5-CH3-H4folate fortificant from fermented milk and for folic acid from fortified bread. Folic acid, ingested as food fortificant in bread, was significantly less absorbed compared with an isolated supplement. We conclude that all tested foods were suitable matrices for folate fortification. However, dairy products, fortified with the new candidate fortificant (6S)-5-CH3-H4folate, are suitable if no active FBP is present.

Orange juice is a good folate source in respect to folate content and stability during storage and simulated digestion

BackgroundEstimated average folate intake in Sweden is less than 55% of the recommended daily intake (RDI) for women of childbearing age (Becker and Pearson in Riksmaten 1997–1998 Kostvanor och näringsintag i Sverige. National Food Administration, Uppsala, pp 34, 44, 121, 2002). Because a good folate status reduces the risk of neural tube defects, mandatory folic acid fortification is discussed in some European countries. This however, could lead to exposure to unintentionally high amounts of folic acid for some population groups, therefore targeted folic acid fortification could be an alternative.AimsTo (1) determine natural folate content in three popular brands of orange juice sold in Sweden, (2) determine stability of natural folate and folic acid fortificant during shelf life in a folic acid/iron fortified orange juice, (3) determine folate stability in four juices during simulated household consumption for one week and (4) determine the in vitro bioaccessibility of natural folate in one brand of orange juice using the TNO gastroIntestinal Model (TIM).MethodsNatural folate content in juices was determined using RP-HPLC-FL. To determine folic acid content and confirm RP-HPLC-FL values LCMS was used. Stability during shelf life was determined in unopened bottles of a folic acid/iron fortified juice and for one week in four popular juices under household consumption conditions with reopening of bottles daily. For an in vitro folate bioaccessibility experiment in orange juice the TNO TIM Model was used.Results5-CH3-H4folate was the dominant natural folate form in the juices with contents ranging from 16–30 µg/100 g. Shelf life losses of folic acid fortificant were 1–4%. During one week simulated household consumption 5-CH3-H4folate content decreased by up to 7% (n.s). Bioaccessibility of natural folate in orange juice was almost 100%. Most folate was released for absorption in jejunum between 60–120 min after trial start.ConclusionOrange juice may be considered a good source of natural folate in respect to content and stability during storage and simulated digestion. Moreover, added folic acid fortificant in a folic acid/iron fortified orange juice was stable during shelf life.