Phytochemicals and health

There is now a consensus that a diet rich in fruit and vegetables can reduce the risk of chronic illnesses such as cardiovascular disease, certain cancers and osteoporosis

Considerable evidence suggests that naturally-occurring chemicals in fruits and vegetables may be of particular importance in health protection. These phytochemicals are of diverse chemical structures, and contribute to the flavour and colour of fruits and vegetables. There are four major components to IFR research:–

• We quantify the types and amounts of phytochemicals in both commonly consumed fruits and vegetables, and also some processed food products. We also investigate the chemical content of some novel foods and wild plants that are sometimes consumed
• We quantify the ‘bioavailability’ of these compounds following consumption. By this we mean the relative amount of the phytochemical that is absorbed into the body from the gastointestinal tract, how these compounds are metabolised, and the rates at which they are subsequently excreted
• We are seeking to understanding how a selected number of these compounds may provide health protection, particularly against cancers of the gastrointestinal tract and cardiovascular disease
• We are assessing the importance of human genetic variation in determining the bioavailability and biological activity of several phytochemicals, partly through our involvement in an EU-funded Network of Excellence.

From these studies we hope to be able to further refine current dietary advice to consume ‘5-a-day’, possibly by recommending certain groups of fruits and vegetables that have particular benefits, and assessing the importance of factors such as processing and human genetic variation that complicate the interpretation of epidemiological studies. We are also seeking to develop certain ‘functional foods’ that have added health benefits when consumed as part of a balanced diet.

While we analyse a wide range of phytochemicals – using a variety of methods, but with increasing emphasis on routine mass spectrometry as a means of detection – we are particularly interested in three classes of compounds. Firstly, polyphenols, which are ubiquitous in the plant kingdom; secondly, glucosinolates, which are restricted to cruciferous vegetables and related wild plants; and thirdly, folates, that are high in certain nuts and green leafy vegetables. For all of these classes of compounds, we have substantial data on their bioavailability, and are exploring their role in health protection. In addition, we are exploring the manner by which human genetic variation may influence the bioavailability of these compounds, and the extent to which they may exert a positive health benefit.

Polyphenols and health

Polyphenols contribute to the colour, taste and aroma of fruits and vegetables. We are engaged in a series of human intervention studies, funded by the Food Standards Agency, to quantify polyphenols in a range of frequently consumed fruits and vegetables (apples, oranges, blackcurrants, broccoli and tomatoes) and a variety of processed products derived from these, such as fruit juices and tomato sauce. We are quantifying the ‘bioavailability’ of these compounds from the fresh and processed products. This will provide important information on the relative benefits of, for example, eating an apple or drinking a glass of apple juice. Moreover, through our analyses of human plasma we now have accurate information on the precise levels and types of metabolites that occur following eating these foods. This enables us to explore the biological effects of these compounds.

Evidence from human clinical studies suggests that polyphenols improve cardiovascular function and reduce risk of cardiovascular disease (CVD). We are exploiting new analytical methods and unique synthetic human conjugates to investigate the mechanisms by which dietary polyphenols alter cardiovascular function. Isolated human polyphenol conjugates are being used to treat cell types crucial to the development of CVD (endothelial cells and monocytes) in order to determine which polyphenols / conjugates are active, and define dose-responses. We also plan to measure physiological parameters in whole arterial tissue and, in the longer term, to validate the results with human studies.

Glucosinolates and health

Glucosinolates occur in cruciferous vegetables such as broccoli, cabbages and Brussels sprouts. When we eat these vegetables, the glucosinolates produce isothiocyanates (‘mustard oils’ or ITCs). Consumption of these vegetables is associated with reduction in risk of some forms of cancer. This may be due to biological activity of the isothiocyanates. As with the work on polyphenols, human intervention studies have enabled us to quantify the precise level and types of metabolites that are found in the body after eating these vegetables. We have exposed a variety of human cell cultures to appropriate physiological levels of ITCs and their human metabolites, and measured changes in gene expression with the use of microarrays. ITCs result in about 200 genes being turned on, and about 100 genes being turned off, and many of these changes in expression are consistent with a protective effect.

	Broccoli – standard cultivar (Iron) and our new, ITC-enriched variety.

An important component of our research is the development of broccoli with enhanced levels of isothiocyanates. These novel cultivars have been developed through conventional breeding and are now ready for commercial production. We are using these in human intervention studies in collaboration with the Queens Medical Centre, University of Nottingham, and comparing changes in gene expression in the gastrointestinal tract following eating either standard or ITC-enriched broccoli. One aspect of this project to to study the effect of variation at the human GSTM1 gene. Forty percent of us lack this gene through an ancestral deletion from the genome. Epidemiological evidence suggests we may gain greater protection from cancer following broccoli consumption if we lack this gene. Thus, each of the volunteers are ‘genotyped’ to see if they have GSTM1, and we will correlate these data with the types of ITC metabolites found, the rate at which they are excreted and the types of genes which ITC switch on.

Folates and health

Folates in the diet before and after conception are known to protect against spinal tube defects in babies. However, many studies now suggest that a diet rich (or sufficient) in folates may also contribute to protection against a range of chronic disease, including CVD, cancer and loss of cognitive function in such conditions as Alzheimer’s disease.

The emphasis of studies at IFR is to quantify the bioavailabilty of both natural folate, as found in a range of foods such as leafy vegetables, and folic acid supplements, and to investigate the role of human genetic polymorphisms on folate absorption and metabolism. These studies have utilised isotope-labelled folates produced by feeding ¹⁵N to spinach plants.

Results from a series of human studies have suggested that current models of human uptake and metabolisim of folic acid from supplements and naturally occurring folates in food may need to be re-evaluated. We have subsequently developed a simple one-compartment kinetic model to estimate apparent folate absorption. Initial results obtained using our novel analytical methods suggest that the liver sequesters a greater proportion of newly absorbed folate when folic acid is consumed, as compared to a reduced folate, thus resulting in a decreased systemic plasma response, and that the site of folic acid metabolism into folates is in the liver, not the absorptive mucosa of the GI tract.

Current studies are also focused on how body folate status can be enhanced through consuming a mixed diet rich in folate-containing foods.

Single compartment model demonstrating the absorption of folate

Diet-gene interactions, nutritional genomics and NUGO

Studies of diet-gene interactions are a key facet of research at IFR. Powerful techniques such as microarrays, real time PCR and proteomics, provide unprecedented scope to unravel the complexity of the many parallel processes regulated within the tissues of our bodies by food components and their metabolites. The application of such techniques within the field of nutrition is termed nutritional genomics or nutrigenomics.

IFR is making significant contributions to this new and fast developing field both by undertaking fundamental studies in-house, such as our baseline gene expression study, and through substantial involvement in the European Nutrigenomics Organisation (NuGO), a Network of Excellence funded under the EU Framework VI programme and launched January 2004.

Baseline Gene Expression Study
This ongoing work is making use of microarrays, to define the normal degree of variation in patterns of gene expression (analysed at the level of RNA) for many thousands of different genes at the same time. We are examining how these patterns vary in blood samples obtained from different individuals, and in sets of samples obtained over several weeks from the same individual. This information is crucial for determining the scope and design of many future nutritional studies. It will also produce a description of normal, apparently healthy, patterns of gene expression in human blood cells.

NuGO
Scientists from IFR have been intimately involved in the development of this new project, which has been awarded €17.4m over 6 years. NuGO brings together 22 expert partner organisations from 7 EU member states, with the aim of spreading excellence in the field of nutrigenomics and driving forward this area of research. The project addresses the scientific and technical challenges faced in key areas of nutrition research and with new and developing genomic and computing research tools. IFR staff are key contributors, not only playing prominent roles in project management and in work packages on Gut Health, Risk-Benefit Analysis, Bioinformatics, and Science and Society but also leading work packages on Technology Innovation & Standardisation, and Communications.

Further reading

Kroon, P. A., Clifford, M. N., Crozier, A., Day, A. J., Donovan, J. L., Manach, C. & Williamson, G. (2004) How should we assess the effects of exposure to dietary polyphenols in vitro? American Journal of Clinical Nutrition 80 15-21

Kern, S. M., Bennett, R. N., Mellon, F. A., Kroon, P. A., Garcia-Conesa, M. T. (2003) Absorption of hydroxycinnamates in humans after high-bran cereal consumption. Journal of Agricultural & Food Chemistry 51 6050-6055

Petri, N., Tannergen, C., Holst, B., Mellon, F. A., Bao, Y., Plumb, G. W., Bacon, J., O’Leary, K. A., Kroon, P. A., Knutson, L., Forsell, P., Eriksson, T., Lennernas, H. & Williamson, G. (2003) Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejenum in vivo. Drug Metabolism & Disposition 31 805-813

Sanderson, P., McNulty, H., Mastroiacovo, P., McDowell, F. W., Melse-Boonstra, A., Finglas, P.M. & Gregory, J. F. (2003) Folate bioavailability: UK Food Standards Agency workshop report. British Journal of Nutrition 90 473-479

Wright, A. J. A., Finglas, P. M., Dainty, J. R., Hart, D. J., Wolfe, C. A., Southon, S. & Gregory, J. F. (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 bioavailablity studies. British Journal of Nutrition 90 363-371