Nutritional and Clinical Relevance of Lutein in Human Health

British Journal of Nutrition (2003), 90, 487–502 DOI: 10.1079/BJN2003927 q The Authors 2003

Review article

Nutritional and clinical relevance of lutein in human health

F. Granado*, B. Olmedilla and I. Blanco Unidad de Vitaminas, Servicio de Endocrinologıá y Nutricioń, Hospital Univesitario Puerta de Hierro, C/ San Martıń de Porres, 4, 28017-Madrid, Spain

(Received 6 February 2003 – Revised 7 April 2003 – Accepted 29 April 2003)

Lutein is one of the most widely found carotenoids distributed in fruits and vegetables frequently consumed. Its presence in human tissues is entirely of dietary origin. Distribution of lutein among tissues is similar to other carotenoids but, along with zeaxanthin, they are found selectively at the centre of the retina, being usually referred to as macular pigments. Lutein has no provitamin A activity in man but it displays biological activities that have attracted great attention in relation to human health. Epidemiological studies have shown inconsistent associations between high intake or serum levels of lutein and lower risk for developing cardiovascular disease, several types of cancer, cataracts and age-related maculopathy. Also, lutein supplementation has provided both null and positive results on different biomarkers of oxidative stress although it is effective in increasing macular pigment concentration and in improving visual function in some, but not all, subjects with different eye pathologies. Overall, data suggest that whereas serum levels of lutein have, at present, no predictive, diagnostic or prognostic value in clinical practice, its determination may be very helpful in assessing compliance and efﬁcacy of intervention as well as potential toxicity. In addition, available evidence suggests that a serum lutein concentration between 0·6 and 1·05 mmol/l seems to be a safe, dietary achievable and desirable target potentially associated with beneﬁcial impact on visual function and, possibly, on the development of other chronic diseases. The use of lutein as a biomarker of exposure in clinical practice may provide some rationale for assessing its relationship with human health as well as its potential use within the context of evidence-based medicine.

Lutein: Biomarkers: Macular pigments: Evidence-based medicine

Lutein is a plant pigment that belongs to the well-known In foods, lutein can be found either in its free form, group of carotenoids. Man is not capable of synthesizing bound to proteins, or esteriﬁed as a monoester or di-ester carotenoids de novo and, thus, their presence in human (Klaui & Bauerfeind, 1981; Goodwin & Britton, 1988). tissues is entirely of dietary origin, although man is capable After being released from the food matrix, it is incorpor- of modifying some of them to some extent. Lutein is, along ated into micelles to be absorbed by passive transport by with b-carotene, one of the most widely distributed enterocytes and, along with other carotenoids and other carotenoids in fruits and vegetables frequently consumed fat-soluble dietary components, is incorporated into nas- by different populations (Granado et al. 1996; O’Neill et al. cent chylomicrons for transport to the liver. In blood, 2001). Chemically, lutein and its structural isomer zeax- lutein is transported by lipoproteins with an even distri- anthin are the dihydroxy derivatives of a-carotene and bution among the different classes (i.e. LDL and HDL) b-carotene, respectively, presenting two hydroxyl groups (Parker, 1996). In addition, because of its polarity, it is at the terminal rings of the molecule (Fig. 1), and thus are assumed to be located at the lipoprotein surface and, referred to as xanthophylls. The presence of substituted thus, is more readily transferred among the different terminal b-rings in the molecule, however, also confers a classes of lipoproteins even during postprandial metab- higher polarity, which determines, in part, distinctive olism (Parker et al. 1999). characteristics during absorption, transport, metabolism Although distribution of lutein among tissues is similar and deposition in tissues (Erdman et al. 1993; Castenmiller to that of other carotenoids and is determined at least & West, 1998; Parker et al. 1999). partly by LDL-receptor density, both lutein and zeaxanthin

Abbreviations: ARMD, age-related macular degeneration; CVD, cardiovascular disease; FCT, food composition table; MPOD, macular pigment optical density. * Corresponding author: Dr F. Granado, fax þ34 91 3737667, email [email protected] 488 F. Granado et al.

Nevertheless, since the 1950s, the quest for optimal health, not the mere avoidance of deficiency, combined with the development of analytical techniques and nutritional epidemiology, which showed the potential effects of many food components as preventive or risk factors in relation to chronic and degenerative diseases, changed this context. Nowadays, nutritional interest in lutein is based not on its ‘essentiality’ but on: (1) the biological activities it shows, which may be potentially relevant to human health; (2) its presence in foods frequently consumed and, thus, the possibility of manipulating its intake by several dietary approaches, with a potentially important impact on human health, disease prevention and cost savings. For example, concerning age-related eye diseases where lutein may play a beneficial role, pooled data from North America, Europe and Australia provide estimates of about 0·2 % of individuals aged 55 to 64 years and 13 % of those aged 85 years or older to have age-related macular degeneration (ARMD; Flood et al. 2002). Since the elderly is an increasing proportion of the population, it is estimated that in the USA about 6·3 million individuals will have ARMD by the year 2030 (Seddon & Hennekens, 1994). So, identifying preventive factors for ARMD is especially important because treatment possibilities are limited both in scope and effec- tiveness. Similarly, cataract is one of the major causes of preventable blindness throughout the world with an increase in prevalence (in the USA) from approximately 5 % at age 65 years to about 50 % for individuals older Fig. 1. Chemical structures of a-carotene (a), b-carotene (b), than 75 years. In this sense, it is estimated that a delay lutein (c) and zeaxanthin (d). in cataract formation of about 10 years would reduce the prevalence of visually disabling cataract by about 45 %, enhancing the quality of life for much of the world’s older population and substantially reducing the economic are selectively accumulated in different parts of the human burden (US$ 5–6 billion) due to cataract-related disability eye (Bone et al. 1985; Handelman et al. 1988; Yeum et al. and cataract surgery (Taylor & Hobbs, 2001). 1995; Bates et al. 1996; Rapp et al. 2000). Lutein and According to the criteria of Bendich & Olson (1989), to zeaxanthin constitute by far the major carotenoids present establish the potential role of lutein in human health, its in these tissues where, in addition, highly specific binding biological activities can be characterized as functions, proteins for these two carotenoids have recently been actions and associations. Functions relate to the essential detected (Yemelyanov et al. 2001). Lutein and zeaxanthin role the nutrient plays at physiological concentrations. In are especially abundant at the centre of the retina (macula) terms of nutrition, functions refer to the essentiality of a and both xanthophylls are usually referred to as macular nutrient and thus its capacity to prevent deficiency states. pigments. Since the only proven function of carotenoids in man is the provitamin A activity of some of them, lutein is not considered an essential nutrient for man. In fact, to our Nutritional relevance: is lutein biologically active in knowledge, there is no clinical condition reported in man man? specifically associated with lutein deficiency or toxicity, For a long time, nutrients have been widely considered in apart from the (reversible) hypercarotenaemia, with or terms of their essentiality to man, in the interests of avoid- without carotenodermia (skin pigmentation), usually due ing nutritional deficiencies. Traditionally, the nutritional to an excessive intake, although it may also be associated importance of carotenoids in man has rested on their provi- with some pathological conditions (i.e. diabetes mellitus, tamin A activity (the capacity to be converted into vitamin anorexia nervosa) (Rojas-Hidalgo, 1987). A) exhibited by some of them (i.e. b-carotene, a-carotene Actions usually refer to effects (beneficial or adverse) and b-cryptoxanthin). However, because of the presence of displayed by a given component when tested under non- hydroxylated terminal rings in its structure, lutein does not physiological conditions (usually using ‘pharmacological’ fit the structural requirements for this activity and, thus, doses) in vitro, ex vivo, in cell cultures or in animal shows no such activity in man (Zechmeister, 1962; models. As observed with other carotenoids, a number of Simpson, 1983), a fact that probably constitutes the biological actions have been reported for lutein under major reason its potential relevance in human health had different assay conditions including cell–cell communi- long been overlooked. cation, inhibition of cell transformation, inhibition of the Lutein in human health 489 monocyte-mediated inflammatory response, immune modification of a disease process (structural alterations, enhancement, in vitro antioxidant activity, inhibition of reduced disease risk) that is causally related to the health LDL resistance to oxidation and macula protection outcome (Van’t Veer & Kok, 2000). (Bendich & Olson, 1989; Sies et al. 1992; Chopra & Traditionally, carotenoid (namely lutein) exposure or Thurnham, 1993; Bertram, 1994; King et al. 1997; nutritional status assessment in human subjects has been Park et al. 1998; Beatty et al. 1999; Van den Berg et al. routinely performed by dietary or biochemical methods, 2000; Collins, 2001; Dwyer et al. 2001). both of which have advantages and limitations. More Many aspects of the metabolism of carotenoids in man recently, for research purposes, non-invasive psychophysi- remain largely unknown. However, dehydration products cal tests using heteroflicker photometry (Hammond et al. (anhydrolutein), geometric isomers and ester forms of 1997; Landrum et al. 1997; Beatty et al. 2001) and lutein have been reported in human serum and tissues, as Raman spectroscopy (Bernstein et al. 1998, 2002) is have several putative oxidative metabolites, referred to as being used to measure macular pigment optical density ketocarotenoids (Khachik et al. 1992, 1997a,b). Since (MPOD), which provides information on long-term lutein these ketocarotenoids are not widely found in foods fre- (and zeaxanthin) exposure. However, while these new quently consumed and their concentrations in serum tools may be highly valuable, at present, the instruments increase upon lutein supplementation, it is suggested that are not commercially available for routine nutritional or these carotenoids are formed in vivo (Khachik et al. ophthalmological assessment. In addition, because of the 1995; Olmedilla et al. 1997a). In this respect, it has been selective deposition of these carotenoids in this tissue proposed that lutein and zeaxanthin (macular pigments) (retina), its relevance to other tissues, intermediate bio- may prevent light-initiated oxidative damage to the retina markers and (non-ocular) clinical conditions remains to and retinal pigment epithelium and thus protect against be established. age-related deterioration (Hammond et al. 1998; Beatty et al. 1999). However, it must be pointed out that while Dietary intake assessment these actions provide biological mechanisms by which lutein may exert its action, extrapolation to in vivo situ- Although about forty to fifty carotenoids are available in the ations is difficult and these actions must be demonstrated diet to be absorbed, metabolized or utilized by man, only in human subjects and their relevance established under five or six are routinely measured in foods and human in vivo physiological conditions. serum and tissues. Foods traditionally considered as good The term associations refers to epidemiological evidence sources of lutein are green vegetables, especially dark of a correlation between nutrient exposure (lutein) and green varieties such as spinach, broccoli, beet, lettuce, etc. health or disease outcomes. Epidemiological associations (Heinonen et al. 1989; Granado et al. 1992; Chug-Ahuja have been reported between lutein and several highly et al. 1993; Hart & Scott, 1995). As occurs with other caro- prevalent diseases in developed countries, namely cardio- tenoids, dietary intake of lutein shows a high variability vascular diseases (CVD), several types of cancer and within and between subjects and among populations age-related cataracts and macular degeneration (see later, (Thurnham, 1988; Ito et al. 1990; Hercberg et al. 1994; p. 492). These associations, however, do not provide a Granado et al. 1996; Agudo et al. 1999; National Academy ‘causal’ link but basically statistical or epidemiological of Sciences Institute of Medicine, 2000; O’Neill et al. 2001; relationships. Johnson-Down et al. 2002), which relates to both natural or Summarising, regardless of the lack of provitamin A intrinsic factors (dietary changes, seasonality) and methodo- activity, the biological actions displayed by lutein, along logical aspects (Granado et al. 1997; Rodriguez-Amaya, with the epidemiological evidence in relation to chronic 1997; Deharveng et al. 1999). and degenerative diseases, has triggered the interest in Regardless of the confidence in the method used for diet- this carotenoid and its consideration as a potentially ary assessment, evaluation of nutrient exposure by dietary beneficial phytochemical with relevance to human health. means is ultimately based on the availability of reliable In addition, the differential characteristics of lutein (and data on food composition. Since the nutritional interest in zeaxanthin), namely the selective accumulation in the carotenoids was largely due to their pro-vitamin A activity, human eye and the presence of binding proteins with traditionally, food composition tables (FCT) and databases high affinity for these xanthophylls, support the biological did not include values for individual carotenoids in foods, plausibility of a relevant role in human health. although they were considered for vitamin A (retinol equivalents) content. However, the increasing evidence of the potential role of several constituents of fruits and Assessment of lutein exposure vegetables (carotenoids) in human health led to a revision Epidemiological associations are based on the relationships of former data and the inclusion of non-provitamin A between nutrient exposure and intermediate endpoints carotenoids (i.e. lutein) in the new FCT and databases during the course of disease or clinical outcomes. To estab- during the 1990s (Table 1). lish a causal relationship between a nutrient and the clini- However, in addition to the variability observed in lutein cal endpoint, it is assumed that an increased exposure to intake, several methodological factors compromise the that nutrient, usually measured by intake or serum levels, homogeneity and comparability of the carotenoid content is capable of enhancing a relevant biochemical indicator in foods, even in the most recently developed databases and/or function (enhanced function, reversible), which, and despite the use of highly specific (HPLC) methods. in turn, is considered to be causally related to the Some of the factors affecting the reliability of the available 490 F. Granado et al.

Table 1. Availability of data for lutein and lutein þ zeaxanthin content in foods: nutritional and epidemiological studies

Reference Type of report Country (food origin) Lutein Lutein þ zeaxanthin

Khachik et al. (1989) HPLC report USA Y Heinonen et al. (1989) HPLC report Finland Y Tee & Lim (1991) HPLC report Malaysia Y Granado et al. (1992) HPLC report Spain Y Poorvliet & West (1993) Database Several Y Mangels et al. (1993) Database USA (several) Y Hart & Scott (1995) HPLC report UK Y Olmedilla et al. (1996) Database Spain Y Sommerburg et al. (1998) HPLC report USA Y Holden et al. (1999)* Database USA (USA) Y Murkovic et al. (2000) Database Austria Y O’Neill et al. (2001) Database Europe (several) Y

* Zeaxanthin values reported independently for selected foods. data have been pointed out elsewhere (Granado et al. 1997; use of common standards and ‘common’ extraction proto- Rodriguez-Amaya, 1997; Deharveng et al. 1999), and are cols (Van den Berg et al. 2000). analytical or food-related. Food-related factors basically concern: Analytical factors affecting the reliability of the data are that: the identity of the food (i.e. different origin, different varieties); most reports and databases provided combined data for the part of the plant consumed (i.e. inner or outer lutein plus zeaxanthin, leading to an overestimation of leaves); the ‘true’ content of lutein in several foods, so that while ripeness, which dramatically affects xanthophyll syn- the presence of zeaxanthin may be negligible in most thesis (content), especially in some fruits; green vegetables, this is not so in many fruits frequently food processing, which affects retention and degra- consumed where equal or higher amounts of zeaxanthin dation, isomerization and bioavailability (Granado et al. are present (i.e. oranges); 1997; Rodriguez-Amaya, 1997). in most if not all FCT, it is not indicated whether the value for lutein refers to free or total (saponified) content. When evaluating the amount of a nutrient consumed Since lutein is also present in ester forms, especially in (nutrient exposure), it is important to note that it depends fruits, this constitutes a major source of uncertainty that not just on the content in food but on the amount of the may lead to a substantial underestimation of the ‘true’ food consumed and, most importantly, the frequency of lutein content; consumption. This is essential to identify the major con- despite it being more accurate, precise and expensive tributors of nutrient (lutein) intake. In this regard, Table 2 than other techniques, the use of HPLC does not ensure a shows our own estimations using data obtained from a good performance of the analysis or increase the confi- European multicentre study, where dietary intake was dence of the data. Results from different international inter- estimated using a common food-frequency questionnaire laboratory exercises for carotenoid analysis in foods and database of carotenoids in food (O’Neill et al. showed a wide variability in the data (CV of 18-55 % for 2001). As shown, although green vegetables are import- lutein), even among expert laboratories and despite the ant contributors to lutein intake in five European

Table 2. Ten top contributors (%) to lutein (þzeaxanthin) intake in ﬁve European countries (adapted from AIR Study Final Report (1997) and O’Neill et al. 2001)*

Spain (n 70) France (n 76) UK (n 71) Republic of Ireland (n 76) The Netherlands (n 75)

Spinach (34) Spinach (31) Peas (36) Peas (19) Spinach (30) Lettuce (16) Lettuce (8) Broccoli (8) Broccoli (16) Broccoli (10) Oranges (7) Eggs (8) Eggs (8) Eggs (10) Peas (9) Eggs (7) Mix veg (6) Sweetcorn (7) Carrots (9) Chicory (8) Broccoli (6) Cucumber (6) Lettuce (6) Tomato (8) Lettuce (4) Peas (6) Green beans (4) Carrots (4) Oranges (7) Tomato (4) Potatoes (3) Courgette (4) Tomato (4) Peppers (6) Oranges (4) Tangerines (3) Peas (3) Tangerines (4) Sweetcorn (4) Eggs (4) Peppers (3) Tomato (3) Celery (4) Spinach (3) Green beans (4) Leeks (2) Sweetcorn (2) Spinach (3) Lettuce (3) Potatoes (4) Total (97) Total (75) Total (84) Total (85) Total (81) Green veg (67) Green veg (56) Green veg (57) Green veg (47) Green veg (65)

veg, vegetables. * Assessed in winter. Lutein in human health 491 groups, relative contribution differs substantially among of the carotenoid profile in tissues is not performed or them. It is also worth noting the relative contribution of substantiated for clinical testing except, possibly, in the non-green vegetables and fruits and the fact that non- case of using psychophysical methods to determine the green foods may account for almost half of the total MPOD for research purposes. lutein intake in some groups. More importantly, zeax- In the absence of accepted, specific and validated func- anthin, a xanthophyll probably as important as lutein in tional methods for the assessment of lutein status, the relation to visual function, is obtained almost exclusively serum concentration is widely used as the ‘best available’ from non-green vegetables in certain population groups method to establish the nutritional status of lutein in (i.e. Spaniards; Table 3), as previously reported (Granado human subjects on a large scale. However, despite being et al. 1996). the biochemical marker most widely used, it also presents Major contributors to lutein intake differ widely both several limitations. The serum lutein concentration relates within and between populations, and their misidentification to dietary intake and thus shows a wide variability both may substantially affect the variability of lutein intake. within and between subjects and among populations, Moreover, their inclusion in or exclusion from the ques- reflecting the variation in food (nutrient) intake or subject tionnaires may lead to the over- or underestimation of response. As is the case with other carotenoids, it is con- the ‘true’ intake, providing misleading results in the evalu- sidered to reflect short-term dietary intake, although it is ation of nutrient exposure and contributing to the mis- widely accepted as a good biomarker of fruit and vegetable classification of subjects, to uncertainty and to intake (Ascherio et al. 1992; Hercberg et al. 1994; inconsistencies between observational studies based on Olmedilla et al. 1994; Thurnham et al. 1998). Lutein in dietary methods. serum correlates with lutein intake although the degree of this association (diet–serum) varies widely depending on a number of factors (Olmedilla et al. 1994; Scott et al. Biochemical markers 1996). However, although a correlation between intake, The use of biochemical indicators overcome most of the serum and some tissues exists (Yong et al. 1994; Johnson confounding factors and biases associated with dietary et al. 2000; Curran-Celentano et al. 2001; Broekmans et al. methods. They are considered more reliable since they pro- 2002), intake assessment and serum concentrations do not vide more accurate information on the amount of nutrient necessarily reflect the amounts in tissues or the relative available to tissues in which these nutrients may exert contribution of carotenoids in tissues, especially regarding their biological action. Regarding concentrations of lutein isomer distribution (structural and geometrical isomers) in human tissues, data are scarce and mostly refer to tissues (Stahl et al. 1993; Su et al. 1998). that can be sampled non-invasively (buccal mucosa cells, Specifically, in serum, lutein:zeaxanthin is about 3:1, human milk or adipose tissue). With the exception of whereas in macula, this ratio reaches values of up to 1:2 human retina, where concentrations of lutein and zeax- and shows a specific concentration pattern from the centre anthin may be measured by non-invasive techniques (see to the eccentric region (Bone et al. 1997; Landrum et al. later; p. 489), the available data on tissues subject to invol- 1999). In addition, meso-zeaxanthin, the major form of vement in diseases that have been associated with lutein in zeaxanthin in retina, is probably the result of chemical pro- epidemiological studies (i.e. cancer, CVD) are very scarce cesses occurring within the eye, possibly a conversion pro- and usually refer to reports on very small numbers of duct derived from lutein at the tissue level (Bone et al. 1997). subjects, sometimes at autopsy, or biopsies of different A high analytical variability also exists in serum caro- tissues (normal or tumour) examined under different ana- tenoid analysis (Van den Berg et al. 1993), in addition to lytical conditions, circumstances that highly compromise the simultaneous quantification of lutein and zeaxanthin. their comparability (Kaplan et al. 1990; Nieremberg & Most analytical methods, including many used in epide- Nann 1992; Stahl et al. 1992, 1993; Schmitz et al. 1993). miological studies, do not separate lutein and zeaxanthin Moreover, because sample collection is invasive and its from those referred to as ketocarotenoids (suggested oxi- relevance to the disease uncertain, the determination dation products). Thus, if the latter are considered potential metabolites of the in vivo antioxidant activity of lutein, many analytical methods report luteinþ Table 3. Top contributors (%) from fruit and vegetables to lutein zeaxanthinþoxidation products as a single compound and zeaxanthin intake in Spain (from Granado et al. 1996)* (lutein), leading to the overestimation of the ‘true’ concentration (nutrient exposure) and forfeiting the ability Lutein Zeaxanthin to detect potential markers of the in vivo biological (anti- Spinach (27) Orange (58) oxidant) activity of lutein. Despite the fact that these Beet (22) Potato (27) ‘metabolites’ have been reported to be present in serum Orange (12) Spinach (12) and tissues (retina) (Khachik et al. 1997b; Bernstein Potato (12) Lettuce (11) et al. 2001) and despite their increment upon lutein sup- Green bean (6) plementation (Khackik et al. 1995; Olmedilla et al. Artichoke (4) 1997a), to date they have not been used as potential bio- Tomato (2) markers of lutein (antioxidant) activity or assessed in Total (96) Total (97) relation to any biomarker and/or disease process. There- Green vegetables (70) Green vegetables (12) fore, their physiological relevance, if any, remains to be * Assessed in winter. elucidated. 492 F. Granado et al.

Evidence of the relationship between lutein and inaccuracies in nutrient exposure assessment (diet and human health serum levels), lack of biomarkers of nutritional status with relevance in tissues, a ‘temporal’ gap as confounding Epidemiological evidence supporting a potential role of factor (serum levels reflect short-term exposure), the pre- lutein in preventing chronic and degenerative diseases sence of the disease affecting the biomarker and the fact comes from different types of studies, mostly observational, that the potential effect of a nutrient is specific for a cer- while intervention trials are experimental and on a (very) tain disease, stage of a disease and/or ‘target’ or suscep- small scale. Few observational studies have examined tible groups. associations between exposure to lutein (individually), assessed by dietary intake or serum levels, and diseases Supplementation studies in human subjects with high prevalence in developed countries, and most are of case-control and cross-sectional design. On interpret- Although not considered the ‘gold standard’, intervention ing the available evidence, several points should be kept in trials add strong support to a nutrient–health relationship, mind: (1) despite the huge number of studies dealing with make it possible to establish temporality and specificity the protective effect of fruit and vegetable consumption, in the causal pathway and allow interpretation of results few have dealt specifically with lutein and, in these, the in terms of cause and effect. However, although concen- clinical endpoints differ substantially; (2) most of these trations of lutein and zeaxanthin in serum and tissues studies have been carried out within the last 10 years due (i.e. macula) have been shown to increase significantly to the availability of accurate information about lutein con- upon ingestion of lutein-rich foods and/or lutein capsules tent in food databases and the use of HPLC analysis on a (Hammond et al. 1997; Landrum et al. 1997; Olmedilla large scale; (3) interpretation of these studies should be et al. 1997a; Granado et al. 1998; Johnson et al. 2000), done with some caution because of the uncertainties associ- lutein has not been used on a large scale in intervention ated with the assessment of nutrient exposure, as mentioned trials to test its efficacy in relation to chronic diseases. earlier (p. 489). Based on the hypothesis that oxidative stress is involved Studies dealing with cancer are scarce and refer to differ- in tissue damage and the development of chronic diseases, ent types and locations. Results show an inverse association in the mid-1990s a European multicentre, placebo- between high lutein intake and/or higher serum levels and controlled supplementation study was conducted to test risk for lung cancer (Michaud et al. 2000; Ratnasinghe whether dietary antioxidants (i.e. carotenoids) could pro- et al. 2000; Voorrips et al. 2000) whereas no association vide protection against oxidative stress and thus reduce or inconsistent results are reported for dietary or serum the risk of oxidative tissue damage. In this study, to our levels and cancer at other locations (prostate, breast, knowledge the largest and widest performed to date, a colon, bladder and stomach) (Giovannucci et al. 1995; total of 400 non-smokers (200 men, 200 women, aged Dorgan et al. 1998; Yeum et al. 1998; Garcıá et al. 1999; 25–45 years) from five European countries, with different Botterweck et al. 2000; Slattery et al. 2000; Toniolo et al. diets and lifestyles, were assigned to supplement their diets 2001; Zeegers et al. 2001). For CVD, results regarding for 4 months with one of three carotenoids (lutein, a- þ b- early atherosclerosis and CHD are controversial, with null carotene or lycopene) or placebo (100 subjects/group). (Kohlmeier et al. 1997; D’Odorico et al. 2000; Klipstein- Because of the long-term progression of chronic and Grobush et al. 2000) and protective associations (Street degenerative diseases, several biomarkers of oxidative et al. 1994; Howard et al. 1996; Iribarren et al. 1997; damage associated with CVD and cancer were assessed Dwyer et al. 2001), whereas for stroke the data suggest a during the study, including DNA, lipids and protein oxi- protective role (Ascherio et al. 1999; Hirvonen et al. dative damage, as well as enzymic and soluble antioxidants 2000); the number of studies, however, is small and those in plasma and serum. In this study, lutein supplementation available refer to case-control analyses where nutrient was followed, on average, by a five- to six-fold increase in exposure may be altered due to the disease process. In serum lutein, with maximum values observed 3 to 4 weeks relation to age-related eye diseases, the results suggest later, after which a steady-state (plateau) level was reached both a protective and no association whatsoever between (Olmedilla et al. 2002). Ketocarotenoid and zeaxanthin lutein intake or serum levels and the development or preva- concentrations in serum also increased and, interestingly, lence of cataracts (Mares-Perlman et al. 1995a; Brown et al. above certain serum lutein concentrations (1·05 mmol/l; 1999; Chasan-Traber et al. 1999; Lyle et al. 1999; Gale et al. 600 mg/l), ester forms of lutein appeared in serum 2001; Taylor et al. 2002), and those from studies based on (Granado et al. 1998). However, the effect of lutein serum levels are highly inconsistent. Similarly, inconsistent supplementation provoked no changes in several bio- associations have been reported between dietary intake and markers of oxidative damage to DNA, lipids and proteins serum levels, and the presence of ARMD (Eye Disease or in other soluble and enzymic antioxidants including Case-Control Study Group 1993; Seddon et al. 1994; ascorbic acid, a-tocopherol, glutathione, superoxide Mares-Perlman et al. 1995b; Beatty et al. 2001; Flood dismutase or glutathione peroxidase (Collins et al. 1998a,b; et al. 2002). Hininger et al. 2001). Thus, on the basis of these data, In general, results from observational studies are some- it seems that, in those apparently healthy subjects, lutein what scarce and rather inconsistent, a fact that may be supplementation increases serum levels of certain carote- related to a number of factors including the use of differ- noids, but this effect is not associated with any change in ent clinical endpoints, populations assessed and study different biomarkers of oxidative stress (Olmedilla et al. designs (inclusion criteria, duration, amounts ingested), 2002). Lutein in human health 493

Another study, however, using other intermediate bio- improvements in clinical indicators relevant to disease markers does not confirm this apparent lack of effect. (visual acuity in patients with cataracts), providing some Recently, on the basis of epidemiological evidence of an valuable information on the dose, timing and methods to inverse association between serum lutein levels and pro- assess treatment efficacy. gression of the intima-media thickness of carotid arteries (in human subjects) it was shown that lutein inhibited the Relevance of lutein in clinical practice and community- inflammatory response of monocytes to LDL trapped in based interventions the artery wall and reduced monocyte migration (co-cul- ture), while indicators of oxidative stress (lipid hydroper- The nutritional relevance of lutein in human health may be oxides and erythrocyte fragility) were reduced and LDL summarized as follows: it is a non-provitamin A carotenoid was markedly resistant to oxidation by lutein supplements but a biologically active phytochemical in man. Conse- (in mice) (Dwyer et al. 2001). So, while these findings are quently, interest in lutein is increasing. Since there are no very encouraging, suggesting a potential role of lutein in human pathological conditions associated with deficiency the prevention of the development of early atherosclerosis, or toxicity specifically related to lutein and because they also highlight the inconsistency of results from studies lutein metabolism in man is still largely unknown, lutein with different designs and raise important concerns about status is not assessed in clinical laboratories these days. the usefulness, methodology, relevance and validity of Furthermore, recommended or maximum tolerable intakes the different biomarkers of oxidative stress in relation to for lutein in human consumers have not been established the disease process. In this regard, since no one animal (National Academy of Sciences Institute of Medicine model completely mimics human absorption and metab- 2000), despite the increasing commercial availability and olism of carotenoids, and for studies using biomarkers of use of lutein-containing supplements and the recommended heart disease primates and gerbils are probably more intake by some physicians. appropriate than rats and mice (Lee et al. 1999), extrapol- Lutein displays different biological actions in vitro and ation of these results and their relevance to man should be ex vivo (Chopra & Thurnham, 1993; Bertram 1994; King considered with caution. et al. 1997; Collins 2001). However, in vivo actions need Lutein has been used in the treatment of eye diseases and to be proven in human subjects under physiological con- to protect visual function since the 1950s (Nussbaum et al. ditions. The consistency and strength of the associations 1981). Small supplementation studies have been per- may be considered relatively high and strong for age- formed, mostly in subjects with compromised visual func- related eye diseases but rather low and weak for CVD tion, and the results appear to be promising, although the and cancer. To date, other causality criteria such as tempor- studies differ in terms of design, endpoints measured and ality and specificity are scarce and based on small sup- subjects involved (Table 4). In control subjects and patients plementation trials for eye diseases. with minimally compromised visual function, lutein supplementation provokes an increase in lutein concentrations Significance and usefulness of serum lutein in serum and in macula, as measured by MPOD, although concentrations it is not observed in all subjects. Similarly, effects on visual function are also variable with improvements and Interpretation of concentrations of lutein in serum is lim- no effect being reported. These individuals, however, ited because of the variability and the lack of widely may clear the lutein from serum very quickly, making accepted reference ranges in (control) populations. In the rise and fall difficult to detect in serum and, in addition, addition, lutein levels in serum are not (routinely) deter- the retina may not be the main body pool or the levels are mined in clinical settings, constituting an important gap tightly regulated in this tissue. In subjects with visual regarding the distribution of lutein in serum under patho- impairment, promising results have been obtained but, logical conditions. In this context, Fig. 2 shows the distri- again, the studies involved few subjects and differing pro- bution of lutein in serum of 1800 patients clinically tocols. Many of these studies refer to subjects suffering diagnosed with different conditions analysed in our lab- from retinitis pigmentosa, an inherited disease leading to oratory. The results are grouped and broadly classified retinal degeneration and blindness, in whom lutein sup- based on the major clinical condition (diagnosis) as plementation led to a better performance of visual function, received in the laboratory. Two observations, however, but not in all subjects (Aleman et al. 2001). In a few must be made regarding this classification: first, the patients with age-related cataracts, lutein supplementation, samples are not necessarily representative of a wide but not a-tocopherol, improved visual acuity (Olmedilla range of clinical conditions since they were received et al. 2003), whereas not all ARMD patients showed only for (fat-soluble) vitamin analysis; second, it is prob- improvement in visual function, possibly because their able that in many subjects other clinical or pathological visual function was highly compromised (Olmedilla et al. conditions coexist with the major diagnosis reported for 2001a; Flood et al. 2002). vitamin analysis. It is important to note that, in several studies involving Fig. 2, while not intended to be exhaustive and detailed, different supplementation protocols (Hammond et al. shows that the concentrations of lutein in serum display a 1997; Landrum et al. 1997; Johnson et al. 2000; Olmedilla non-parametric (biased to the lower end) wide distribution et al. 2001a), serum lutein concentrations reached and regardless of the condition (control or pathological) of the time required to observe a response (lutein in serum and group. In relative terms, two groups may be considered in MPOD) were consistent and that there were parallel Fig 2: (1) patients with malabsorption syndromes, surgical 494 Table 4. Supplementation studies with lutein and visual function

Characteristics and state of Biomarker of modiﬁed function or modiﬁed subjects n Dose and time disease process Effect Reference

Control subjects Normal dark adaptation, 14 Lutein dipalmitate (Helenien, adaptinol) Dark adaptation, visual acuity Transiently improved Monje et al. (1948)* visual acuity 2–6 months Normal patients Helenien Dark adaptation Improved Klaes & Riegel (1951)* Normal patients Helenien Threshold sensitivity Improved Mosci et al. (1956)* Normal patients Helenien Dark adaptation Improved Andreani et al. (1956)* Controls Helenien Serum lutein levels Increased Hayano et al. (1959)* Dark adaptation Improved Controls 11 60 g spinach ^ 150 g maize for Serum lutein and zeaxanthin levels Increased Hammond et al. (1997) 4–15 weeks MPOD Increased (in 4 weeks) Controls 2 30 mg/d for 140 d Serum lutein/zeaxanthin levels Increased Landrum et al. (1997) MPOD Increased Controls 7 Spinach þ maize for 15 weeks Lutein in serum, BMC, adipose Increased Johnson et al. (2000) MPOD Increased Controls 8 10 mg/d for 12 weeks Serum lutein levels Increased Tos et al. (2000) MPOD Increased Controls 8 20 mg lutein/d for 6 months Serum lutein and zeaxanthin Increased Aleman et al. (2001) MPOD Unchanged Visual acuity Unchanged Foveal sensitivity Unchanged . Elderly controls 52 4 mg/d (regularly consumed) MPOD (Raman spectroscopy) Similar to younger adults Bernstein et al. (2002) Granado F. Patients with visual impairment Retinitis pigmentosa Helenien Threshold sensitivity Improved Mosci et al. (1956)* Retinitis pigmentosa Helenien Dark adaptation Improved Andreani et al. (1956)* Retinitis pigmentosa Helenien Serum lutein levels Increased Hayano et al. (1959)*

Dark adaptation Improved al. et Retinitis pigmentosa 18 Helenien Electroretinograms No change Muller et al. (1961)* Retinitis pigmentosa 21 20 mg lutein/d for 6 months Serum lutein and zeaxanthin Increased Aleman et al. (2001) MPOD Increased in 50 % of patients Central vision (visual acuity, foveal sensitivity) Unchanged Retinitis pigmentosa 13 20 mg lutein/d for 9 weeks Visual acuity Improved (not all subjects) Dagnelie et al. (2000) Visual ﬁeld area Improved Progressive myopia, 50 Helenien Luminous and chromatic sensitivity Improved Asciano et al. (1974)* chorioretinopathy Cataract patients 5 15 mg £ 3/week for 2 years Serum lutein and zeaxanthin levels Increased Olmedilla et al. (2001a) (nine Lutein esters in serum Undetected eyes) ‘Oxidative’ lutein products Increased Visual acuity Improved Contrast sensitivity Improved Glare sensitivity Improved ARMD patients 6 Spinach, lutein Visual acuity Improved Richer (1999) Contrast sensitivity Improved Glare recovery Improved ARMD patients 5 15 mg £ 3/week for 1 year Serum lutein and zeaxanthin Increased Olmedilla et al. (2001a) (nine ‘Oxidative’ lutein metabolites Increased eyes) Lutein esters in serum Undetected Visual acuity No effect or slight improvement ARMD patients 15 .4 mg/d (regularly consumed) MPOD (Raman spectroscopy) Improved (similar to non-ARMD Bernstein et al. (2002) patients)

MPOD, macular pigment optical density; BMC, buccal mucosa cells; ARMD, age-related macular degeneration. * Data taken from Nussbaum et al. (1981). Lutein in human health 495

Fig. 2. Distribution (5–95th percentiles; A) of serum concentrations of lutein (lut) in control groups, subjects supplemented with lutein under different protocols and groups of patients clinically diagnosed with different conditions (see p. 493 and 495). (||| ), Median values; (B), safe level; AAIR, Agroindustrial Research Programme; *, range; IMT, intima-media thickness; **, lutein þ zeaxanthin; BMT, bone marrow transplan- tation; EN, enteral nutrition. (Data taken from Olmedilla et al. 1997b, 2001a,b; Granado et al. 1998; National Academy of Sciences Institute of Medicine, 2000; Dwyer et al. 2001; F Granado, B Olmedilla and I Blanco, unpublished results.) stress (laryngeal cancer, before and after enteral nutrition) ARMD, retinitis pigmentosa) (Aleman et al. 2001; or receiving radiotherapy (before bone marrow trans- Olmedilla et al. 2001a), although this effect does not plantation) show ranges and median concentrations in necessarily mean that the disease process has been modi- the lower part of reference values in control groups; fied as a result of the intervention (i.e. cataracts still (2) patients who have lutein levels (range and median) develop) (Olmedilla et al. 2001a). While the functional close to (type 1 diabetes mellitus, other endocrinological effect (i.e. visual performance) is the primary objective syndromes, neurological disorders) or slightly above in these interventions, the assessment of serum levels those observed in control groups (rheumatic conditions, may be very useful in terms of checking compliance and after acute myocardial infarction, senile cataracts and thus efficacy of the intervention, as well as for monitoring other clinical conditions). and establishing relationships between changes in serum Based on these data and in comparison with apparently and those in functional and intermediate biomarkers rel- healthy controls, the serum lutein concentrations observed evant to the disease. Moreover, information on serum in these groups are of uncertain clinical relevance. Lutein changes may also be important in order to establish and levels in serum should be carefully interpreted since they adjust effective doses, thresholds for treatment efficacy, may be altered because of the disease process and thus timing of the intervention (required for a functional do not provide reliable information about the implication, effect), risk evaluation (saturation or potential adverse if any, in the onset and/or progression of the disease. effects) derived from long-term interventions and preven- Moreover, because of its transport by plasma lipoproteins, tion of unforeseen additional risks particularly with lutein levels in serum may be misleading as they may be regard to supplementation. secondary to other physiological and biochemical events Measurement of lutein exposure is also relevant on a associated with certain conditions such as the acute- community basis. Nowadays, one of the major strategies phase response (i.e. myocardial infarction) or drug use in health promotion is focused on adequate strategies in (i.e. lipid-lowering drugs) or with certain populations preventive medicine, that is, to avoid risk factors and pro- (i.e. elderly individuals). From a clinical perspective, it mote healthy behaviours. Increased lifespan and incidence seems clear that lutein concentrations in serum have of several chronic diseases run in parallel and, thus, pre- little or no predictive, diagnostic or prognostic value in a vention is the most convenient, sustainable and cost-effec- variety of disease processes; this is also applicable when tive strategy to delay the onset and progression of these the potential role of lutein as a risk or preventive factor conditions on a population basis. is examined in epidemiological (i.e. case-control) studies. Dietary assessment methods may be appropriate and Nevertheless, despite these constraints, lutein concen- useful for estimating lutein exposure on a group or commu- trations in serum may be useful as a ‘marker’ both on nity basis, although, because of the relevant constraints the clinical and the community level. For example, lutein especially regarding FCT and the misuse of these data, supplementation increases MPOD and is associated with this approach may not be suitable on an individual level. a better functional (visual) performance in some, but not Serum lutein, when assessed repeatedly, prospectively on all, patients with a number of clinical conditions (cataracts, a long-term basis, may be a good indicator of healthy 496 F. Granado et al.

(eating) habits related to chronic diseases. On a community in those subjects reaching serum levels above 1·05 mmol/l level, screening of lutein concentrations in serum would (.600 mg/l) was consistently observed (Granado et al. allow the identification and establishment of appropriate 1998). This was an unexpected finding given that lutein cir- cut-off points with physiological (functional) relevance culates in the free form and that carotenoid esters have not both in the lower (risk factor) and the upper levels (preven- been observed with lycopene or a- þ b-carotene sup- tive factor and adverse effects) of reference distribution. plementation at the same doses (Olmedilla et al. 2002). This approach should be, in any case, a preliminary and These ester forms were present regardless of the total caro- necessary step for any evidence-based decision on the tenoid concentrations reached in serum, and appeared in use of lutein supplements in at-risk groups as well as to serum before and independently of the presence of carote- evaluate the need, adequacy and efficacy of any nutritional nodermia (clinical sign) and were not related to any change interventions with clinical (and economic) impact on in biochemical or haematological parameters of the sub- disease prevention. jects and they disappeared upon discontinuation of lutein (Granado et al. 1998). It must be noted, however, that the lutein esters represented 3 % of lutein levels achieved Risk–benefit assessment in serum. Therefore, the clinical significance could be Nowadays, the use of supplements (i.e. antioxidant vita- questionable. Interestingly, in another study using the mins, lutein-containing supplements) is increasing. This same capsules but three times/week for more that 2 years behaviour may stem from the desire to bypass the rec- (Fig. 2, cataracts þ lutein), levels of lutein in serum ommended changes in dietary and lifestyle habits aimed reached and remained at values of between 0·6 and at achieving disease prevention and/or healthy ageing. 1·0 mmol/l without the appearance of ester forms, caroteno- This behaviour mostly derives from the (assumed) healthy dermia or changes in the haematological or biochemical benefits derived from specific nutrients contained in these profile of the subjects (Olmedilla et al. 2001a, 2003). supplements and the conviction that these compounds are At present, there is almost a complete lack of infor- ‘natural’ and, thus, they are ‘safe’. However, simul- mation regarding the short- and long-term potential adverse taneously, the growing concern about their safety has pro- and/or toxic effects derived from a high consumption (by voked the development of methods for risk assessment and supplementation) of most carotenoids, including lutein. the establishment of safe levels of intake (National Acad- The selective accumulation of lutein in patients with emy of Sciences Institute of Medicine, 2000; Lindsay, systemic amyloidosis (Bruch-Gerharz et al. 2001) and the 2002). reduced mobilization of retinoids from the liver upon In any intervention, the benefits should outweigh the lutein supplementation reported in rats (Jenkins et al. risks. However, contrary to non-dietary compounds 2001) must be taken into account, but should be considered (i.e. drugs), evaluation of risks with nutrients and food as preliminary findings to be confirmed. The premise that components is compromised by factors related to the nutri- these compounds are safe when consumed at levels ent (lack of information on nutrient metabolism, different above those provided by dietary means is unsubstantiated. bioavailability depending on the matrix, interaction with In fact, it should not be forgotten that this premise other nutrients) and the subjects (nutritional status, (‘safety’) was also assumed in the 1980s when large inter- between-subject variability, presence of other risk factors, vention trials using a-tocopherol and b-carotene were applicability to gender and age groups, etc.). Risk charac- initiated, and the results of these studies have demonstrated terization associated with lutein intake and that this assumption was false, at least for certain risk supplementation is difficult because of the lack of well- groups (smokers, alcohol drinkers, asbestos workers) controlled large-scale supplementation trials, lack of (Albanes 1999; Kushi 1999). homogeneity of the studies (variability in sources used, Lutein, in the same way as other phytochemicals, may doses, protocols, endpoints and groups assessed), lack of display a positive risk-benefit ratio depending on the dose-response studies (Lindsay, 2002) and suitable dose and response elicited and the cumulative effect animal models (Lee et al. 1999). when consumed on a long-term basis (Omenn, 1998; However, as with b-carotene, an approximation can be Lindsay, 2002). In addition, it should be considered that made using serum concentrations. In Fig. 2, ranges of when supplied orally, it will have a systemic distribution lutein concentrations in serum in control groups with and that the deposition and the biological effect (i.e. gene different dietary habits are shown (Olmedilla et al. expression, apoptosis) in remote tissues is, at present, 2001b; National Academy of Sciences Institute of Medi- uncertain and needs to be determined (Collins, 2001). cine 2000; F Granado, B Olmedilla and I Blanco, unpub- This is especially relevant for lutein, taking into account lished results). Also shown are the ranges achieved under its selective distribution in certain tissues (i.e. eye), the pre- different protocols of lutein supplementation (Olmedilla sence of biologically active oxidation products in serum et al. 2002) and those associated with functional effects and tissues, the small but unusual transport forms under (blockage of intima-media thickness progression, a bio- non-dietary conditions (i.e. ester forms) and its higher marker of CVD; Dwyer et al. 2001) and improvement of potential for provoking carotenodermia compared with visual acuity (Olmedilla et al. 2001a). Fig. 2 also shows other carotenoids (Olmedilla et al. 2002). At present, the lutein concentrations achieved in serum in a group of factors determining the impact of lutein supplementation apparently healthy non-smokers supplemented with lutein in remote tissues are unknown and may be related to (15 mg/d for 4 months; Olmedilla et al. 2002). In this the target group, including subject variability (i.e. genetic study, the (reversible) presence of lutein esters in serum susceptibility), clinical condition and/or the stage of the Lutein in human health 497 disease. This lack of knowledge and the variability in Thus, concentrations of lutein in serum above 0·6 mmol/l response translates into uncertainty when considering the (.340 mg/l), achievable by diet, seem to be consistently potential impact of intervention on a community level. associated with lower risks in epidemiological studies and lower levels of different biochemical and intermediate markers (i.e. DNA damage biomarkers, reduced intima- Approaching cut-off points with physiological relevance media thickness progression, higher MPOD), as well as At present, there is no widely accepted ‘normal’ or refer- with an improvement in physiological function (i.e. ence range for lutein in serum, although some attempts visual performance) and, thus, quality of life of the sub- have been made in certain populations (Thurnham, 1988; jects. On the other hand, based on the available evidence, Ito et al. 1990; Sharpless & Duewer, 1995; Olmedilla serum levels of lutein of up to 1·05 mmol/l (,600 mg/l) et al. 1997b, 2001b; National Academy of Sciences Insti- seem to be indicative of ‘safety’ since they are not associ- tute of Medicine, 2000). As shown in Fig. 2, concentrations ated with an unusual serum lutein profile, biochemical or of lutein show wide distributions within and between popu- haematological changes or carotenodermia. Thus, concen- lations. However, the wider the distribution of an analyte in trations of lutein in serum between 0·6 and 1·05 mmol/l a population is the greater the number of intervals that can could be considered an achievable and desirable ‘bio- be defined as having potential physiological significance chemical target’ on a community level, probably associated (Sharpless & Duewer, 1995). with improved health outcomes. These proposed cut-off In this respect, evidence from both epidemiological and points, however, must be considered as ‘tentative’, testable supplementation studies may provide valuable data. While and applicable starting points to be confirmed and vali- for the lower range of concentrations it is difficult to reach dated in relation to different physiological and clinical end- any consensus on cut-off points with functional significance, points, as well as on a community level for disease in the upper part of the distribution the possibility of defining prevention (i.e. prevention of early atherosclerosis, pro- them seems to be more promising, especially considering the motion of visual health). potential impact on a community level. Despite the analytical variability and the differences among populations, con- Nutritional strategies for action centrations .0·60 mmol/l (.340 mg/l) are consistently at or above the 95th percentile for the lutein distribution in Due to the insufficient evidence and the lack of nutrient populations with different dietary habits (Fig. 2), as well specificity (lutein), especially in relation to prevention of as those associated with lower levels of oxidative markers cancer and CVD, a food-based rather than a compound- (Collins et al. 1998a,b) and lower risk for chronic diseases based approach is recommended. Translation of these in epidemiological studies (Dwyer et al. 2001). Similarly, guidelines for the general population means encouraging short- and long-term studies using different protocols, a balanced diet rich in a variety of fruits and vegetables, doses and sources of lutein (diet and capsules) have shown including green and yellow. Since the effect of diet is supplementation to be effective in increasing serum concen- cumulative, this is a prudent and safe approach that will trations above these levels and in maintaining serum lutein simultaneously provide other phytochemicals with poten- concentrations above the 95th percentile of a reference tial positive effects in disease prevention. population (Hammond et al. 1997; Landrum et al. 1997; Available dietary strategies to increase lutein intake in Johnson et al. 2000; Olmedilla et al. 2001a). In addition, the population are shown in Table 5. As can be seen, all serum concentrations achieved in these studies of them could be compatible within a balanced diet and (.0·60 mmol/l) are also consistent with those associated achievable at a relatively low cost. In addition, some of with lower risk for ARMD and cataracts (Eye Disease them are safe and applicable on a community basis, Case-Control Study Group 1993), with an increase in whereas other dietary approaches could be of interest and MPOD upon lutein supplementation (Hammond et al. helpful in at-risk groups, once their safety had been 1997; Landrum et al. 1997; Johnson et al. 2000) and with proven. In this respect, it should also be considered, how- an improvement in visual performance in subjects with ever, that in a proportion of subjects at high risk for devel- compromised visual function (Olmedilla et al. 2001a). oping such diseases (i.e. genetic predisposition), presenting

Table 5. Dietary strategies to increase lutein intake

Strategy Balanced diet Applicability Safety Cost

To encourage consumption of lutein-rich fruit Compatible Public health Yes Low and vegetables To enhance lutein content in foods Compatible Public health Yes Medium–Low (traditional breeding techniques or plant biotechnology) Groups at risk To be proved through ‘Biofortification’ To optimize industrial processing to increase Compatible Public health Yes Low lutein retention and bioavailability Functional foods (fortification and supplementation of Compatible Groups at risk To be proved Low foods, newly designed foods) Use of lutein-rich natural extracts or Compatible High-risk groups To be proved Low lutein supplements 498 F. Granado et al. special nutritional demands (i.e. malabsorption syndromes) supplemented foods could become commercially available and/or having some pathological eye disease compromis- (Surai et al. 2000). The consumption of these products is ing visual function (i.e. retinitis pigmentosa, cataracts, frequently driven by the publicity regarding ‘unproven ARMD), the use of ‘biofortification’ and/or lutein sup- properties’ of this compound, and is even recommended plements, within a balanced diet, would probably help to by physicians who, in the absence of data about the effi- ameliorate the disability and improve the quality of life. cacy of the treatment and the apparently lack of adverse Some of these subjects at high risk could be considered a effects, make an intuitive decision based on the ethical ‘target’ group for immediate intervention (i.e. elderly indi- principle of beneficence (Guerra-Romero, 1996), rather viduals, subjects at initial stages of macular degeneration, than following the principles of evidence-based medicine. and/or cataracts). The claims often state that lutein is safe merely on the In considering the use of lutein supplements in disease basis of the lack of adverse effects reported to date. prevention (i.e. CVD, age-related eye disease), decisions There is at least one argument to refute this assertion: should be based on the available evidence and the potential the lack of reports of significant adverse or side effects risks should be evaluated, keeping in mind the unexpected to date (that is, the lack of knowledge) is not, in essence, results from previous intervention trials in human subjects a proof of safety. This ‘safety’ should be considered, at using single nutrients (i.e. b-carotene). However, the varia- least, unsubstantiated and based only on the lack of bility in response to lutein supplementation suggests that, evidence, since no specific studies have been performed in terms of applicability on a community basis, efficacy to test it and lutein has not been used on a large scale, in of intervention would have to be considered in terms of groups with different characteristics, at different doses or probability, in addition to safety and cost savings. In any for a sufficient time to provoke any significant and measur- case, well-designed, randomized clinical trials are needed able potential adverse effect. This, in fact, reflects two to evaluate the benefits and risks of supplementation with different approaches in public health, that is, to prove lutein in relation to human health and disease and, until that a compound is unsafe rather than to prove it is safe such effects are proven and safety established, the indiscri- for human health. minate use of lutein supplements cannot be justified.

Final considerations Acknowledgements Despite the increasing amount of literature dealing with This work was partially presented at the International Sym- different aspects of lutein in man, our aim was to briefly posium on Microalgal Products and Health (Fundacioń review the available evidence and provide, within the con- Ramoń Areces, Aguadulce, Almerıá, May 2002). It was text of evidence-based medicine, some rationale for the supported in part by a grant from the Instituto de Salud decision-making process regarding the potential use of Carlos III, RCMB (C03/08), Madrid, Spain. The authors lutein in clinical practice and public health. are indebted to Martha Messman for preparing the manu- At present, there are no definitively established, physio- script. The authors declare that they have no competing logically significant cut-off points for lutein in serum financial interests. above which ‘protection’ or prevention against chronic diseases is ensured or provided. Similarly, there are no recommendations for optimal nutritional intake of lutein. There is little available information on lutein supplemen- References tation in human consumers. No extra benefits or harmful Agudo A, Amiano P, Barcos A, et al. (1999) Dietary intake of effects in terms of the onset or progression of chronic vegetables and fruits among adults in five regions of Spain. and degenerative diseases in apparently healthy subjects Eur J Clin Nutr 53, 174–180. have been demonstrated. Nevertheless, available evidence AIR Study Final Report (1997) AIR2-CT93-0888 (DG12), CEC, suggests that serum concentrations of lutein in the range Final Consolidation Report Project. Brussels, Belgium. of 0·6–1·05 mmol/l (350–600 mg/l) may be achievable by Albanes D (1999) B-carotene and lung cancer: A case study. Am J Clin Nutr 69, Suppl., 1345S–1351S. dietary means and could be considered a potential Aleman TS, Duncan JL, Bieber ML, et al. (2001) Macular ‘target’ for health promotion and disease prevention. pigment and lutein supplementation in retinitis pigmentosa Although this range of serum concentrations may be con- and Usher syndrome. Invest Ophthalmol Vis Sci 42, sidered too ‘cautious’, in concentrations of lutein above 1873–1881. this cut-off point (.1·05 mmol/l), unusual lutein ester Ascherio A, Rimm EB, HernańM,et al. (1999) Relation of con- forms appear in plasma and have not been associated sumption of vitamin E, vitamin C and carotenoids to risk for with extra benefits with regard to different oxidative mar- stroke among men in the United States. Ann Intern Med 130, kers related to chronic diseases. In this respect, there 963–970. should be awareness that, similar to the case of b-carotene, Ascherio A, Stampfer M, Colditz GA, Rimm EB, Litin L & the range between benefits and harm may be narrow Willett WC (1992) Correlations of vitamin A and E intakes with plasma concentrations of carotenoids and tocopherols (Van den Berg et al. 2000) and, thus, there should be cau- among American men and women. J Nutr 122, 1792–1801. tion when establishing recommendations for lutein intake Bates CJ, Chen SJ, MacDonald A & Holden R (1996) above levels achievable by dietary means. The percentage Quantitation of vitamin E and a carotenoid pigment in catarac- of the population consuming lutein-containing supple- tous human lenses and the effect of a dietary supplement. ments is increasing and a variety of lutein-fortified and Int J Vitam Nutr Res 66, 316–321. Lutein in human health 499

Beatty S, Boulton M, Henson D, Koh HH & Murray IJ (1999) Curran-Celentano J, Hammond BR, Ciulla TA, Cooper DA, Pratt Macular pigment and age-related macular degeneration. Br J LM & Danis RB (2001) Relation between dietary intake, serum Ophthalmol 83, 867–877. concentrations and retinal concentrations of lutein and zeax- Beatty S, Murray IJ, Henson DB, Carden D, Koh HH & Boulton anthin in adults in a Midwest population. Am J Clin Nutr 74, ME (2001) Macular pigment and risk for age-related macular 796–802. degeneration in subjects from a Northern European population. Dagnelie G, Zorge IS & McDonald TM (2000) Lutein Invest Ophthalmol Vis Sci 42, 439–446. improves visual function in some patients with retinal Bendich A & Olson JA (1989) Biological actions of carotenoids. degeneration: a pilot study via the Internet. Optometry FASEB J 3, 1927–1932. 71, 147–164. Bernstein PS, Khachik F, Carvalho LS, et al. (2001) Identification Deharveng G, Charrondiere UR, Slimani N, Southgate DAT & and quantitation of carotenoids and their metabolites in the Riboli E (1999) Comparison of nutrients in the Food tissues of the human eye. Exp Eye Res 72, 215–223. Composition Tables available in the nine European countries Bernstein PS, Yoshida MD, Katz NB, McClane RW & participating in the EPIC. Eur J Clin Nutr 53, 60–79. Gellermann W (1998) Raman detection of macular carotenoid D’Odorico A, Martines D, Kiechl S, et al. (2000) High plasma pigments in intact human retina. Invest Ophthalmol Vis Sci levels of alpha- and beta-carotene are associated with a lower 39, 2003–2011. risk of atherosclerosis: Results from the Bruneck Study. Bernstein PS, Zhao DY, Wintch SW, Ermakov IV, McClane RW Atherosclerosis 153, 231–239. & Gellermann W (2002) Resonance Raman measurement of Dorgan JF, Sowell A, Swanson CA, et al. (1998) Relationships of macular carotenoids in normal subjects and in age-related macu- serum carotenoids, retinol, a-tocopherol and selenium with lar degeneration patients. Ophthalmology 109, 1780–1787. breast cancer risk: Results from a prospective study in Bertram JS (1994) The chemoprevention of cancer by dietary Columbia, Missouri (United States). Cancer Causes Control carotenoids: Studies in mouse and human cells. Pure Appl 9, 89–97. Chem 66, 1025–1032. Dwyer JH, Navab M, Dwyer KM, et al. (2001) Oxygenated Bone RA, Landrum JT, Friedes LM, et al. (1997) Distribution of carotenoid lutein and progression of early atherosclerosis. lutein and zeaxanthin stereoisomers in the human retina. Exp The Los Angeles Atherosclerosis Study. Circulation 103, Eye Res 64, 211–218. 2922–2927. Bone RA, Landrum JT & Tarsis SL (1985) Preliminary Eye Disease Case-Control Study Group (1993) Antioxidant status identification of the human macular pigment. Vision Res 25, and neovascular age-related macular degeneration. Arch 1531–1535. Ophthalmol 111, 104–109. Botterweck AAM, van den Brandt PA & Goldbohm RA (2000) Erdman JW Jr, Bierer TL & Gugger ET (1993) Absorption and Vitamins, carotenoids, dietary fiber, and the risk of gastric car- transport of carotenoids. Ann N Y Acad Sci 691, 76–85. cinoma. Cancer 88, 737–748. Flood V, Smith W, Wang JJ, Manzi F, Webb K & Mitchell P Broekmans WMR, Berendschot TTJM, Klo¨pping-Ketelaars IAA, (2002) Dietary antioxidant intake and incidence of early et al. (2002) Macular pigment density in relation to serum and age-related maculopathy: the Blue Mountains Eye Study. adipose tissue concentrations of lutein and serum concen- Ophthalmology 109, 2272–2278. trations of zeaxanthin. Am J Clin Nutr 76, 595–603. Gale CR, Hall NF, Phillips DIW & Martyn CN (2001) Plasma Brown L, Rimm EB, Seddon JM, et al. (1999) A prospective antioxidant vitamins and carotenoids and age-related cataract. study of carotenoid intake and risk of cataract extraction in Ophthalmology 108, 1992–1998. US men. Am J Clin Nutr 90, 517–524. Garcıá R, Gonza´lez CA, Agudo A & Riboli E (1999) High intake Bruch-Gerharz D, Stahl W, Gerharz CD, et al. (2001) Accumu- of specific carotenoids and flavonoids does not reduce the risk lation of the xanthophyll lutein in skin amyloid deposits of of bladder cancer. Nutr Cancer 35, 212–214. systemic amyloidosis (al type). J Invest Dermatol 116, Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA 196–197. & Willett WC (1995) Intake of carotenoids and retinol in Castenmiller JJM & West C (1998) Bioavailability and biocon- relation to risk of prostate cancer. J Natl Cancer Inst 87, version of carotenoids. Annu Rev Nutr 18, 19–39. 1767–1776. Chasan-Traber L, Willett WC, Seddon JM, et al. (1999) A pro- Goodwin TW & Britton G (1988) Distribution and analysis of spective study of carotenoid and vitamin A intakes and risk carotenoids. In Plant Pigments, pp. 61–127 [TW Goodwin, of cataract extraction in US women. Am J Clin Nutr 70, editor]. New York, NY: Academic Press Limited. 509–516. Granado F, Olmedilla B, Blanco I, Gil-Martıńez E & Rojas- Chopra M & Thurnham DI (1993) In vitro antioxidant activity of Hidalgo E (1997) Variability in the intercomparison of food lutein. In Food and Cancer Prevention: Chemical and Biologi- carotenoid content data: A user’s point of view. Crit Rev cal Aspects, pp. 125–129 [KW Waldron, IT Johnson and GR Food Sci Nutr 37, 621–633. Fenwick, editors]. London: Royal Society of Chemistry. Granado F, Olmedilla B, Blanco I & Rojas-Hidalgo E (1992) Chug-Ahuja JK, Holden JM, Forman MR, Mangels AR, Carotenoid composition in raw and cooked Spanish vegetables. Beecher GR & Lanza E (1993) The development and appli- J Agric Food Chem 40, 2135–2140. cation of a carotenoid database for fruits, vegetables and Granado F, Olmedilla B, Blanco I & Rojas-Hidalgo E selected multicomponent foods. J Am Diet Assoc 93, 318–323. (1996) Major fruit and vegetables contributors to the main Collins AR (2001) Carotenoids and genomic stability. Mutat Res serum carotenoids in Spanish diet. Eur J Clin Nutr 50, 475, 21–28. 246–250. Collins AR, Gedik CM, Olmedilla B, Southon S & Bellizzi M Granado F, Olmedilla B, Gil-Martıńez E & Blanco I (1998) (1998a) Oxidative DNA damage measured in human lympho- Lutein ester in serum after lutein supplementation in human cytes: large differences between sexes and between countries, subjects. Br J Nutr 80, 445–459. and correlations with heart disease mortality rates. FASEB J Guerra-Romero L (1996) La medicina basada en la evidencia: 12, 1397–1400. Un intento de acercar la ciencia al arte de la praćtica clıńica Collins AR, Olmedilla B, Southon S, Granado F & Duthie S (Medicine based on the evidence: An attempt to approach (1998b) Serum carotenoids and oxidative DNA damage in science with the art of clinical practice). Med Clin (Barc) human lymphocytes. Carcinogenesis 9, 2159–2162. 107, 377–382. 500 F. Granado et al.

Hammond BR Jr, Jonhson EJ, Russell RM, et al. (1997) Dietary Khachik F, Bernstein PS & Garland DL (1997a) Identification of modification of human macular pigment density. Invest lutein and zeaxanthin oxidation products in human and monkey Ophthalmol Vis Sci 38, 1795–1801. retinas. Invest Ophthalmol Vis Sci 38, 1802–1811. Hammond BR, Wooten BR & Snodderly DM (1998) Preservation Khachik F, Spangler CJ, Smith JC, Canfield LM, Steck A & of visual sensitivity of older subjects: association with macular Pfander H (1997b) Identification, quantification and relative pigment density. Invest Ophthalmol Vis Sci 39, 397–406. concentrations of carotenoids and their metabolites in human Handelman GJ, Dratz EA, Reay CC & van Kuijk JGM (1988) milk, and serum. Anal Chem 69, 1873–1881. Carotenoids in the human macula and whole retina. Invest King TJ, Khachik F, Bortkiewicz H, Fukushima LH, Morioka S & Ophthalmol Vis Sci 29, 850–855. Bertram JS (1997) Metabolites of dietary carotenoids as Hart DJ & Scott KJ (1995) Development and evaluation of an potential cancer preventive agents. Pure Appl Chem 69, HPLC method for the analysis of carotenes in vegetables and 2135–2140. fruits in the UK. Food Chem 54, 101–111. Klaui H & Bauerfeind JC (1981) Carotenoids as food colors. In Heinonen M, Ollilainen V, Linkola EK, Varo PT & Koivistoinen Carotenoids as Colorants and Vitamin A Precursors. Techolo- PE (1989) Carotenoids in Finnish foods. Vegetables, fruits and gical and Nutritional Applications, pp. 48–292 [JC Bauerfeind, berries. J Agric Food Chem 37, 655–659. editor]. New York, NY: Academic Press. Hercberg S, Preziosi P, GalańP,et al. (1994) Vitamin status of a Klipstein-Grobush K, Launer LJ, Geleijnse JM, Boeing H, healthy French population: dietary intakes and biochemical Hofman A & Witteman JC (2000) Serum antioxidant and markers. Int J Vitam Nutr Res 64, 220–232. atherosclerosis. The Rotterdam Study. Atherosclerosis 148, Hininger I, Meyer-Werner A, Moser U, et al. (2001) No 49–56. significant effects of lutein, lycopene or b-carotene supple- Kohlmeier L, Kark JD, Go´mez-Aracena E, et al. (1997) Lycopene mentation on biological markers of oxidative stress and LDL and myocardial infarction risk in EURAMIC Study. Am J oxidability in healthy adult subjects. J Am Coll Nutr 20, Epidemiol 146, 618–626. 232–238. Kushi LH (1999) Vitamin E and heart disease: A case study. Am J Hirvonen T, Virtamo J, Khorhonen P, Albanes D & Pietinen P Clin Nutr 69, Suppl., 1322S–1329S. (2000) Intake of flavonoids, carotenoids, vitamins C and E Landrum JT, Bone RA, Joa H, Kilburn MD, Moore L & and risk of stroke in male smokers. Stroke 31, 2301. Sprague K (1997) A one year study of the macular pigment: Holden JM, Eldridge AL, Beecher GR, et al. (1999) Carotenoid The effect of 140 days of a lutein supplement. Exp Eye Res content of U.S. foods: An update of the database. J Food 65, 57–62. Comp Anal 12, 169–196. Landrum JT, Bone RA, Moore LL & Gomez CM (1999) Analysis Howard AN, Williams NR, Palmer CR, et al. (1996) Do hydroxy- of zeaxanthin distribution within individual human retinas. carotenoids prevent coronary heart disease? A comparison Meth Enzymol 299, 457–467. between Belfast and Toulouse. Int J Vitam Nutr Res 66, Lee CM, Boileau AC, Boileau TWM, et al. (1999) Review of animal 113–118. models in carotenoid research. J Nutr 129, 2271–2277. Iribarren C, Folsom AR, Jacobs DR Jr, Gross MD, Belcher JD & Lindsay DG (2002) Food chemical safety. Limitations in current Eckfeldt JH (1997) Association of serum vitamin levels, approaches to its assessment. Free Radic Res 36, Suppl. 1, LDL susceptibility to oxidation and autoantibodies against 25–27. MDA-LDL with carotid atherosclerosis. A case-control study. Lyle BJ, Mares-Perlman JA, Klein BEK, Palta M, Bowen P & Arterioscler Thromb Vasc Biol 17, 1171–1177. Greger JL (1999) Serum carotenoids and tocopherols and inci- Ito Y, Ochiai J, Sasaki R, Suzuki S, Kasuhara Y & Morimitsu Y dence of age-related nuclear cataract. Am J Clin Nutr 69, (1990) Serum concentrations of carotenoids, retinol and a-toco- 272–277. pherol in healthy persons determined by high performance Mangels AR, Holden JM, Beecher GR, Forman MR & Lanza E liquid chromatography. Clin Chim Acta 194, 131–144. (1993) Carotenoid content of fruits and vegetables: An evalu- Jenkins MY, Mitchell GV & Grundel E (2001) A dietary sup- ation of analytical data. J Am Diet Assoc 93, 284–296. plement containing lutein and natural tocopherols modulates Mares-Perlman JA, Brady WE, Klein BE, et al. (1995a) Serum tissue levels of retinoids. 2001 FDA Science Forum. http// carotenoids and tocopherols and severity of nuclear and cortical www.cfsan.fda.gov/frf/forum01/A143PPO.htm opacities. Invest Ophthalmol Vis Sci 36, 276–288. Johnson E, Hammond BR, Yeum KJ, et al. (2000) Relation among Mares-Perlman JA, Brady WE, Klein R, et al. (1995b) Serum serum and tissue concentrations of lutein and zeaxanthin and antioxidants and age-related macular degeneration in a popu- macular pigment density. Am J Clin Nutr 71, 1555–1562. lation-based case-control study. Arch Ophthalmol 113, Johnson-Down L, Saundy-Unterberger H & Gray-Donald K 1518–1523. (2002) Food habits of Canadians: lutein and lycopene intake Michaud DS, Feskanich D, Rimm EE, et al. (2000) Intake of in the Canadian population. J Am Diet Assoc 102, 988–991. specific carotenoids and risk of lung cancer in 2 prospective Kaplan LA, Lau JM & Stein EA (1990) Carotenoid composition, US cohorts. Am J Clin Nutr 72, 990–997. concentrations and relationships in various human organs. Clin Murkovic M, Gams K, Draxl S & Pfannhauser W (2000) Devel- Physiol Biochem 8, 1–10. opment of an Austrian Carotenoid Database. J Food Comp Khachik F, Beecher GR, Goli MB, Lusby WR & Smith JC Jr Anal 13, 435–440. (1992) Separation and identification of carotenoids and their National Academy of Sciences Institute of Medicine (2000) Diet- oxidation products in the extracts of human plasma. Anal ary Reference Intake for Vitamin C, Vitamin E, Selenium and Chem 64, 211–222. Carotenoids. Washington, DC: National Academy Press. Khachik F, Beecher GR & Lusby WR (1989) Separation, Nieremberg DW & Nann SL (1992) A method for determination identification and quantification of the major carotenoids of concentrations of retinol, tocopherol and five carotenoids in in extracts of apricots, peaches, cantaloupe and pink human plasma and tissue samples. Am J Clin Nutr 56, grapefruit by liquid chromatography. J Agric Food Chem 37, 417–426. 1465–1473. Nussbaum JJ, Pruett RC & Delori FC (1981) Macular yellow Khachik F, Beecher G & Smith JC Jr (1995) Lutein, lycopene pigment. The first 200 years. Retina 1, 296–310. and their oxidative metabolites in chemoprevention of cancer. Olmedilla B, Granado F, Blanco I, Gil-Martıńez E & Rojas- J Cell Biochem 22, 236–246. Hidalgo E (1996) Contenido de Carotenoides en Verduras y Lutein in human health 501

Frutas de Mayor Consumo en Espanã. Instituto Nacional de la vegetables and fruits and blood plasma concentrations in a Salud (INSALUD). Madrid, Spain: Secretarıá General. group of women aged 50–66 years in the UK. Br J Nutr 75, Olmedilla B, Granado F, Blanco I & Rojas-Hidalgo E (1994) 409–418. Seasonal and sex-related variations in six serum carotenoids, Seddon JM, Ajani UA, Sperduto RD, et al. (1994) Dietary caro- retinol and a-tocopherol. Am J Clin Nutr 60, 106–110. tenoids, vitamins A, C and E and age-related macular degener- Olmedilla B, Granado F, Blanco I, Vaquero M & Cagigal C ation. Eye disease case-control study group. J Am Diet Assoc (2001a) Lutein in patients with cataracts and age-related macu- 272, 1413–1420. lar degeneration: A long-term supplementation study. J Sci Seddon JM & Hennekens CH (1994) Vitamins, minerals and Food Agric 81, 904–909. macular degeneration. Promising but unproven hypothesis. Olmedilla B, Granado F, Blanco I & Vaquero M (2003) Lutein, Arch Ophthalmol 112, 176–179. but not a-tocopherol, supplementation improves visual function Sharpless D & Duewer DL (1995) Population distributions in patients with age-related cataracts: A 2-y double blind, and intralaboratory reproducibility for fat-soluble vitamin- placebo-controlled study. Nutrition 19, 21–25. related compounds in human serum. Anal Chem 67, Olmedilla B, Granado F, Gil-Martıńez E & Blanco I (1997a) 4416–4422. Supplementation with lutein (4 months) and a-tocopherol Sies H, Stahl W & Sundquist A (1992) Antioxidant functions of (2 months), in separate or combined oral doses, in control vitamins: Vitamins E and C, b-carotene and other carotenoids. men. Cancer Lett 114, 179–181. Ann N Y Acad Sci 669, 7–21. Olmedilla B, Granado F, Gil-Martıńez E, Blanco I & Rojas- Simpson KLS (1983) Relative value of carotenoids as precursors Hidalgo E (1997b) Reference levels of retinol, a-tocopherol of vitamin A. Proc Nutr Soc 42, 7–17. and main carotenoids in serum of control and insulin-dependent Slattery ML, Benson J, Curtin K, Ma K-N, Schaeffer D & Potter diabetic Spanish subjects. Clin Chem 43, 1066–1071. JD (2000) Carotenoids and colon cancer. Am J Clin Nutr 71, Olmedilla B, Granado F, Southon S, et al. (2001b) Baseline serum 575–582. concentrations of carotenoids, vitamins A, E and C in control Sommerburg O, Keunen JEE, Bird AC & van Kuijk FJGM (1998) subjects from five European countries. Br J Nutr 85, 227–238. Fruits and vegetables that are sources for lutein and zeaxanthin: Olmedilla B, Granado F, Southon S, et al. (2002) A European the macular pigment in human eyes. Br J Ophthalmol 82, multicentre, placebo-controlled supplementation study with 907–910. a-tocopherol, carotene-rich palm oil, lutein or lycopene: Anal- Stahl W, Schwarz W & Sundquist A (1992) Cis-trans isomers of ysis of serum responses. Clin Sci 102, 447–456. lycopene and b-carotene in human serum and tissues. Arch Omenn GS (1998) Chemoprevention of lung cancer: the rise and Biochem Biophys 294, 173–177. demise of b-carotene. Ann Rev Public Health 19, 73–99. Stahl W, Sundquist AR, Hanusch M, Schwarz W & Sies H (1993) O’Neill ME, Carroll Y, Corridan B, et al. (2001) A European Separation of b-carotene and lycopene geometrical isomers in Carotenoid Database to assess carotenoid intakes and its use biological samples. Clin Chem 39, 810–814. in a five-country comparative study. Br J Nutr 85, 499–507. Street DA, Comstock GW, Salkeld RM, Schuep W & Klag M Park JS, Chew BP & Wong TS (1998) Dietary lutein from (1994) Serum antioxidants and myocardial infarction: Are marigold extract inhibits mammary tumor development in low levels of carotenoids and a-tocopherol risk factors for myo- BALB/c mice. J Nutr 128, 1650–1656. cardial infarction? Circulation 90, 1154–1161. Parker RS (1996) Absorption, metabolism, and transport of caro- Su LC, Bui M, Kardinaal A, et al. (1998) Differences between tenoids. FASEB J 10, 542–551. plasma and adipose tissue biomarkers of carotenoids and toco- Parker RS, Swanson JE, You C, Edwards AJ & Huang T (1999) pherols. Cancer Epidemiol Biomarkers Prev 7, 1043–1048. Bioavailability of carotenoids in human subjects. Proc Nutr Soc Surai PF, MacPherson A, Speake BK & Sparks NHC (2000) 58, 155–162. Designer egg evaluation in a controlled trial. Eur J Clin Nutr Poorvliet EJ & West CE (1993) The Carotenoid Content of Foods 54, 298–305. with Special Reference to Developing Countries. Vitamin A Taylor A & Hobbs M (2001) 2001 Assessment of nutritional Field Support Project (VITAL). Arlington, VA: International influences on risk for cataract. Nutrition 17, 845–858. Science and Technology Institute, Inc. Taylor A, Jacques PF, Chylack LT, et al. (2002) Long-term intake Rapp LM, Maple SS & Choi JH (2000) Lutein and zeaxanthin of vitamins and carotenoids and odds of early age-related cor- concentrations in rod outer segment membranes from perifo- tical and posterior subcapsular lens opacities. Am J Clin Nutr veal and peripheral human retina. Invest Ophthalmol Vis Sci 75, 540–549. 41, 1200–1209. Tee ES & Lim CL (1991) Carotenoid composition and content of Ratnasinghe D, Forman MR, Tangrea JA, et al. (2000) Serum Malaysian vegetables and fruits by AOAC and HPLC methods. carotenoids are associated with increased lung cancer risk Food Chem 41, 309–339. among alcohol drinkers, but not among non-drinkers in a Thurnham DI (1988) Do higher vitamin A requirements in men cohort of tin miners. Alcohol Alcohol 35, 355–360. explain the difference between sexes in plasma provitamin A Richer S (1999) Part II. ARMD-pilot (case series) environmental carotenoids and retinal? Proc Nutr Soc 47, 181. intervention data. J Am Optom Assoc 70, 24–36. Thurnham DI, Northrop-Clewes CA & Chopra M (1998) Bio- Rodriguez-Amaya DB (1997) Carotenoids and Food Prep- markers of vegetable and fruit intakes. Am J Clin Nutr 68, aration: The Retention of Provitamin A Carotenoids in Pre- 756–757. pared, Processed and Stored Foods. Arlington, VA: U.S. Toniolo P, Van Poppel AL, Akhmedkhanov A, et al. (2001) Agency for International Development. Serum carotenoids and breast cancer. Am J Epidemiol 153, Rojas-Hidalgo E (1987) Alteraciones metabo´licas (Metabolic 1142–1147. alterations). In La Mano del Diabe´tico, pp. 57–61 Madrid, Tos TJM, Berendschot R, Goldbohm A, et al. (2000) Influence of Spain: ARAN Ediciones. lutein supplementation macular pigment assessed with two Schmitz HH, Poor CL, Gugger ET & Erdman JW (1993) Analysis objective techniques. Invest Ophthalmol Vis Sci 41, of carotenoids in human and animal tissues. Methods Enzymol 3322–3326. 213, 102–117. Van den Berg H, Faulks R, Granado F, et al. (2000) The potential Scott KJ, Thurham DI, Hart D, et al. (1996) The correlation for the improvement of carotenoid levels in foods and the likely between the intake of lutein, lycopene and b-carotene from systemic effects. J Sci Food Agric 80, 880–913. 502 F. Granado et al.

Van den Berg H, Heseker H, Lamand M, et al. (1993) Flair con- women with benign breast tumor or breast cancer. J Nutr 128, certed action No 10 status papers. Introduction, conclusions and 1920–1926. recomendations. Int J Vitam Nutr Res 63, 247–251. Yeum K-J, Taylor A, Tang G & Russell RM (1995) Measurement Van’t Veer P & Kok FJ (2000) Human studies to substantiate of carotenoids, retinoids and tocopherols in human lenses. health effects of antioxidants: What is needed. Free Radic Invest Ophthalmol Vis Sci 36, 2756–2761. Res 33, S109–S115. Yong L-Ch., Forman M, Beecher GR, et al. (1994) Relationship Voorrips LA, Goldbohm A, Brants HAM, et al. (2000) A prospec- between dietary intake and plasma concentrations of carotenoids tive cohort study on antioxidant and folate intake and male lung in premenopausal women: Application of the USDA-NCI cancer risk. Cancer Epidemiol Biomarkers Prev 9, 357–365. carotenoid food-composition database. Am J Clin Nutr 60, Yemelyanov AY, Katz NB & Bernstein PS (2001) Ligand-bind- 223–230. ing characterization of xanthophyll carotenoids to solubilized Zechmeister L (1962) Cis-trans Isomeric Carotenoids and Aryl- membrane proteins derived from human retina. Exp Eye Res polyenes. New York, NY: Academic Press. 72, 381–392. Zeegers MPA, Goldbohm RA & van den Brandt PA Yeum K-J, Ahn S-H, Rupp de Paiva SA, Lee-Kim Y-Ch, (2001) Are retinal, vitamin C, vitamin E, folate and Krinsky NI & Russell RM (1998) Correlation between caroten- carotenoids intake associated with bladder cancer risk? Results oid concentrations in serum and normal breast adipose tissue of from the Netherlands Cohort Study. Br J Cancer 85, 977–983.