Nutrients and Phytochemicals: from Bioavailability to Bioefficacy Beyond

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Nutrients and phytochemicals: from bioavailability to bioefficacy beyond antioxidants Birgit Holst1 and Gary Williamson1,2

The effect of any dietary compound is influenced by the active certain lipids and carbohydrates. As the latter is caused bioavailable dose rather than the dose ingested. Depending on by socio-economical factors rather than by a lack of the individual predisposition, including genetics and scientific knowledge, modern nutrition research has medication, a bioavailable dose may cause different changed focus. Traditionally, defining nutrient require- magnitudes of effects in different people. Age might affect the ments, identifying and correcting nutrient deficiencies, predisposition and thus the requirements for nutrients including and chemical and microbial contamination-related food phytonutrients (e.g. phytochemicals such as flavonoids, safety issues received most attention. Current emphasis is phenolic acids and glucosinolates). These are not essential for directed towards the development of functional, health growth and development but to maintain body functions and promoting foods and dietary recommendations for health health throughout the adult and later phases of life; they are maintenance and well-being throughout life, as well as ‘lifespan essentials’. Major mechanisms involved in chronic, diets for special groups of the population. On that basis, age-related diseases include the oxidant/antioxidant balance, significant research effort is focused on minor dietary but the latest research indicates indirect effects of dietary constituents, vitamins and trace elements, phytochem- bioactives in vivo and adaptive responses in addition to direct icals (carotenoids, flavonoids, indoles, isothiocyanates, radical scavenging. and so on), zoochemicals (conjugated linoleic and n À 3 Addresses fatty acids, and so on), fungochemicals and bacteriochem- 1 BioAnalytical Science Department, Nestle´ Research Center, Nestec icals (formed during food fermentations and by the gut Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland microflora). 2 Chair of Functional Food, Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Vitamins have been known for a long time to be essential Corresponding author: Holst, Birgit ([email protected]) for our body, and, during evolution, humans have lost the capacity to synthesize these compounds. They are specifically taken up, transported, metabolized and Current Opinion in Biotechnology 2008, 19:73–82 excreted depending on body requirements. Selected This review comes from a themed issue on vitamins have been proposed and intensively studied Food Biotechnology as being responsible for the health benefits assigned to Edited by Hannelore Daniel and Martin Kussmann fruits and vegetables. Available online 9th April 2008 Today, new functions of well-known micronutrients are 0958-1669/$ – see front matter studied as well as traditional ethnic plant foods, herbal # 2008 Elsevier Ltd. All rights reserved. extracts but also well-known and commonly consumed DOI 10.1016/j.copbio.2008.03.003 fruits and vegetables containing less well characterized active components designated as phytochemicals or phy- tonutrients. Introduction As early as in the first weeks of life but especially Among the well-known micronutrients, selenium and throughout adulthood and in the elderly populations, vitamin E are currently of interest for a range of func- our diet has a significant impact on health and well-being. tionalities beyond their vitamin function. One example is Examples range from neural tube defects caused by folate the increased viral pathogenicity and virulence of benign deficiencies in the unborn to age-related, chronic diseases viruses in deficiency conditions of these micronutrients, such as diabetes, cancer, cardiovascular and Alzheimer’s which may be related to an increased mutation rate in the disease. For diabetes, the metabolic syndrome and clini- virus population [1]. cally manifested deficiencies, an unbalanced nutrition or an inadequate diet are already established as key risk Although not designated as vitamins, there is a large group factors. On the basis of well-defined macronutrient and of compounds in fruits and vegetables, teas and herbal mineral requirements, possible interventions are known extracts which might not be essential throughout life or by scientists, authorities and the general population. cause clinically manifested deficiencies, but are essential Nevertheless, a large proportion of the world population for health and well-being in adulthood and in the elderly still suffers from severe deficiencies of major nutrient population. These compounds – phytochemicals – are groups while other populations are affected by diseases plant secondary metabolites, which protect the plant related to an over-consumption of nutrients such as against a variety of stresses. When consumed with the diet, www.sciencedirect.com Current Opinion in Biotechnology 2008, 19:73–82 74 Food Biotechnology

they may reduce the risk of age-related chronic diseases. This review discusses mainly phytochemicals as these are a Examples of classes of phytochemicals include flavonoids, major focus of current functional food development. phenolic acids, glucosinolate-derived compounds such as Furthermore, their uptake into the body and bioavailability isothiocyanates, terpenes, and low-molecular weight can be much lower than the bioavailability of ‘classical sulphur-containing compounds. nutrients’ but critical for their biological effects. Key lessons from the vitamins and trace elements will be Many of the phytochemicals and some vitamins have utilized to guide future research on phytochemicals. antioxidant activity in vitro, which has led to the use of the general term ‘antioxidants’. These have been linked What is bioavailability? to the health benefits of fruit and vegetables but too much On oral consumption, the uptake of micronutrients and of a ‘good’ thing can be bad, as shown from data examin- phytochemicals into the body is not complete, and a certain ing the effects of high doses of antioxidants individually percentage is not absorbed. To quantify the amount that is or in combination: results have been disappointing, even actually absorbed, distributed to the tissue, metabolized alarming. Thus, recent meta-analyses concluded that and eventually excreted, the term bioavailability was high, pharmacological doses of individual antioxidant introduced. Bioavailability describes the concentration of vitamins exert no health benefits, and might even a given compound or its metabolite at the target organ; increase the risk for certain diseases in some groups of however, no single definition exists that accurately takes the population [2,3]. into account the multi-factorial nature of the term. The Food and Drug Administration defines bioavailability as What went wrong, since these studies were designed to ‘the rate and extent to which the therapeutic moiety is absorbed show a positive effect on health? The apparent contro- and becomes available to the site of drug action’. Because of versy can be related to the high doses used, and thus difficulties in accessing organ sites in vivo in humans, closely to the bioavailability of antioxidants. Most effects attempts to use the term with quantitative precision or of micronutrients in general and dietary antioxidants in to calculate exact values in humans are challenging. As particular follow a U-shaped curve (Figure 1), with low or a consequence, the term ‘absolute bioavailability’is deficiency levels causing an increased disease risk, an often used by clinical pharmacologists to describe the optimal, protective amount (which may be a narrow or exact amount of a compound that reaches the systemic broad range), and excessive levels causing again an circulation, calculated as the fraction of the area under the increased disease risk. curve (AUC) after oral ingestion compared with the AUC after intravenous administration. In nutrition, however, Figure 1 ‘relative bioavailability’ is commonly used to describe the bioavailability of a compound from one source compared with another. The ‘classical’, pharmacological definition of bioavailability covers several linked and integrated processes; specifically, liberation, absorption, distribution, metabolism and excretion (generally presented under the acronym LADME, Figure 2).

L = Liberation, processes involved in the release of a compound from its matrix. A = Absorption, the diffusion or transport of a compound from the site of administration into the systemic circulation. D = Distribution, the diffusion or transportation of a compound from the intravascular (systemic circulation) to the extra-vascular space (body tissues). M = Metabolism, the biochemical conversion or biotransformation of a compound. E = Excretion, the elimination of a compound, or its metabolite, from the body via renal-, biliary- or pulmonary processes.

Many factors affect the bioavailability of a compound; Theoretical risk of chronic disease with dietary deficiency or excess these may be divided into exogenous factors such as the (‘mega-dose’). The dose–response of nutrients in general and complexity of the food matrix, the chemical form of phytochemicals in particular is not linear. At low concentrations deficiencies may be observed. Following a physiological dose range, the compound of interest, structure and amount of which can be relatively narrow, a further increase of the dose may lead to co-ingested compounds [4] as well as endogenous factors adverse effects. including mucosal mass, intestinal transit time, rate of

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Figure 2

Basic events describing the fate of nutrients: (1) liberation, the release and dissolution of a compound to become available for absorption (bioaccessibility); (2) absorption, the movement of a compound from the site of administration to the blood circulation; (3) distribution, the process by which a compound diffuses or is transferred from the intravascular (blood) to the extra-vascular space (body tissues); (4) metabolism, the biochemical conversion or transformation of a compound into a form that is easier to eliminate; and (5) excretion, the elimination of unchanged compound or metabolites from the body, mainly via renal, biliary, or pulmonary pathways. The complex connection between the luminal and body content results in a web of possible pathways for each compound. Indicated in blue are possible biological specimens for biomarker measurements. (With kind permission from John Wiley & Sons Limited [39]). gastric emptying, metabolism and extent of conjugation, in the lumen that is in the right form to be absorbed) is and protein-binding in blood and tissues. When taken critical and could limit mineral and trace element absorp- together, these factors can cause large inter-individual tion if bound to other food constituents or present in a and intra-individual variations in bioavailability, some- non-soluble form. The bioavailability of minerals and times spanning the entire range from 0% to 100% of the trace elements is under strict homeostatic regulation, ingested dose. which modulates active transport as well as metabolism and excretion from the body. More recently, similar Bioavailability and interaction with drug transporters for absorption and distribution as well as metabolism metabolic enzymes have been described for selected For nutrients, especially macronutrients, bioavailability is vitamins, for example vitamin C and E. generally high. Active transport mechanisms for fatty acids, amino acids, small peptides and sugars enable Unlike macronutrients, some vitamins, such as vitamin E, uptake into and transport within the body. Mineral and undergo first pass phase I and II metabolism in the small trace element absorption in the gut also involves active intestine and liver, which is generally recognized for transport mechanisms. However, these transporters are handling of xenobiotics, including phytochemicals. often less specific, as it is the case for the transporter of divalent cations, which transports several minerals, but With a regular diet, humans consume up to several grams only soluble forms of the free cations. Therefore, bioac- per day of phytochemicals. Nevertheless, because of the cessibility (the amount of a given compound or element large number of structurally different compounds, the www.sciencedirect.com Current Opinion in Biotechnology 2008, 19:73–82 76 Food Biotechnology

limited uptake and bioavailability of some phytochemicals, majority of drugs. On this basis, phytochemicals may their occurrence as parent compound or metabolites compete with drugs for enzymes, co-factors and trans- at the systemic and tissue levels is relatively low and porters and therefore have a significant potential to influ- in the micromolar range [5]. The reason for the low ence drug uptake, metabolism and clearance from the bioavailability of certain phytochemicals, especially when body. Although drug–nutrient interactions during absorp- compared to macronutrients, is based on their recognition tion and metabolism are very likely to occur and may have and handling by the body as xenobiotics. Thus, the enormous health implications, these have rarely been estimated absorption of polyphenols when based on considered, even for well studied vitamins like vitamin urinary data from healthy volunteers and from ileostomist E. Different tocopherols are metabolized by CYP3A, a studies (ileostomy involves bringing the ileum to the phase I cytochrome P450 enzyme, which also metabolises abdominal surface by surgical intervention; ileostomy almost 30% of all drugs. Simultaneously, CYP3A is patients are often enrolled into studies on small intestinal induced by a-tocopherol via the activation of the preg- absorption and metabolism) is between 1% and 60% [5,6]. nane-X-receptor, a member of the family of nuclear Already during absorption, phytochemical bioavailability is receptors which are activated by a large number of further decreased by extensive phase I and especially lipophilic xenobiotics [11]. Drug–nutrient are most likely phase II biotransformation reactions that produce conju- at high, supplemental doses. gates and metabolites with sometimes increased, but usually decreased, biological activity compared to the Examples for which drug–nutrient interactions have been parent compound. The resulting water soluble and stable studied include St. John’s Wort, a medicinal herb, indu- conjugates are rapidly excreted by the body [7]. On the cing cytochrome P450 enzymes and the corresponding basis of drug trials using high, pharmacological doses, the transporters. The resulting decreased plasma concen- liver was originally considered to be the major site of trations of prescribed drugs are described for cyclosporin, xenobiotic metabolism. However, small intestinal enter- where sub-therapeutic levels resulted in transplant organ ocytes express a significant capacity for phase I and II rejection, and reduced efficacy of oral contraceptives metabolism and efflux pumps, for example p-glycoprotein [12,13]. The best-known example of drug–food inter- and ABCC2. Thus, for nutritional doses of micronutrients actions concerns grapefruit juice. Grapefruit contains in general and in particular phytochemicals, small intesti- compounds that inhibit CYP3A, thus altering the metab- nal metabolism and efflux could significantly limit the olism of many drugs, reducing their clearance and increas- uptake of compounds for which this cannot be explained ing their bioavailability significantly [14]. by molecular properties, for example size and polarity [5]. Because of the difficulty in accessing the small intestine as Clinically significant drug–nutrient interactions have also a site of absorption and first pass metabolism (only human been described for tyramine, a monamine oxidase inhibi- in situ perfusion or ileostomist studies allow in vivo data to tor-based anti-depressant. A large range of foods contain- be obtained [8]), the pathways are not well characterized. ing high amounts of tyramine (e.g. avocados, bananas, beans, cheese, chocolate, coffee, fish, processed meat, Unabsorbed phytochemicals may be active in the raspberries, soy beans, wines and yogurt) have been stomach or gut, rather than systemically [9]. As the gut shown to strongly affect drug efficacy [15]. These are is the major organ involved in immune response, local prominent examples but drug–nutrient interactions con- effects within the gastrointestinal tract may still affect cern a far broader range of food, especially food of plant systemic health parameters and overall health indirectly, origin containing high concentrations of phytochemicals, but significantly. since most of the phytochemicals have the potential to increase or decrease efficacy of certain drugs. Currently, Reaching the colon, phytochemicals are broken down by drug–nutrient interactions present a threat since they are the gut microflora to form a wide range of bioactive not well characterized and understood. If sufficient products. Some of these could be responsible for the health knowledge becomes available, modulation of drug meta- benefits ascribed to the parent phytochemical, such as bolising enzymes and transporters (e.g. P-gp expression equol, which is formed by the gut microflora. Increasing and/or activity) may be a useful strategy to improve the evidence shows that the clinical effectiveness of soy pharmacological profile of drugs [12]. isoflavones depends partly on the microbial biotransforma- tion of isoflavones into the stronger oestrogen, equol [10]. Bioaccessibility is necessary for Processes (in red) and organs (in white) involved in bioavailability nutrient and phytochemical absorption, distribution, A prerequisite for bioavailability of any compound is metabolism and excretion are summarized in Figure 2. their bioaccessibility in the gut, defined as the amount that is potentially absorbable from the lumen (Figure 2). Metabolism and elimination of phytochemicals follows Depending on the food matrix, for example location in the same biochemical pathways and employs the same the plant, food processing, gastric and luminal digestion, metabolic enzymes, transporters and efflux pumps as the in addition to the chemical form and properties of

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nutrients and phytochemicals, a low bioaccessibility may requires a broad range of protective mechanisms. Indeed, limit their bioavailability significantly. Good examples if accumulated at specific, suitable sites or specialized are the minerals, which, in general, need to be presented compartments (e.g. a-tocopherol in plasma LDL or cell to the enterocytes in a soluble form to be absorbed. membranes) or at appropriate concentrations (e.g. urate The formation of insoluble salts, such as chelates with and ascorbate), small molecule antioxidants may take part phytic acid, reduces their solubility and finally their directly in redox reactions in vivo. bioavailability. Dietary antioxidants may furthermore function as free Antioxidants are by nature and function subject to oxi- radical scavengers in food and/or during digestion, redu- dation, which limits their stability in the product during cing the formation of advanced lipoxidation end products storage, food processing and digestion, and thus their (ALEs) and advanced glycation end products (AGEs). bioaccessibility. Good examples are green tea catechins. The debate on the health implication of extensive ALE and AGE consumption with processed food, as well as of Most phytochemicals are present in the food as precur- their formation during digestion, is still ongoing [9,23,24]. sors, for example as glycosides, but are predominantly A recent publication by Gorelik et al. suggest that red absorbed as aglycones. The cleavage of the glucose wine polyphenols exert a beneficial effect by preventing moiety is catalysed in the gut lumen by the brush border ALE production in food and by inhibiting absorption of enzyme lactase phloridzin hydrolase (LPH) [16]. For cytotoxic lipid peroxidation products. They showed that phytochemicals with different sugar moieties, for after consumption of a turkey meal, plasma and urinary example hesperidin (occurs naturally as a rhamnoside), malondialdehyde (MDA) levels were increased, whereas LPH does not catalyse the hydrolysis of the sugar moiety. consumption together with red wine significantly alle- For these phytochemicals, the site and kinetics of absorp- viated this increase [25]. However, MDA is a controver- tion and finally their bioavailability can be modulated by sial biomarker and more research in this area is needed enzymatic release of the attached sugar, for example [26]. rhamnose during processing [17]. Despite a large number of publications on the classical Bioefficacy: beyond free radical scavenger hydrogen-donating activity of dietary antioxidants, antioxidants especially of phytochemicals, it is now considered that Many diseases with a strong dietary influence include this function is unlikely to be the sole explanation for oxidative damage as an initial event or at an early stage of their effects in vivo. This premise is on the basis of several disease progression. Therefore, a major focus in dietary lines of reasoning: disease prevention is placed on antioxidant intervention. According to research over the last decade, including 1. The in vivo redox status is under strict homeostatic many human intervention studies, antioxidants in general control by a complex antioxidant network consisting of and phytochemicals in particular play an outstanding role antioxidant compounds and antioxidant enzymes in lowering chronic disease risk [7,18]. For decades, the involved in the efficient recycling of endogenous beneficial role of antioxidants was related to the reduction antioxidants. It involves three levels of protection: of unwanted and uncontrolled production of reactive prevention, interception and repair. The role of dietary oxygen species which would lead to a status designated antioxidants in these processes is still not known. The as oxidative stress. Today, the scientific community topical question whether small molecule antioxidants, increasingly recognizes that their mechanism of action for example phytochemicals, micronutrients and in vivo might be far more complex [19,20,21]. vitamins act as free radical scavengers in vivo is still not answered [22]. The antioxidant dilemma 2. Only micromolar or nanomolar concentrations of The human body is well adapted to the consequences of dietary antioxidants, especially phytochemicals and aerobic life, especially the generation of reactive oxygen their metabolites, have been detected in vivo, for species. The appropriate level of free radicals versus example in plasma or in organs such as the brain, which antioxidants at specific cell and tissue level is essential are one or more orders of magnitude lower than those since, at low concentrations, free radicals serve as sig- recorded for endogenous antioxidants such as gluta- naling molecules and, at somewhat higher concentrations, thione and albumin. Defence against hydroxyl radicals they are involved in immune defence. Interference with by small molecules, however, requires concentrations these processes by excessive administration of antioxi- in the high millimolar range. Consequently, at the dants may not be desirable. whole body level, dietary antioxidants are unlikely to express a beneficial action in vivo by out-competing As oxidation generates a diverse range of free radical endogenous antioxidants, which are present at signifi- species in all cells and cell compartments and thus in cantly higher concentrations. Hence, an increase in very different environments, the antioxidant defence total antioxidant capacity as measured by currently www.sciencedirect.com Current Opinion in Biotechnology 2008, 19:73–82 78 Food Biotechnology

popular assays such as total reactive antioxidant selenium levels, explaining the synergism between Nrf2 potential (TRAP), ferric reducing ability of plasma activators and selenium [34]. Nrf2-regulated classes of (FRAP), trolox equivalent antioxidant capacity genes affect not only electrophile detoxification and free (TEAC) is, in most cases, not related to a direct radical metabolism but also glutathione homeostasis, radical scavenging effect of the supplemented anti- generation of reducing equivalents, solute transport and oxidant [22]. enhanced proteasome function. Nrf2 intersects other sig- 3. Current evidence suggests that the cellular effects of naling pathways such as NF-kB in inflammation and AhR, dietary antioxidants may be mediated by their the aryl hydrocarbon receptor, which regulates xenobiotic interactions with specific proteins central to intra- detoxification [35,36]. The Nrf2/Keap1 system is now cellular signaling cascades. These are detailed in the recognized as one of the major cellular defense mechan- paragraph adaptive response/hormesis. isms against oxidative and xenobiotic stresses and contrib- utes to the protection against various pathologies, Even the in vivo radical scavenging activity of the ‘clas- including carcinogenesis, liver toxicity, respiratory distress sical’ lipid soluble antioxidant a-tocopherol (vitamin E) is and inflammation. currently strongly debated. A major argument supporting a role of a-tocopherol beyond radical scavenging is the Given the large number of antioxidants and especially significant difference in the molecular function between phytochemicals and possible target organs and functions, individual tocopherols as well as tocopherols and toco- there is a need to focus on and examine the most prom- trienols [27]. Furthermore, a-tocopherol has been shown ising actions of a few selected compounds and end-points. to affect transcriptional gene regulation, cell proliferation, Consideration of realistic effects needs to take into platelet aggregation, and monocyte adhesion, and may account the dose, since some effects are only seen at play a specific biological role in risk reduction of chronic ‘mega-doses’ in vitro. Commonly, a phytochemical only diseases [28]. modulates one or two biochemical reactions in vivo which consequently affect several pathways and endpoints. For Adaptive response/hormesis example, the inhibition of one specific enzyme, COX-2, Phytochemicals are processed by the body as xenobiotics affects inflammation, and consequently the development since it does not distinguish between beneficial, neutral of a larger number of chronic diseases. Another example is or toxic compounds but only between nutrients and epicatechin, a polyphenol found in cocoa, green tea and compounds which are not nutrients. Many activate specific many fruits. It affects nitric oxide formation via nitric or generalized adaptive cellular response pathways to oxide synthase and NADPH oxidase, hence modifying oxidative stress and environmental toxin exposure. Thus, vascular biomarkers such as flow mediated dilation, phytochemicals may exert at least part of their beneficial an important diagnostic marker of cardiovascular risk effects by acting as ‘low-dose stressors’ or pro-oxidants [37]. that may prepare cells to resist more severe stress [29]. Characteristic of such a hormetic mechanism of action is a We propose that in vitro work should be driven by in vivo biphasic dose response on cells [30]. Low phytochemical results, and not vice versa. In nutrition, it is unrealistic to doses activate signaling pathways that result in increased extrapolate results from in vitro studies to a protective expression of genes encoding cytoprotective proteins, but effect in vivo. Once a compound or food is proven to exert high doses are cytotoxic. The most prominent example is an effect in vivo, then mechanisms can be tested in vitro; the activation of the Nrf2/Keap1 pathway (nuclear factor this circumvents the ‘what concentration to use?’ argu- erythroid-2 related factor 2/kelch-like ECH-associating ment in vitro and avoids disappointments when testing in protein 1) by phytochemicals such as sulforaphane, curcu- vitro concepts in vivo in humans. The proposed approach min, dithiolethiones and triterpenoids [31,32]. Once is the opposite of that used for drug discovery, where in dissociated from the Nrf2/Keap1 complex, Nrf2 regulates vitro screening precedes in vivo tests. For a complete basal and inducible expression of a large number of genes picture, there must be a combination of in vitro mechan- encoding enzymes involved in antioxidant functions or istic studies and in vivo interventions, preferably together xenobiotic detoxification (e.g. thioredoxin reductase-1 with epidemiological support. We conclude that mechan- (TrxR1), gastrointestinal glutathione peroxidase (GI- istic in vitro data using higher concentrations than found GPx, GPx2), heme oxygenase-1, NAD(P)H:quinone in vivo (within reason) should only been considered if oxidoreductase or glutathione S-transferase) via the stress- they can be supported by an in vivo effect. or antioxidant-response elements (StRE/ARE). The num- ber of compounds which interact with the Nrf2/Keap1 Biomarkers to link bioavailability and complex is large and recent in vitro data even propose that bioefficacy coffee polyphenols stimulate endogenous defense mech- Assigning health benefits to a particular dietary pattern, anisms against electrophilic and oxidative insults [33]. individual food or food constituents is difficult because Whether increased protein synthesis follows the enhanced of the complexity of the diet, the co-occurrence and transcripts depends, for some proteins, on adequate interactions of nutrients and phytochemicals in foods,

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interactions between diet and the genetic background of avert disease but with the least certainty; later markers are the individual and environmental factors. Food frequency most closely related to the disease but offer fewer chances questionnaires, food diaries, and 24 hour recalls are often for dietary intervention [41]. used but by no means present an accurate measure of the dietary intake of micronutrients and phytochemicals. The Biomarkers need to be responsive, specific, easy to use, information obtained is combined with food composition applicable and relevant. tables and databases to calculate intake of specific nutri- ents. Food composition databases are now being estab- Biomarkers for exposure and effect can be studied in a lished for vitamins and phytochemicals, for example by hypothesis-driven manner, in a targeted way or at global the USDA National Nutrient Databank System (NDBS; level where proteomics (global protein analysis) and meta- http://www.ars.usda.gov/ba/bhnrc/ndl). However, in rea- bolomics (global metabolite analysis) have become major lity, levels of micronutrients and especially of phyto- tools. Proteomic platforms have the advantage of deliver- chemicals are highly variable as they are affected by ing both markers for disposition and efficacy as well as the seasonal and agronomic factors, plant variety, age and targets of intervention [42]. By contrast, metabolomics part of the plant used, food preparation conditions, and so enables a quantitative, non-invasive analysis of easily on. The lack of accuracy using this approach can lead to accessible human body fluids (urine, blood, saliva, tears) serious problems of interpreting epidemiological studies and has therefore gained popularity in current nutrition or human intervention trials [38]. A more accurate research [43,44]. A critical discussion concerning the measure of exposure to a dietary bioactive compound application of these technologies in the nutrition and can be obtained from biomarkers of exposure, provided health context is given in the chapter ‘Profiling techniques these have been sufficiently validated [39]. Biomarkers in nutrition and health research’ of this Journal. can also be used to estimate or identify changes and effects occurring within an organism, and to assess its The problems of applying biomarkers to nutrition research underlying susceptibility. are less related to accuracy and sensitivity of the analytical techniques but more to a lack of validation in terms of (i) Biomarkers in general are defined as observable proper- understanding bioavailability and mode of action of bioac- ties of an organism that indicate variation in cellular or tive food components; and (ii) the application in large biochemical components, structure, or function, and that population studies. Therefore, future research should can be measured in biological systems or samples [40]. A focus on development, validation and application of valid biomarker could also be considered as a key measure appropriate biomarkers of exposure, effect and suscepti- linking a specific exposure of a dietary compound to a bility which should be combined and applied consistently health outcome, as well as early signals of biological in well designed intervention trials, preferably registered effect. Additionally, biomarkers of susceptibility consider on a site such as http://www.clinicaltrials.gov/ to improve host, environmental and lifestyle factors, as well as the transparency. We would like to emphasise here that a genetic polymorphisms, which cause some individuals common registration not only increases the visibility but to be more prone to diseases and inter-individual differ- also the credibility of clinical trial outcomes. ences in the response to a dietary regime or intervention. The processes involved can be envisioned as a continuum For micronutrients in general and for phytochemicals of that links exposure, dose, and effect (Figure 3), where dietary origin specifically, the interpretation of studies biomarkers for disease prevention and intervention may either with the purified compound or its dietary source is be measured anywhere along the pathway. Earlier mar- further complicated by their low levels of ingestion and kers (to the extent that they are measurable at low often weak biological activity in the short-term. Phyto- exposure or dose) have the greatest potential utility to chemicals may have multiple targets and, depending on

Figure 3

Classes of biomarkers to describe the pathway from exposure to health/disease outcome. (With kind permission from John Wiley & Sons Limited [39]). www.sciencedirect.com Current Opinion in Biotechnology 2008, 19:73–82 80 Food Biotechnology

their dose, they can be both beneficial and deleterious. need to be carefully evaluated and the bioavailability of the This poses particular problems in determining the net active component measured and linked to the clinical effect of individual foodstuff and their constituents. outcome. For example, quercetin cannot be given as Further key points to consider when assessing the use powder in a capsule since it will not be solubilized in of biomarkers in studies of nutrition and health include: the gut and therefore not be absorbed and effective at timing of a measurement in relation to the response, systemic or tissue level. health status and genetic background of the individual, homeostatic regulation of nutrient levels and environ- For dietary interventions, we strongly support the trend to mental factors [39]. fund research related to prevention rather than cure, because the latter should remain in the medical and Conclusions pharmaceutical area. Nevertheless, a realistic goal for risk Time for paradigm changes reduction of chronic diseases such as cardiovascular disease The Food and Nutrition Board (FNB) of the IOM/NAS is and some cancers by dietary means could be as high as 30%. currently expanding the list of nutritional substances, establishing Dietary Reference Intakes (DRIs) rather than Can there be a beneficial effect of phytochemicals in an nutrient intakes. The FNB recognizes that there may be already healthy organism? dietary substances other than the classic nutrients for This depends on the definition of healthy. Biomarkers which recommendations should be given. The DRI com- may define the status of an apparently healthy individual, mittee emphasizes that (i) functional endpoints, other than but the apparent ‘health-range’ of a given biomarker can clinical manifested deficiency, might be important in be large and also change, for example with age; there establishing dietary recommendations and that (ii) biologi- might be no ‘correct’ value for a given biomarker in all cally active dietary substances, including nutrients, population groups and all conditions. might have substantially different functional outcomes at different intake levels. These functional outcomes could In general, it is difficult to further improve the nutritional include toxic or other adverse responses, even for nutrients. and health status in a well-nourished and healthy person. This is particularly true for nutrients, including micronu- Functional food development trients, were homeostatic regulation of their concentration While dietary recommendations mainly concern the appro- may affect the outcome of a dietary intervention. Homeo- priate provision of nutrients, the development of func- stasis in individual tissues further complicates the tional food places specific emphasis on potential health interpretation of biomarkers measured in easily accessible benefits of ‘antioxidant’ phytochemicals. For a final func- body fluids, for example the blood. Nevertheless, even if tional food product, it is important to guarantee consistent tissue concentrations are not yet affected, low plasma levels and profiles of phytochemicals, and to be backed up levels may reflect increased requirements and/or decreased by data on their safety and clinical relevance. Since long- dietary uptake, which can be corrected by dietary means. term effects of increased doses are difficult to study and Thus, for ascorbate, the body tightly regulates the plasma thus to foresee, the development of sustainable functional level, but if ascorbate is low, vitamin C in the diet can foods should only consider the same level of phytochem- increase this level. However, as for the definition of a icals as the best dietary source can deliver at dietary doses. ‘healthy person’, it is difficult to define deficient, low or This will avoid the ‘mega-dose’ problems recently seen for sufficient plasma levels. A low or deficient plasma level in the antioxidant vitamins. Within this framework, proces- one subject might present a normal status in another. A sing has a big role to play in optimising the preservation of good example is the highly variable level of plasma ascor- the active components throughout the manufacturing bate found in different, healthy individuals. process while current by-products of fruit and vegetable processing (e.g. pomace), if treated and stored as food, Are phytochemicals adult vitamins? could provide rich sources of bioactive phytochemicals. Although phytochemicals might not be essential for growth and development or the maintenance of major Key lessons from the vitamins body functions, there is increasing knowledge concerning Recent meta-analyses reveal different major issues con- their potential for health maintenance or disease risk cerning intervention trials using high doses of antioxidant reduction throughout adulthood and during aging. This vitamins and trace elements. To avoid this situation in the means that they are essential to fulfil the maximum future for phytochemicals, a most comprehensive knowl- individual lifespan, and so we propose that they are edge base concerning bioavailability, bioefficacy and the ‘lifespan essential’. This does not necessarily include effect of predisposition need to be assembled and carefully an increase of the maximum potential lifespan, but rather considered when establishing a hypothesis for a clinical an increase of the chance of reaching the genetically trial and the study design. Future intervention trials need determined lifespan and an increase in the quality of life to enrol a sufficient number of well-defined subjects to during aging by reducing the incidence of chronic, age- ensure statistical power. The dosage form and quantity related diseases (Figure 4).

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Figure 4 supplementation may increase all-cause mortality. Ann Intern Med 2005, 142:37-46. 3. Bjelakovic G, Nikolova D, Simonetti RG, Gluud C: Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis. Lancet 2004, 364:1219-1228. 4. Scholz S, Williamson G: Interactions affecting the Bioavailability of dietary polyphenols in vivo. Int J Vit Nut Res 2007, 77:224-235. 5. Manach C, Williamson G, Morand C, Scalbert A, Remesy C:  Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005, 81:230S-242S. This review summarizes published human bioavailability data on poly- phenols and is the first to give a quantitative estimation of the absorption and metabolism from different foods and from different studies. 6. Kahle K, Kraus M, Scheppach W, Richling E: Colonic availability of apple polyphenols – A study in ileostomy subjects. Mol Nutr Food Res 2005, 49:1143-1150. 7. 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