N05013: Bioavailability of folic acid and natural folates - studies using the functional marker plasma homocysteine

Study Duration: June 1998 to June 2002

Contractor: University of Ulster

Folate is attracting major interest in recent years as having an established role in the prevention of neural tube defects (NTD) and possible preventive roles against cardiovascular disease, certain cancers and neuro-psychiatric conditions such as dementia and depression. However, it is well recognised that there is widespread under-provision of folate in the diets of many people even in developed countries. Thus, for the prevention of NTD, various official bodies worldwide recommend women to take an additional 400 micrograms per day of folate before conception and in early pregnancy. There are three ways to achieve this recommendation: folic acid supplementation; folic acid fortification; or increased intake of folate rich foods. Although folic acid supplements are very effective in optimising folate status in women who take them, they do not offer an effective means of primary prevention of NTD in the general population because of poor compliance. Fortification is also a highly effective strategy to increase folate status, but it is controversial if applied on a population basis (as introduced recently in the US) because in order to ensure that the required folic acid levels are delivered to the target group, a substantial proportion of the general population are inevitably exposed to very high levels. The third approach to increase folate status, which does not have the health concerns associated with food fortification, is by increased consumption of foods naturally rich in folate. However, the potential to increase folate status by this means has been found to be somewhat limited, which is considered to be primarily due to the poor bioavailability of natural food folates compared with the synthetic vitamin, folic acid.

Bioavailability may be defined as the fraction of an ingested nutrient that is utilized for normal physiological functions or storage. A great deal of uncertainty exists in our knowledge of the bioavailability of folate from natural food sources. In order to attempt to improve food folate bioavailability, more needs to be known about it and the factors which affect it, but studies in this area are notoriously difficult. Long-term intervention studies in free-living subjects, involving the provision of folate rich foods for comparison with equivalent amounts of the vitamin in the synthetic form, folic acid, are problematic for various reasons. First, natural food folates are unstable and may undergo considerable losses during preparation and cooking. In contrast, subjects assigned to receive folic acid treatment may be required to simply ingest a tablet daily in addition to their usual diet, and are, therefore, likely to be much more compliant with intervention protocols. Such differences can confuse the interpretation of many folate bioavailability studies.

Thus, the underlying issues may be summarised as follows: 1. There is widespread under-provision of folate which is contributing to an increased risk of neural tube defects and other diseases; 2. This under-provision is attributed to the instability and poor bioavailability of natural food folates compared with the synthetic vitamin, folic acid. 3. Available methods to study food folate bioavailability, the factors that affect it, and thus attempts to improve them, are inadequate.

The overall aim of this research project was to study food folate bioavailability in a meaningful way. In a series of long-term (6 to12 week) studies we examined the response in healthy subjects to intervention with folic acid (both as supplements in tablet form or as folic acid-fortified food) or natural food folates. The aim was to feed the vitamin in a sufficiently concentrated form so as to elicit a biological response, not only in the usual markers of folate status, plasma and red cell folate levels, but additionally in the functional biomarker (index) of folate status, plasma homocysteine. Because the normal metabolism of homocysteine requires folate, the rationale was that a change in this biomarker in response to the intervention would be indicative of the extra amount of folate available for utilisation by cells.

We had 6 objectives in total, 5 of which corresponded to intervention studies: Objective 1 was aimed at confirming the quantitative response of plasma homocysteine to increasing levels of folic acid provided in tablet form (4 doses in the range 100 to 400 micrograms per day), and establishing the optimal dose of folic acid above which further increases in intake produced no further response in plasma homocysteine. We then determined the bioavailability of folic acid from fortified bread compared with that in tablet form (Objective 3) at the dose which was shown to be optimal in Objective 1. Objectives 4, 5 and 6 were all aimed at comparing the bioavailability of folic acid in tablet form with an equivalent amount of the vitamin provided as natural food folate sources, either extracted from their normal food matrix (Objective 4), or with the food matrix intact and fed either alone as a drink with no other foods present (Objective 5) or as part of a meal (Objective 6). The laboratory support for the above intervention studies was provided in a series of smaller investigations (collectively conducted as 'Objective 2'), which included the measurement of folates in foods and tablets prior to their administration in the various intervention studies. The biggest task faced under Objective 2 was to develop a protocol for the production of concentrated extracts of food folates of known total folate content and folate type, suitable for human consumption in defined amounts for comparison with an equivalent dose of folic acid. Spinach, yeast and egg yolk were selected because they encompass a wide range of the different types (derivatives) of folates found in foods and hence may be considered representative of the normal diet. In addition, under Objective 2, an in vivo study to examine the acute absorption of food folate extracts was conducted prior to using the extracts in a long-term intervention (Objective 4). Finally, as part of the wider issue surrounding natural food folates, Objective 2 included a series of experiments designed to examine the effect of typical cooking methods on folate retention in various foods which are important sources of the vitamin in the UK diet.

The novelty of our overall approach to the question of folate bioavailability was to examine the effect of natural food folates administered in various ways in sufficiently concentrated – but physiological - amounts so as to elicit a response in the folate biomarkers, plasma folate and homocysteine. Our rationale was that the provision of natural food folates under supervision in a concentrated product of pre-determined folate content would overcome the problems of poor compliance and the confounding effects of different cooking methods/duration on folate losses prior to ingestion.

Our results may be summarised as follows:

1. The method and duration of cooking of green vegetables (broccoli & spinach) have marked effects on folate retention from this major food folate source prior to ingestion.

2. The lowering of plasma homocysteine appears to be a valid indicator of folate bioavailability in subjects who have not been previously folate-supplemented (tablets or fortified foods) and in whom any homocysteine-lowering owing to vitamin B-6 or B-12 has been corrected by pre-treatment with physiological doses of these vitamins.

3. The bioavailability of folic acid from fortified bread was found to be highly bioavailable producing equal responses to that of free folic acid.

4. Once ingested, the bioavailability of green vegetables (spinach), was found to be only about 38% compared with folic acid at the same dose. We have no evidence that foods which have a greater percentage of folate in the conjugated (polyglutamate) form were less bioavailable than spinach in which about 50% is conjugated folate. Yeast, for example, contains folates which are virtually all polyglumates; the bioavailability of folate from yeast tended to be greater than that for spinach. The food matrix did not exert an effect on folate bioavailability, in that similar results were found whether or not the food was administered with the food matrix intact. However, we found some evidence to suggest that the presence of other (non-folate) foods ingested at the same time adversely affects the bioavailability of folate from food sources.

5. Using newer food folate methodology, we found much higher values for food folate compared with published values. This may have major implications for the calculation of dietary folate intake (and therefore folate recommendations) and requires further investigation.

Benefits of the research to the consumer: The loss of folate from foods during cooking is highly dependent both on the food in question and the method of cooking. Folates of animal origin (i.e. beef) were found to be stable to cooking even for prolonged periods. Likewise, folate was well retained in potatoes during boiling and we found no evidence (from a folate perspective) to support the popular view that 'the goodness is in the skin'. However, the method and duration of cooking of green vegetables were found to have marked effects on folate retention from this major folate source. Public health efforts to increase folate intakes should, therefore, incorporate practical advice on cooking. For example, steaming in preference to boiling could be promoted as a means of doubling the folate content of cooked green vegetables. In addition, consumers choosing to boil green vegetables, should be strongly discouraged from doing so for prolonged periods and should be advised to minimise the cooking water and utilise it for soups or gravy. These practical measures could make a substantial impact on folate intake from natural food sources and play a potential role in the prevention of folate related diseases by helping to optimise folate status.

Benefits of the research to the Agency: Food folate bioavailability and the factors which effect it are poorly understood. We have used a robust methodological approach to provide the Agency with quantifiable data, both on food folate stability and bioavailabilty. Furthermore, we have highlighted that there is a marked degree of underestimation of food folates by the traditional methodology used to generate many of the folate values for staple foods in current Food Tables. This has clear implications for the calculation of dietary folate intake and therefore folate recommendations.

Sanderson P, McNulty H, Mastroiacovo P, McDowell IF, Melse-Boonstra A, Finglas PM, Gregory JF 3rd (2003) Folate bioavailability: UK Food Standards Agency workshop report. Br J Nutr. 90, 473-479.

Wright AJ, Finglas PM, Dainty JR, Hart DJ, Wolfe CA, Southon S, Gregory JF. (2003) 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. Br J Nutr. 90, 363-371.

Contact: Dr Alison Tedstone
Tel: 020 7276 8929
Email: alison.tedstone@foodstandards.gsi.gov.uk