Review of the Food Standards Agency's Antioxidants in Food Programme (N04): Executive Summary

This Report describes a review by the British Nutrition Foundation (BNF) which sets in an international context the findings of 52 research projects funded by MAFF and FSA as part of the Antioxidants in Food programme (AN04/N04). These projects addressed biomarkers of oxidative damage, supplementation (in vitro and human studies), bioavailability and gene expression. The review has been conducted with the support and guidance of a Steering Group of experts (see below). The Report sets out BNF's recommendations about the way forward for the Antioxidants in Food programme of work.

It is worth noting that one of the Programme Objectives was to prepare for a sufficiently powered multi-centre intervention study. BNF was specifically asked to assess whether the work conducted to date within the Programme provides a robust scientific case to proceed with this Programme Objective.

The project design included a Steering Group. Most of the consultation with the Steering Group was by e-mail or telephone, but towards the end of the project, the Steering Group met at a Workshop in London .

The research Programme has mainly focused on development of biomarkers of oxidative damage that might be suitable for intervention trials in human subjects. It has not attempted, in a systematic way, to understand the mechanisms that involve antioxidants and which are associated with chronic diseases such as CVD and cancer. Instead, the assumption has been that if antioxidants can be shown to reduce oxidative damage, then there is likely to be a beneficial effect on disease risk.

Also included within the Programme are projects that have focussed on bioavailability or gene expression. A number also included small human intervention studies, the primary purpose of which was to test the validity of biomarkers developed or refined within the Programme. This mixture of projects and themes has shaped the form of the Report.

The findings of this review should be considered in the context of other recently completed and on-going reviews, which include: The National Academy of Sciences (USA) review of vitamin C, vitamin E, selenium and carotenoids; the EU-funded EUROFEDA project; the EU-funded ESCODD project; the work of the Food Standard Agency's Expert Group of Vitamins and Minerals; and the review conducted by BNF of the FSA's Optimal Nutrition Status research programme.

It can be concluded that although there is associative evidence from observational and intervention studies in human subjects that a diet rich in plant foods (particularly fruit and vegetables) conveys health benefits, as do high plasma levels of several nutrients found in these foods and known to be antioxidants, there is no evidence that any particular nutrient or class of bioactive substances makes a special contribution. The Steering Group confirmed that there is still a lack of understanding of the mechanisms underpinning the apparent protective effect of plant foods and that, as yet, there is no clear picture of which components are effective and hence no way of predicting whether all or just some plant foods are important in this respect.

To date much attention has focused on the potential for prevention by antioxidants of oxidative damage to DNA, in particular, but also to proteins and lipids. Oxidative damage to these molecules is now recognised as a process that occurs routinely in cells, as well as being associated with disease and ageing processes. Little is yet known about what might be regarded as a normal level of endogenous oxidative damage or what level might signify increased risk of disease. Furthermore, it has often been taken for granted that generation of oxygen and nitrogen free radicals is a detrimental event that should be prevented, but it is now recognised that ROS and RNS function as signalling molecules in cells and may also be important in triggering events such as apoptosis. In this context, there is the potential for the requirements of antioxidant molecules within cells to be very closely linked with critical functions such as signal pathways and gene expression, disruption of which could be detrimental to cell functioning and survival.

The establishment by MAFF of the Antioxidants in Food Programme (AN04/N04) was ground-breaking and the research that has arisen from the Programme has been very important as it has focussed attention on disease prevention rather treatment. The Programme has helped to progress the identification of biomarkers, in particular.

In the wider context, the EU-funded ESCODD project, which included contractors from the AN04/N04 Programme, has helped to clarify reasons for discrepancies in the measurement of oxidative DNA damage; it has helped in the optimisation of detection methods and devising of standard protocols; and has provided a platform for reaching a consensus about the basal level of damage in human cells. As a result, it is now apparent that the results of some of the earlier human studies need to be re-evaluated in the light of awareness of the artefacts produced during sample preparation for chromatographic analysis of oxidative DNA damage. In other words, some of the earlier studies probably over estimated levels of DNA damage as a result of the nature of the sample preparation techniques; on the other hand it is also possible that some of the studies that have produced a low estimate of endogenous damage are underestimates owing to the methodologies used.

Whilst there is now a small number of robust biomarkers, the biological relevance of these (e.g. a direct association with a disease) remains an issue. Furthermore, methods need to be validated in biological terms, not just against one another. Whilst attempts to do this were included in the AN04/N04 Research Programme, the expectations associated with these small intervention studies were probably over-ambitious. One way of taking this requirement forward would be to employ a nested case control study within an existing prospective study, perhaps using lymphocytes. Again, the applicability of measurement of, say, DNA oxidation in lymphocytes to tissue specific disease needs to be verified.

Another useful way forward might be collaborative studies with surgical departments able to supply biopsy material. This would enable site specific studies to be undertaken. In this context, the sensitivity of existing assays may be a relevant concern.

It is apparent from the Review that a focus on antioxidant mechanisms in isolation is no longer appropriate and diversification is needed. It is also clear that interest in measurement of oxidative damage and the impact on this of antioxidants has run ahead of the basic and necessary underpinning research on the functional effects of antioxidants, their bioavailability and their tissue distribution.

A major objective of this Review has been to establish whether or not there is sufficient robust scientific evidence to justify funding a multi-centre intervention trial. It is BNF's view (and that of the Steering Group) that there remain too many unanswered questions to progress to this stage at this point in time.

Nevertheless, we believe there is ample scope for a refocused programme of work that concentrates on establishing the functional effects and bioavailability of various plant food components, including the so-called antioxidant nutrients, but which takes a broader view on mechanisms of action, e.g. the interactions between dietary factors and the immune system, markers of endothelial damage (e.g. ICAM-1, VCAM-1, P-selectin), modulation of Phase I and Phase II enzymes, and effects on gene expression and cell signalling.

The extent of knowledge about the bioavailability of many of the substances of interest is still very limited. For example, little is known about the proportion of the intake of a particular dietary component that reaches the tissues; why some substances, e.g. the carotenoids lycopene and zeaxanthin, are actively concentrated in the prostate gland and macular region of the eye respectively; whether particular metabolites are important and their fate.

Future studies in humans should focus on groups with low intakes/status of the substances in question and perhaps those people more likely to be susceptible to oxidative damage (e.g. smokers, those with certain polymorphisms, or those with established disease) or more likely to benefit from an increase in supply of the plant constituents (e.g. because of previous low intake or because of prior priming with a stressor – a high PUFA intake was suggested at the Workshop). The Programme has been hampered by its focus on healthy non-smokers and younger people.

Future studies should also clarify safety issues e.g. whether or not some antioxidant nutrients act as pro-oxidants in vivo in some circumstances.

Although it is the FSA's expressed intention to focus on foods rather than supplements, there may be merit in comparing the effects of foods, plant extracts and supplements so as to compare the effects of the whole food versus components, and to establish whether a dose response exists. (Some believe there is a role for specific animal experiments in this context.)

• It would not be appropriate to begin a multi-centre intervention trial at this point in time.
• The FSA Research Programme should be refocused to consider a wider range of potential mechanisms of action, not just prevention of oxidative damage, with the ultimate aim of improving understanding of whether or not the link between plant food consumption and reduced chronic disease risk is causative, by identifying mechanisms of action for substances within the foods.
• More emphasis should be placed on studying the bioavailability (absorption, metabolism and turnover, tissue and cellular distribution) of a range of plant derived substances, including polyphenols as well as recognised vitamins and minerals, to establish whether in vitro effects are applicable to the in vivo situation.
• More emphasis should also be directed to understanding the functions of plant derived nutrients and other bioactive substances at a tissue and cellular level, and the impact on these (and on bioavailability) of factors such as genotype, age and ill health. A starting point for this work could be a review of the published literature to establish currently available information about a range of key components of plant foods, e.g. certain nutrients and classes of polyphenols, including interaction between them, dose response relationships, and groups of the population likely to have low intakes of these.
• Any human studies should involve subject groups in whom a response might be anticipated, e.g. those with low plasma levels of the substance of interest.
• There is a need for further collaborative studies (perhaps involving EU funding), such as the ESCODD study (DNA oxidation), which compare and validate different methods of measurement of biomarkers. In addition, the EU-funded EUROFEDA study may also provide useful information to aid decision making.
• In parallel with this, work is needed on the factors than influence successful modification of food selection. There is also an important role for product innovation, particularly in relation to vegetables. (It is clear from work in the USA and recent work in Britain that it is possible to increase fruit consumption to a modest degree, but success with vegetable consumption has been much less.) Perhaps relevant to this is the need for an improved understanding of how food preparation and processing influences the availability of food components for absorption in the human gut.

Such an approach should help the Agency to formulate more specific and focussed dietary messages, both in terms of population sub groups and in terms of specific food items.

British Nutrition Foundation, May 2002

View the FSA response to this review.