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Accumulation and bioavailability of dietary carotenoids in vegetable crops

Dean A. Kopsell1 and David E. Kopsell2
1 2

Plant Sciences Department, The University of Tennessee, Knoxville, TN 37996, USA School of Agriculture, The University of Wisconsin Platteville, Platteville, WI 53818, USA

Carotenoids are lipid-soluble pigments found in many vegetable crops that are reported to have the health benets of cancer and eye disease reduction when consumed in the diet. Research shows that environmental and genetic factors can signicantly inuence carotenoid concentrations in vegetable crops, and that changing cultural management strategies could be advantageous, resulting in increased vegetable carotenoid concentrations. Improvements in vegetable carotenoid levels have been achieved using traditional breeding methods and molecular transformations to stimulate biosynthetic pathways. Postharvest and processing activities can alter carotenoid chemistry, and ultimately affect bioavailability. Bioavailability data emphasize the importance of carotenoid enhancement in vegetable crops and the need to characterize potential changes in carotenoid composition during cultivation, storage and processing before consumer purchase. Vegetable crops are important sources of dietary carotenoids Consumption of vegetable crops provides the human diet with many essential vitamins and minerals important for health maintenance. Vegetables also contain other food components, or phytochemicals, that provide benets beyond normal health maintenance and nutrition. One important class of phytochemicals is the carotenoids. Carotenoids are lipid-soluble pigments found in all photosynthetic organisms. Among the naturally occurring plant pigments, carotenoids are widely distributed, with a high degree of structural diversity and large variations in biological functions [1,2]. There are >600 carotenoids found in nature, with 40 dietary carotenoids regularly consumed in the human diet [3]. Pro-vitamin A activity is the classical biological function of b-carotene, a-carotene and cryptoxanthins in mammalian systems. Health benets attributed to carotenoids include prevention of certain cancers [46], cardiovascular diseases [7] and eye diseases [8,9], as well as enhanced immune system functions [10,11]. Current US Department of Agriculture (USDA) dietary guidelines recommend eating 79 servings of fruit and vegetables per day. However, the average consumption by adults in the USA is only 4.4 servings per day, with an estimated 42% of Americans eating less than
Corresponding author: Kopsell, D.A. (dkopsell@utk.edu). Available online 1 September 2006.
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two daily servings of fruit and vegetables (see http:// www.healthierus.gov/dietaryguidelines). Research into carotenoid enhancement of vegetable crops to benet human health has paralleled efforts to increase consumption of fruit and vegetables in the diet. Current methods to assess bioavailability (see Glossary) include serum measurements and in vitro digestion models. Release of carotenoid from the membranes of plant tissues facilitates intestinal absorption; however, changes in carotenoid chemistry by biotic (e.g. oxidation) and abiotic (e.g. temperature) factors can inuence bioavailability. Programs to improve carotenoid levels in vegetable crops must successfully link plant physiology with accurate bioavailability assessments in human subjects. In this review, we discuss our current knowledge of carotenoid chemistry, carotenoid contributions to human health, bioavailability assessments, how pre- and postharvest practices can alter carotenoid levels and inuence potential bioavailability, and recent vegetable carotenoid enhancement efforts. Carotenoid chemistry and plant biosynthetic pathway Carotenoids are C40 isoprenoid polyene compounds that form lipid soluble yellow, orange and red pigments [12,13]. Carotenoids are secondary plant compounds and can be divided into two groups: the oxygenated xanthophylls such as lutein (3R,30 R,60 R b,e-carotene-3,30 diol), zeaxanthin (3,30 R-b,b-carotene-3,30 diol) and violaxanthin (3S,5R,6S,30 S,50 R,60 S-5,5,50 ,60 -depoxy-5,6,50 ,60 -tetrahydrob, b-carotene-3,30 diol), and the hydrocarbon carotenes such as b-carotene (b, b-carotene), a-carotene (60 R, b,e-carotene) and lycopene (c, c-carotene) [12]. In higher plants, carotenoid compounds are synthesized and localized in cellular plastids where they are associated with light-harvesting complexes (LHCs) in the thylakoid membranes, or present as semicrystalline structures derived from the plastids [14,15]. Terpenes are a diverse group of plant secondary metabolites formed via acetyl-CoA (mevalonic acid pathway) or from glycolytic intermediates (methylerythritol phosphate pathway). The basic structural element of all terpenes is the 5-carbon branched isopentane. Terpenes are classied by the number of isoprene units present. Carotenoids are composed of 40 carbons and are thus classied as tetraterpenes. The carotenoid biosynthetic pathway was elucidated in the mid-1960s [15]. Currently, genes and cDNAs for the major enzymes functioning in carotenoid biosynthesis have been cloned from plant, algal and microbial sources [16]. Carotenoids are produced in

1360-1385/$ see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2006.08.006

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Glossary
Bioavailability: the extent to which a nutrient in a food constituent can be absorbed and used by the body after ingestion. Chromophore: any light-absorbing molecular group that produces color in a compound. Chylomicrons: the largest of the blood lipids consisting mostly of triglycerides and small amounts of cholesterol, phospholipids, proteins and fat-soluble vitamins. They are synthesized in the intestinal mucosal cells and serve to transport lipids to tissues. Epoxide: an organic molecule containing a chemical group consisting of an oxygen molecule bound to previously connected atoms, usually carbon (C). High-density and low-density lipoproteins: lipidprotein combinations with the solubility characteristics of a protein. They are classified as to their density and are mainly involved in lipid transport in the aqueous blood plasma. Hydrophobic: having little or no affinity for water. Isomerization: biochemical or chemical reaction involving an intra-molecular shift resulting in a change in orientation of functional groups within a molecule. cis (Z) isomers and trans (E) isomers often have different physical properties, which arise from differences in the shape of the molecule or the overall dipole moment. Lipophilic: having a strong affinity for lipids. Reactive oxygen species (ROS): chemical species that participate in oxidative reactions with polyunsaturated membrane phospholipids, protein thiol groups, non-protein thiol groups and DNA with the potential to alter cellular functions. Reactive species include singlet oxygen (1O2), superoxide (O2), hydroxyl radical (OH), nitric oxides (NO) and peroxyl (HOO).

Carotenoids function to help harvest light energy, mostly in the bluegreen wavelength range, which is transferred to the photosynthetic reaction centers (RCs). In the PSII complex, b-carotene is highly concentrated close to the reaction center, and lutein is present in several lightharvesting antennae components [19,20]. When the absorption of light radiation exceeds the capacity of photosynthesis, excess excitation energy can result in the formation of triplet excited chlorophyll (3Chl) and reactive singlet oxygen (1O2). Carotenoid pigments protect photosynthetic structures by quenching 3Chl to dissipate excess energy [21] and binding 1O2 to inhibit oxidative damage [20,22,23]. Xanthophylls might also be involved in structural stabilization of LHCs and reduction of lipid peroxidation [21]. Carotenoids and human health One of the most important physiological functions of carotenoids in human nutrition is to act as vitamin A precursors. Pro-vitamin A carotenoid compounds support the maintenance of healthy epithelial cell differentiation, normal reproductive performance, and visual functions [24]. Both pro-vitamin A carotenoids (b-carotene, a-carotene and cryptoxanthins) and non-pro-vitamin A carotenoids (lutein, zeaxanthin and lycopene) function as free-radical scavengers, enhance the immune response, suppress cancer development, and protect eye tissues [25]. Humans cannot synthesize carotenoids and, therefore, must rely on dietary sources to provide sufcient levels. Fruits and vegetables are primary sources of carotenoids in the human diet and their consumption has been associated with numerous health benets [26,27]. Carotenoids can reduce the risk of cardiovascular disease through reductions in low-density lipoprotein oxidation and oxidative stress at locations of plaque formations. Recent studies have established a link between increased intake of fruits and vegetables that are high in carotenoids with reduced incidences of mortality related to cardiovascular disease. Cohort studies involving a food frequency questionnaire and serum carotenoid data have established the preventative effects of dietary carotenoids on cardiovascular diseases in separate studies of populations in Italy [28], Japan [29], Europe [30] and Costa Rica [31]. Cardiovascular disease reductions were mostly associated with increased intakes of a-carotene, b-carotene and b-cryptoxanthin. Carotenoids can inhibit cell proliferations, cell transformations, and modulate the expression of gene determinants in the prevention of certain types of cancers. There is a great deal of research on the anticarcinogenic effects of individual carotenoids on specic types of cancers. Data from cohort studies reveal protective effects from b-carotene intakes against the risk of tobacco-related cancers in non-smokers; however, b-carotene can increase cancer risks among smokers [32]. The protective effects of carotenoids against lung cancers differ between the sexes, with men beneting more from increased carotenoid intakes [33]. Higher carotenoid intakes can also lower risk of certain lymphomas [34]. By contrast, cohort and epidemiological study data reveal that increased levels of dietary carotenoids have no

the plastids and are derived from isopentenyl diphosphate (IPP). In the rst step in biosynthesis, IPP is isomerized to dimethylallyl diphosphate (DMAPP), which becomes the substrate for the C20 geranylgeranyl diphosphate (GGPP) [17]. The enzyme GGPP synthase catalyzes the formation of GGPP from IPP and DMAPP [16]. The rst step unique to carotenoid biosynthesis is the condensation of two molecules of GGPP to form the rst C40 carotenoid, phytoene, via phytoene synthase [1]. Two structurally similar enzymes, phytoene desaturase and j-carotene desaturase, make the conversions of phytoene to lycopene [2,16,17]. These desaturase enzymes create the chromophore present in the carotenoid pigments, and change the colorless phytoene into colored lycopene. The carotenoid pathway branches at the cyclization reactions of lycopene to produce carotenoids with either two b-rings (e.g. b-carotene, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin) or carotenoids with one b-ring and one e-ring (e.g. a-carotene and lutein) [16,18]. The pathway advances with the additions of oxygen moieties, which convert the hydrocarbon a- and b-carotenes into the subgroup referred to as the xanthophylls. Further steps in xanthophyll synthesis include epoxydation reactions. The reversible epoxidationde-epoxidation reaction converting violaxanthin back to zeaxanthin via the intermediate antheraxanthin is collectively referred to as the violaxanthin cycle and is vital for energy dissipation from incoming solar radiation [15,17] (Figure 1). Some vegetables have a modied carotenoid biosynthetic capacity and produce compounds exclusively associated with their respective genus, or even species. Lettuce (Lactuca species) contains lactucaxanthin, along with other chloroplast carotenoids. Zeinoxanthin is found in yellow corn (Zea mays) grains. Peppers (Capsicum species) contain capsanthin, capsorubin and cryptocapsin, which are responsible for the vibrant fruit coloration within the genus [1]. Within the thylakoid membranes of chloroplast organelles, carotenoids are found bound to specic protein complexes of photosystem I and photosystem II (PSII).
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Figure 1. Precursor compounds and major carotenes and xanthophylls in the carotenoid biosynthetic pathway in plants. Abbreviations: DMAPP, dimethylallyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate.

preventative effects on incidences of bladder cancers [35]. The preventative effects of lycopene on prostate cancer are unclear: positive results have been demonstrated in vivo, but recent results from screening trials show that lycopene levels were not associated with prostate cancer risk [36].
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Lutein and zeaxanthin are two of seven major carotenoids found in human blood serum; however, they are the only carotenoids present in the retina and lens [37]. In the retina, lutein and zeaxanthin are chiey responsible for the yellow pigmentation collectively referred to as macular pigment (MP) [38]. The yellow pigments are

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Figure 2. Carotenoid carbon positions and structure of zeaxanthin, antheraxanthin, violaxanthin, lutein and 5,6-epoxylutein. Conjugated double bonds at 5,6 and 50 ,60 positions are highly effective at quenching singlet oxygen [45,46].

postulated to participate in photoprotection, and diminished MP might be related to retinal damage [39,40]. Factors that increase MP include increased antioxidant ingestion, high levels of fruit and vegetable consumption, high levels of dietary carotenoid intake and the resultant elevated serum carotenoid concentration, normal body mass index, and a history of no tobacco use. Many of these same factors are also associated with a decreased risk of developing age-related macular degeneration, which suggests that there might be a causal relationship [41,42]. Epidemiological evidence has been inconsistent regarding the potential relationships between diet, genetics and environment and development of age-related macular degeneration. A direct correlation between MP levels and development of macular disease has not been established [39,43], although strong associative relationships are reported. Studies indicate that a high intake of a variety of vegetables, providing a mixture of carotenoids, is more strongly associated with reduced eye disease risk than intake of individual carotenoid supplements [9]. Carotenoid epoxidation and isomerization In the human body, oxidants produced during normal metabolism and immune defense against infectious and chemical agents are responsible for damage to DNA, proteins and cellular tissues [26,44]. This harmful oxidative damage is considered the major cause of aging and degenerative diseases such as cancer, cardiovascular disease, immune-system decline and cataracts. Compounds such as ascorbate, a-tocopherol and carotenoids are examples of antioxidants that have the ability to quench reactive oxygen species [44]. The physical properties of carotenoid molecules, particularly the conjugated carboncarbon double bond system, permit the quenching of 1O2. In vivo antioxidant activity is determined by carotenoid structure and concentration, as well as the nature and concentration of the reactive oxygen species. The localization of carotenoid molecules in biological tissues will also inuence their ability to encounter and scavenge free radicals. Indeed, carotenoids are one of the most potent biological quenchers of singlet oxygen [45].
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The antioxidant activity of cyclic carotenoids comes from the susceptibility of 5,6 and 50 ,60 double bonds in their cyclic end groups to undergo epoxidation with 1O2 (Figure 2) [26]. Acyclic carotenoids, such as lycopene, derive potent antioxidant activity from their high number of conjugated dienes [46]. Epoxide forms of carotenoids, with an oxygen bound to the 5,6 or 50 ,60 position, are present in vegetable crop tissues. The ratios of lutein epoxide:all-trans-lutein was 1.0:1.5 in cabbage (Brassica oleracea var. capitata), 1:2 in broccoli (Brassica oleracea var. botrytis), 1:6 in spinach (Spinacia oleracea) and 1:23 in kale (Brassica oleracea var. acephala) [47]. Low doses of lycopene from tomato (Lycopersicon esculentum) products signicantly increased serum lycopene levels and reduced lipid peroxidation in vivo [48]. However, the complexity of measuring in vivo antioxidant behavior, the variability associated with the carotenoid content of vegetables, and the nutritional status of subjects used in human dietary intervention studies all affect the interpretation of the results [14,46]. Based on their chemistry, epoxide isomers are predicted to lack antioxidant activity because they are unable to bind 1O2, and some might even have pro-oxidant activity. The conjugated double-bond systems of the carotenoids create the light-absorbing chromophores that result in the distinctive colors associated with these plant pigments [16]. In the pigmentprotein complexes of photosynthetic organisms, the all-trans congurations of carotenoids are the major component of the LHCs, and the RCs contain the 15-cis congurations [4951]. The all-trans carotenoids in the LHCs provide efcient singlet-energy transfer to chlorophyll molecules and, hence, participate mainly in lightharvesting. The 15-cis carotenoids show a preference for isomerization towards the all-trans congurations upon excitation and, hence, are better suited for photoprotective functions in the RCs [49]. All carotenoids exhibit cistrans isomerization, and both isomeric groups can be found in vegetable crops (Table 1) [47,5254]. Phytoene exists predominately as the 15-cis isomer; however, the predominant isomer of lycopene is the all-trans geometric form. Isomerase activity

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Table 1. Carotenoids identied and quantied in major vegetable crops

Carotenoids identieda All-trans b-carotene, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, all-trans lutein epoxide, 90 -cis neoxanthin, neolutein, all-trans violaxanthin, all-trans zeaxanthin, 9-cis zeaxanthin and 13-cis zeaxanthin. All-trans b-carotene, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, all-trans and Broccoli cis lutein epoxide, neolutein, all-trans neoxanthin, 90 -cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9-cis zeaxanthin and 13-cis zeaxanthin. All-trans a-carotene, all-trans b-carotene, lutein and lycopene Carrot a-Carotene, b-carotene, b-cryptoxanthin, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, Corn all-trans zeaxanthin and 9-cis zeaxanthin. Kale/collards All-trans b-carotene, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, all-trans and cis lutein epoxide, neolutein, all-trans neoxanthin, 90 -cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9-cis zeaxanthin, 13-cis zeaxanthin. All-trans b-carotene, lactucaxanthin, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, all-trans and Lettuce cis lutein epoxide, neolutein, all-trans neoxanthin, 90 -cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9-cis zeaxanthin and 13-cis zeaxanthin. Pepper (bell) a-Carotene, b-carotene, b-cryptoxanthin, capsanthin, lutein and zeaxanthin. All-trans b-carotene, all-trans lutein, 9-cis lutein, 90 -cis lutein, 13-cis lutein, all-trans and Spinach cis lutein epoxide, neolutein, all-trans neoxanthin, 90 -cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9-cis zeaxanthin and 13-cis zeaxanthin. Tomato (raw) All-trans b-carotene, all-trans g-carotene, all-trans d-carotene, j-carotene, all-trans lutein, all-trans lycopene, neurosporene, phytoene, phytouene and lycopene-5,6 diol. Commodity Beans, green
a

Refs [54,98] [54,98]

[67,85] [54,68] [54,63,81,98]

[54,70,98]

[73] [54,82,98]

[55,99,100]

Carotenoids in bold text identify those reported in highest concentrations.

is present in higher plants to mediate cis to trans conversions [15]. All-trans carotenoids in plants are susceptible to photo-, thermal- and chemical-isomerization [49], and cistrans isomers differ in their intestinal absorption in humans (Figure 3). Human blood plasma contains mostly all-trans carotenoids but levels of the cis form of some carotenoids are as high as 50% [55]. As mentioned previously, the majority of lycopene found in fresh and processed tomatoes exists in the all-trans form [56,57]. However, lycopene in human and animal tissues exits predominately as cis-isomers, indicating a preference for cis-lycopene in intestinal absorption [58,59]. By contrast, greater excretions of cis-b-carotene and lower excretions of trans-b-carotene were measured in human subjects after ingestion of both raw and processed carrots (Daucus carota var. sativa), indicating an absorption preference for alltrans b-carotene [60]. Factors that inuence carotenoid accumulation and bioavailability Carotenoid accumulation in plant tissue appears to be shaped by the physiological, genetic and biochemical attributes of a plant species, as well as by environmental growth factors such as light, temperature and fertility [6164]. Signicant differences in carotenoid accumulation among different vegetable crop species have been reported [8,65,66], and signicant genetic variation within species has been found for carrot [67], corn [68], kale [61,63,69], lettuce [70], potato (Solanum tuberosum subsp. tuberosum) [71], pepper [72,73] and soybean (Glycine max) [74]. The genetic variation for carotenoid concentrations within species might be advantageous to vegetable improvement programs. The accumulation of carotenoid in plant foods is also inuenced by environmental growing conditions. Carotenoid accumulations have been shown to increase and decrease in response to environmental manipulations, with different results for different plant species. Changes in the growing air temperature [75], irradiance level [76], irradiance photoperiod [77] and nutritional fertility [78,79]
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all affect plant carotenoid accumulations. The increased coloration in vegetable and fruit tissues associated with maturity is often indicative of increases in carotenoid concentrations [72,73,80]. Carotenoid concentrations increase in leaf tissues with maturity [81,82] but decrease during senescence [1]. Manipulation of cultural growing conditions and time of harvest would therefore affect the carotenoid concentrations of fruit and vegetable crops. The bioavailability of carotenoids from plant foods is highly variable and is inuenced by the species and structures of carotenoids present in the food, composition and release of carotenoids from the food matrix, amount consumed and absorption in the intestinal track, transportation within the lipoprotein fractions, biochemical conversions and tissue-specic depositions, as well as by the nutritional status of the ingesting host [12,14,83]. Carotenes are entirely lipophilic molecules located in the hydrophobic cores of plant membranes. Similarly, xanthophylls are largely hydrophobic molecules with their polar groups at opposite ends of a non-polar carbon skeleton [84]. Because of their lipophilic nature, biotic or abiotic activities that expose carotenoid molecules to potential oxidation, degradation or isomerization will ultimately have an inuence on carotenoid biochemistry and bioavailability. The rst step in carotenoid bioavailability is release from the food matrix. Food processing activities, such as thermal processing, mincing or liquefying result in changes to carotenoid chemistry, probably through isomerization or oxidation reactions [53,60,83,85,86]. However, freezing or low-temperature storage generally preserves carotenoid concentrations by reducing potential enzymatic oxidation [85,86]. Processing activities usually increase bioavailability through increased release of bound carotenoids from the food matrix; however, thermal degradations in carotenoid chemistry might adversely affect bioavailability in some food crops. Absorption of carotenoids in humans is passive and follows digestive pathways similar to those of lipids. Protein- or membrane-bound carotenoids must rst be released from tissues and dissolved in a hydrophobic

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Figure 3. Structures of some common all-trans and cis carotenoid isomers found in fruit and vegetable crops. The occurrence and properties of carotenoid isomers in food crops can influence intestinal absorption.

domain (oils, fats or bulk lipid emulsions) [14]. Because of their hydrophobic nature, carotenoids in the mostly aqueous environment in plant foods must be transferred to bulk lipids or intestinal micelles in the digesta [14]. Carotenoid absorption requires the presence of dietary fat in the small intestine, which stimulates the release of emulsifying bile acids by the gall-bladder [12]. Recent studies have shown that absorption of carotenoids increases when they are ingested with dietary lipids [87,88]. After release from the food matrix, carotenoids are assimilated and oriented into lipid micelles before uptake by intestinal mucosal cells. After release to the enterocyte, carotenoids are incorporated into chylomicrons, which are eventually delivered to the blood stream and, ultimately, to the liver. Carotenoid compounds can remain in the liver, or be transferred to low-density or high-density lipoproteins before eventual tissue-specic deposition [12]. Carotenoid bioavailability is usually assessed in blood serum after ingestion in dietary trials or cohort studies. The relatively simple analysis quanties carotenoid changes in serum at various time intervals following ingestion of whole foods or supplements. Some caveats to interpreting serum carotenoid bioavailability include: (i) serum
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responses to single oral doses of carotenoids are highly variable; (ii) carotenoids measured in serum signify an equilibrium between intestinal absorption, breakdowns, tissue uptake and tissue release; and (iii) high concentrations of endogenous carotenoids (i.e. a-carotene, b-carotene, lycopene and lutein) are already present in serum [25]. Recent studies have also used in vitro Caco-2 human intestinal cell lines to assess carotenoid bioavailability [8991]. In these studies, pure carotenoid compounds and whole food samples are brought through an in vitro digestion and reacted with Caco-2 human intestinal cells. Absorption potential is measured using standard HPLC carotenoid analysis. Current results demonstrate that in vitro Caco-2 cells can be used to predict carotenoid bioavailability from supplements and whole foods. Increases in serum carotenoid concentrations typically result after ingestion of carotenoids from whole food or mono-molecular supplements [25,48,60,83,87,88,91]. Our own research has revealed differences in serum carotenoids and responses to macular pigment optical density (MPOD) evaluations in human subjects administered different doses of lutein from mono-molecular supplements and whole food sources. Serum carotenoid levels were

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higher than the baseline level after ingestion of 10 mg or 30 mg lutein supplements or spinach fortied with 8 mg or 12 mg lutein per 100 g fresh weight. Increases in MPOD from the baseline to the end of a 12 week intervention occurred in subjects administered 30 mg lutein supplements and spinach with 12 mg lutein per 100 g fresh weight (D.A. Kopsell, unpublished). In vitro and in vivo studies show that carotenoid bioavailability is inuenced by source (whole food versus supplement), degree of processing, interactions with other carotenoid compounds, the degree of isomerization before, during and after absorption, transit time in the intestine, and the nutritional status of the human subjects [14]. Recent efforts to increase vegetable carotenoid concentrations Carotenoid enhancement efforts in plant foods have advanced through the use of both traditional breeding and molecular approaches. Assessment of genetic variation in several crop species can be assumed to be the beginning of improvement programs using traditional breeding methods. However, improvements in carotenoid concentrations using traditional breeding efforts are limited because of the high costs associated with analytical laboratory measurements. Recent reviews have chronicled molecular advances in carotenoid pathway manipulation to improve biosynthesis and partitioning [13,15,92]. Successful approaches have centered on modication of the biosynthetic pathway to change the ux and end products, increasing pre-existing carotenoids, and engineering carotenogenic behavior in tissues completely devoid of carotenoid activity [13]. Identication and cloning of genes and cDNAs in the carotenoid pathway have facilitated genetic manipulations. Most studies have demonstrated that phytoene synthase has the greatest control over uxes in the carotenoid pathway; however, success has also been achieved overexpressing phytoene desaturase enzymes [15]. However, genetic manipulations to the carotenoid biosynthetic pathway have resulted in abnormal growth and dwarng in tomato [93]. These results were attributed to metabolic precursor competition between carotenoid and gibberellin (GA), both stemming from the common precursor GGPP. Fluxes of metabolites away from GA resulted in stunted tomato plants with abnormal pigmentation. A two- to threefold increase in tomato fruit-specic carotenoid accumulation has been achieved using a bacterial phytoene synthase (crtB) and a tissue-specic promoter [94]. Genetic strategies have also increased b-carotene production in canola (Brassica napus) using a seed-specic promoter [95] and increased xanthophyll carotenoid production in potato using a tuber-specic promoter (epoxidation reactions were suppressed) [96]. Most notably, xanthophyll and carotene production in rice (Oryza sativa) endosperm (Golden Rice) has been increased using exogenous cyclase and desaturase enzymes [97]. The tomato examples show the need to understand how genetic engineering inuences carotenoid precursor pools, enzyme activities and locations of expression, as well as other isoprenoid pathways and metabolic regulations. There are obvious advantages to genetic engineering techniques, but they must
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be accompanied by a clear understanding of how these manipulations will ultimately affect plant physiology. Advances in carotenoid biosynthesis have been facilitated using molecular approaches, but the question still remains: will the general public accept genetic modication of raw or unprocessed foods? Programs designed to increase carotenoid bioavailability in food crops must begin with a solid understanding of carotenoid chemistry and biosynthesis. Before selecting candidate crops for enhancement efforts, care must be taken to outline specic research impacts, whether combating widespread vitamin A deciency or ghting aging eye diseases. Understanding how carotenoids in the food matrix respond to food processing activities and any subsequent changes to carotenoid chemistry will be of primary importance. Lastly, there is a need for collaborative activities to assess bioavailability in model systems or human subjects to gauge progress in enhancement efforts.
Acknowledgements
We thank Pam Bishop, Joanne Curran-Celentano, Laura Dukach, Evan Ford, Catherine Gerwick, Brooke Gowdy-Johnson, Mark Lefsrud, Scott McElroy, John McLean, Carl Sams, Joe Sheehan and Adam Wenzel for their collaborative participation in our projects. Funding for our carotenoid research came in part from a grant received by the Cooperative State Research, Education, and Extension Service, US Department of Agriculture, under Agreement No. 20015210211254.

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