Color Quality of Fresh and Processed Foods - American Chemical

lycopene. 1.7 + 0.8 ndd nd. 6.1 ±0.6 nd lutein. 0.4 ±0.1 0.3 ±0.1 1.1 ±0.7 0.3 ±0.3 0.51 ±0.27. Total. —f ... e Anthocyanins are cyanidin deri...
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Chapter 12

Role of Color and Pigments in Breeding, Genetics, and Nutritional Improvement of Carrots 1

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P. W. S i m o n , S. A. Tanumihardjo , B. A. Clevidence , and J. A. Novotny 3

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Agricultural Research Service, U.S. Department of Agriculture, Vegetable Crops Research Unit, Department of Horticulture, University of Wisconsin at Madison, WI 53706 Department of Nutritional Sciences, University of Wisconsin at Madison, Madison, WI 53706 Agricultural Research Service, U.S. Department of Agriculture, Diet and Human Performance Laboratory, Beltsville Human Nutrition Research Center, Beltsville, MD 20705 2

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The color of carrots was an important attribute during its domestication as a root crop. Modern carrot researchers continue to study color, and carrot genetic stocks have been developed with not only orange, but also distinctive dark orange, red, yellow and purple color. Genes for 22 carotenoid biosynthetic enzymes have been mapped and cloned, and the α- and β-carotene in typical orange and dark orange carrots, lycopene in red carrots, lutein in yellow carrots, and anthocyanins in purple carrots have been demonstrated to be bioavailable. The function of carrot color genes largely remains unknown and the sources of wide variation in pigment absorption are unexplained, but carrot has been demonstrated to be a sustainable source of dietary provitamin A and other phytonutrients of interest for researchers and consumers.

U.S. government work. Published 2008 American Chemical Society.

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History of Carrot Color Carrot is known around the world today as an orange vegetable rich in provitamin A carotenoids. While this correctly characterizes modern carrot, the first domesticated carrots of about 1000 years ago were only found in Central Asia, its center of diversity, and they were either yellow or purple in color (/, 2, 3). As traders and farmers introduced carrots west and east of carrot's center of diversity, both purple and yellow carrots were known across the Middle East and North Africa and in both Europe and China by the 15 century. Then, in the 16 century orange carrots were first reported in Europe and eventually in Asia. Thus, orange carrots are a relatively recent development. Two other carrot colors became popular shortly thereafter - white carrots in Europe and red carrots in Asia. It is interesting to note that a broad array of carrot color was known 400 years ago, but orange came to be the preferred color in most of the world soon after their first appearance. The yellow, orange and red colors of carrot roots are attributable to carotenoids - pigments important in photosynthesis, while purple carrot color is attributable to anthocyanins. Neither carotenoids nor anthocyanins have a known function in plant roots so their abundance in carrots is likely a consequence of human selection over the course of domestication. While we do not know why yellow, orange, red, and purple carrots became popular, their nutritional implications are now known to be significant. th

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Biochemistry and Genetics of Carrot Pigments Carotene and Anthocyanin Biosynthesis The carotenoid biosynthetic pathway is lightly conserved in plants, fungi, and photosynthetic bacteria with very similar enzymes and genes for those enzymes across a very diverse array of organisms. (4, 5, 6). The structural genes for carotenoid biosynthetic enzymes are well characterized in terms of gene sequence, action, and location in the genome in several plants. The anthocyanin biosynthetic pathway is also well-conserved in higher plants and structural genes in this pathway have been characterized. In some cases variation in plant color is associated with variation in their structural genes. For example, the yl gene of maize that accounts for yellow versus white kernel color is associated with a carotenoid biosynthetic gene, phytoene synthase (7, 8)\ genes accounting for several bell pepper colors are associated with carotenoid structural genes, ( 9 ) ; and several of the Al, A2, Bzl, Bz2, CI, C2, Prl, and Rl genes of maize, that account for red and purple kernel, leaf, and root color are associated with

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anthocyanin structural genes (10). However, some color differences in maize, pepper, tomato, and cauliflower due to accumulation of carotenoid and anthocyanins are not associated with structural genes involved in the biosynthesis of these pigments, but rather with regulatory genes elsewhere in the genome (11, 12, 13).

Carrot Color Genetics At least ten major genes that influence carotenoid and anthocyanin accumulation have been identified in carrot (3) but only two of these genes have been placed in the genetic map; along with approximately 15 more minor QTL (quantitative trait loci, genes associated with a continuously variable trait) (14). These major carrot color genes, Y and Y , account for the distinctive color differences: white (no carotenoids), yellow (primarily lutein), and orange (primarily ot- and P-carotene). The QTL primarily accounts for genetic variation in a- and p-carotene concentration ranging from 10 ppm to over 600 ppm. Typical carrots in the U.S. today contain 170 ppm carotenoids (over 95% a- and P-carotene) and account for about half of the provitamin A carotenoids in the food supply (75). We recently placed 22 carotenoid biosynthetic enzymes on the carrot map and associated them with Y and Y (16). Genetic analysis of carrot pigment accumulation is confounded by environmental and developmental variation. The development of DNA markers to track genetic variation without confounding non-genetic influences will set the stage for more efficient progress in breeding for altered carrot pigment content. 2

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Color and Nutritional Value of Carrot Carotenoids Carotenoids and Health Effects Carotenoids are a group of yellow, orange, and red phytochemicals found in all plants and some microbes that are assumed to be important for overall human health (77, 18). Although hundreds of carotenoids are present in nature, only a few carotenoids circulate in appreciable amounts in human blood and specific carotenoids are localized in certain tissues. For example, lutein and zeaxanthin, which are xanthophylls (oxygen containing carotenoids), are concentrated in the macular region of the eye. Thus, lutein might be an important compound in the prevention of macular degeneration, the leading cause of blindness in the elderly. Over the past decade, several epidemiologic and clinical studies have suggested that carotenoid consumption is associated with lower risk of cardiovascular

154 diseases and cancers; especially lung, oral cavity, pharyngeal, prostate and cervical; as well as eye diseases. However, intervention trials with pharmacological doses of isolated P-carotene have shown either no effect or harmful effects on lung cancer risk among smokers. This suggests that other carotenoids or other components in fruits and vegetables may be responsible for protective influences observed in epidemiological studies. Thus, the overall evidence suggests that diets high in fruit and vegetables are important for optimal health and reduced risk of disease. Carotenoids are one of several components that may confer health benefits yet are not considered essential nutrients (19). Research is very active in this area and identification of non­ invasive ways to assess carotenoid status will be important in moving research forward. In addition to making the world a more colorful place, carotenoids have known nutritional value and antioxidant properties. The essential nutrient that is derived from carotenoids is retinol or vitamin A. The predominant provitamin A carotenoids found in humans are p-carotene, ot-carotene and P-cryptoxanthin. The vitamin A value of provitamin A carotenoids is under debate. The most recent conversion factors that have been assigned by the Institute of Medicine to P-carotene, ct-carotene and p-cryptoxanthin are 12, 24, and 24 jig to 1 \ig of retinol, respectively (20). Recent research in humans and Mongolian gerbils are in accord with these estimates, yet many factors affect these ratios (77). Most notably, these factors include the vitamin A status of the host, the amount or concentration of provitamin A carotenoid fed, and the food matrix. Carotenoids are thought to act as potent antioxidants to neutralize free radicals formed from the natural metabolic processes of cells. Free radicals damage tissues and cells through oxidative processes. Environmental factors such as smoking and pollution can increase free radical concentrations. Carotenoids may counter these influences by functioning as antioxidants and quenching oxygen-containing free radicals. As components of lipoproteins, carotenoids may regenerate the antioxidant form of vitamin E as well as protect it from oxidation.

Carrots of Various Colors as Sources of Carotenoids Carrots are a common vegetable and have gained popularity in the past decade in the United States due to the introduction of prepackaged "cut & peel" carrots. Typical orange carrots contain predominantly a- and p-carotene (Table I). High ct-carotene serum concentrations uniquely indicate carrot consumption. The red carrot color can be attributed to high lycopene content, but a- and Pcarotene are also present in appreciable quantities depending on the type of red carrot. The yellow carrot is generally low in carotenoids and the yellow color

155 can be attributed to lutein, a carotenoid that does not have provitamin A activity, and small amounts of p-carotene.

Table I. Concentrations of carotenoids and anthocyanins in raw carrots of various c o l o r s ab

High P-carotene Orange

Orange

Purple

Red

Yellow

mg/100 g ± S D Total carotenoids a-carotene

28 + 0.8

15 +4.1

18 ± 7

9.8 ±1.4

0.71 ±0.38

3.1 ±2.4

2.2 ±0.8

4.1 ± 1.2

o.ir

0.05

P-carotene

19 ±2.8

13 ±3.3

12 ± 5

3.4 ±0.9

0.18 ± 0.17

nd

6.1 ±0.6

nd

c

lycopene

1.7 + 0.8

nd

lutein

0.4 ±0.1

0.3 ±0.1

1.1 ±0.7

0.3 ±0.3

0.51 ±0.27



133 ± 2 0





Total anthocyanins

f



d

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a

Carotenoid and anthocyanin data are expressed as means ± SD of three determinations on a fresh weight basis. b

Carotenoids data are from reference 26.

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Carotenoid values were found in only one of the three carrots.

d

nd, not detected

e

Anthocyanins are cyanidin derivatives, reference 43.

f

—, not determined

Bioavailability of Carrot Carotenoids in Humans Vegetables in general are often dismissed as a source of vitamin A because of factors that affect carotenoid bioavailability (77). The carrot matrix has a negative effect on P-carotene bioavailability compared with P-carotene beadlets in the ferret (20) and in humans (22). Thus, a series of studies was performed to compare the relative bioavailability of carotenoids from pigmented carrots to carotenoids from other vegetables or from supplements containing equivalent amounts of carotenoids. The first study in young adults determined whether lycopene in red carrots is bioavailable by feeding carrot muffins at 5 mg lycopene/day for 11 days (23). The second study determined the effect of carrot fiber on lycopene absorption by

156 mixing tomato paste with white carrot and feeding 5 mg lycopene/day for 11 days. The results showed that both lycopene and p-carotene are bioavailable from red carrots, but lycopene absorption was negatively affected by carrot fiber. Making inferences from both studies, the lycopene in the red carrot is about 44% as bioavailable as that from tomato paste and the serum concentration of lycopene begins to plateau at about 20 mg dietary lycopene/day. Red carrots provide an alternative to tomato paste as a good dietary source of lycopene and provide more p-carotene than typical red tomatoes. Interestingly, yellow carrots predate orange carrots. Although the lutein concentration in carrots is not high, the bioavailability of 1.7 mg lutein fed as yellow carrot for seven days was tested against an equalized lutein-in-oil supplement in young adults (24). The lutein derived from the yellow carrot was 65% as bioavailable as the supplement. Lutein from this novel food source resulted in a significant increase in serum concentrations and did not result in the decline in P-carotene that accompanied administration of the lutein supplements. A subsequent analysis of a crystalline lutein supplementation trial (25) revealed that in this form, bioavailability varies greatly both within and between subjects. A sustainable intervention to improve vitamin A status may be the promotion of common vegetables that have enhanced P-carotene concentrations. In agriculture, orange carrots may have up to a 5-fold variation in p-carotene concentrations. Carrots are also the most abundant dietary source of ot-carotene (26). Orange and high-P-carotene dark orange carrots in muffins providing 2.6 and 7.0 mg P-carotene/day, respectively, were chronically fed for 11 days to healthy young adults (27). Although serum a- and P-carotene concentrations both responded to carrot treatment, the concentrations did not differ over time making the results difficult to interpret (28). Future studies need to be conducted in groups of people who have marginal vitamin A status in order to assess the impact of different carotenoid concentrations of carrots in at-risk populations.

Bioavailability of Carrot Carotenoids in Gerbils Not all research questions concerning the specialty carrots can be answered with human studies. Therefore, a series of studies was performed in the Mongolian gerbil so that liver storage of the carotenoids could be determined. Freeze-dried carrot powder was fed in some studies and carotenoid supplements in others. In two studies (29, 30), the use of the gerbil as a model for the carotenoid lutein was dismissed as lutein-in-oil supplements were not efficiently absorbed and stored. However, the utility of the model for a- and P-carotene was confirmed. The bioavailability of a- and P-carotene from carrots was determined in gerbils. Liver stores of P-carotene and vitamin A in the gerbils did not differ

157 between the orange and purple carrot treatments and carrots resulted in higher liver vitamin A than P-carotene supplements equalized to the P-carotene jn the carrots (31). Feeding dark orange carrots, as compared with typical orange carrots, resulted in more than two times higher p-carotene content in liver but only 10% greater vitamin A liver stores. Using the vitamin A utilization rate (i.e., 2.5 |ug retinol/day) from another study in gerbils (32), conversion factors are estimated to be 9 to 11 jag p-carotene to 1 ug retinol for the typical orange carrots and -23 jag P-carotene to 1 jag retinol for the dark orange carrots. It is important to note that the gerbils had an adequate vitamin A status and therefore future studies need to be conducted in vitamin A-depleted gerbils and humans to see if conversion factors differ by vitamin A status. Dark orange carrots may be an alternative source of provitamin A to typical carrots in areas of vitamin A deficiency. Moreover these studies showed that phenolics, including anthocyanins and phenolic acids, in purple carrot do not interfere with the bioavailability of p-carotene from purple carrots. Another interesting outcome from the human and gerbil studies revolved around a-carotene. In the gerbils fed varying amounts of ct-carotene and equal amounts of p-carotene from different colored carrots (57), the a-carotene concentration in liver increased dose dependently and did not contribute significantly to the vitamin A stores. Moreover in the human study comparing typical orange and dark orange carrots, the a-carotene serum concentration was identical in both treatments even though the concentration of a-carotene was more than two-times higher in the dark orange carrots. These studies are inconclusive concerning the vitamin A value of a-carotene during sufficient vitamin A status. To follow up this finding, a-carotene was isolated from carrots and 18.8 jug (35 nmol)/day was fed to vitamin A-depleted gerbils for 21 days (32). In the vitamin A-depleted gerbils, purified a-carotene maintained vitamin A status as well as P-carotene supplements when fed at twice the amount of Pcarotene. Conversion factors were -5.5 jug a-carotene or -2.8 fig P-carotene to 1 jug retinol which are slightly higher than those proposed by the Institute of Medicine for oil-based supplements (20). Thus, a-carotene can support vitamin A status when needed by the host.

Color and Nutritional Value of Carrot Anthocyanins Anthocyanins and Health Anthocyanins are water-soluble red, blue, and purple pigments found in fruits, vegetables, and ornamental crops. Promising research has shown that dietary anthocyanins may serve an important role in promoting health.

158 Anthocyanins have been associated with reduced risk of atherosclerosis (33, 34) and cancer (35 36), reduction of inflammation (37, 38), and improved antioxidant status (33, 34, 39). Anthocyanins may be particularly beneficial to brain tissue and function. Rats fed blueberries had improved spatial learning and memory compared to control rats (40). Loren and colleagues recently reported that maternal supplementation with pomegranate juice protects the fetal brain against neonatal hypoxic-ischemic brain injury (41). Andres-Lacueva and colleagues (40) found anthocyanins in brain tissue of rats fed blueberries, suggesting that anthocyanins are able to cross the blood-brain barrier.

Purple Carrots as a Source of Anthocyanins The six common anthocyanidin backbones are cyanidin, malvidin, delphinidin, peonidin, petunidin, and pelargonidin. These backbones can be glycosylated and form linkages with aromatic acids, aliphatic acids, and methyl ester derivatives (42). Anthocyanins found in Daucus carota L. (sometimes referred to as purple carrot or black carrot) are predominantly derivatives of cyanidin, though pelargonidin and peonidin glycosides have also been reported (43). The major forms have been identified as cyanidin-3-xylosyl-galactoside and cyanidin-3-xylosyl-glucosyl-galactoside. The latter can be acylated with ferulic, sinapic, or p-coumaric acid, and the acylated forms are predominant (44). This is in accord with the general finding that vegetable anthocyanins are more likely to be acylated than fruit anthocyanins (45, 46). Analysis of whole carrots showed the following anthocyanin derivatives: cyanidin-3-(2"-xylose-6glucose-galactoside) (Cy3XGG), cyanidin-3-(2"-xylose-galactoside) (Cy3XG), cyanidin-3 -(2"-xylose-6"-sinapoy 1-glucose-galactoside) (Cy3XSGG), cyanidin3-(2"-xylose-6"-feruloyl-glucose-galactoside) (Cy3XFGG), cyanidin-3-(2"xylose-6"-(4-coumuroyl)glucose-galactoside) (Cy3XCGG) (44). Cultured purple carrot cells were also found to contain cyanidin-3-(2"-xylose-6"-(4hydroxybenzoyl) glucose-galactose) (47). Purple storage root flesh of USDA inbred B7262, which is a line with a purple exterior and an orange center, has been analyzed for anthocyanin content. One hundred grams of raw purple storage root material, after removal of the orange cores, contained on average 8.0 mg Cy3XGG, 8.3 mg Cy3XG, 99.8 mg Cy3XSGG, 47.6 mg Cy3XFGG, and 2.8 mg Cy3XCGG, for a total of 166 mg anthocyanin/100 g. Approximately 25% of orange taproot material was removed before analysis, thus the anthocyanin content of the whole taproot was approximately 133 mg anthocyanin/100 g. For comparison, anthocyanin contents of other sources include Bing cherries with on average 38 mg/100 g (48), pomegranate juice with approximately 14 mg/100 g (49), blueberries with on average 230 mg/100 g (50), blackberries with on average 179 mg/100 g (50),

159 and black current with on average 207 mg/100 g (50). In the United States, anthocyanin consumption is estimated at about 215 mg/day during summer months and about 180 mg/day during winter months (57).

Carrot Anthocyanin Bioavailability in Humans Due to their potential health-promoting effects, understanding factors affecting anthocyanin bioavailability has become important. Consideration of carotenoids, a well-studied class of phytonutrients, provides examples of factors that influence phytonutrient bioavailability, such as cooking, dose size, specific forms of the compounds ingested, and concomitant intake of other dietary components. For example, carotenoid absorption is improved when fat is consumed in conjunction with carotenoid-rich foods (52). Lycopene bioavailability is substantially higher from processed tomato paste compared to raw tomatoes (53). The carotenoid lutein appears to be significantly more bioavailable than |3-carotene from kale in humans (54), thus showing different forms must be specifically studied. And lycopene absorption efficiency decreases with increasing dose size (55). Factors influencing bioavailability of anthocyanins are just beginning to be investigated. Our group has studied the bioavailability of purple carrot anthocyanins, including factors that influence their absorption. Bioavailability studies have been performed with Daucus carota USDA inbred B7262 (44). In general, all anthocyanin forms found in the carrots except Cy3XCGG were identified in human blood and urine after carrot consumption, thus confirming that anthocyanins are bioavailable from purple carrots and can be absorbed intact. Previous studies have also shown absorption of intact glycosylated anthocyanins (56-61). Anthocyanins from carrots are quickly absorbed, appearing in plasma by 30 min after dosing, reaching peak plasma levels by 2 h after ingestion, then slowly decreasing, with anthocyanins still detectable in plasma at 8 h after dosing. Carrot anthocyanins can be detected in urine by 2 h after dosing, with the greatest rate of excretion occurring 4 h after dosing and with anthocyanins still detectable in the 16-24 h collection. Recovery of carrot anthocyanins in blood and urine from human volunteers after carrot consumption was similar to recoveries of anthocyanins from other sources. Urinary recoveries of anthocyanins after ingestion of 250 g purple carrots were 0.014% for acylated anthocyanins, 0.19% for non-acylated anthocyanins, and 0.038% for total anthocyanins. Wu and co-workers (57) recovered 0.077% of anthocyanins in urine of volunteers after consumption of 12 g elderberry extract and 0.004% of anthocyanins in urine of volunteers after consumption of 189 g blueberries. Bub and colleagues (62) detected less than 0.03% of malvidin-3-galactoside in urine after a single ingestion of 500 mL of

160 red wine, dealcoholized red wine, and grape juice. Others have found similar recoveries. Since carrots are often served cooked, the effect of microwave cooking on anthocyanin bioavailability has been investigated. The microwave-cooked carrots showed a trend toward lower anthocyanin contents than raw carrots (23% decrease in total anthocyanin content), though this reduction did not reach statistical significance. Other studies have suggested that anthocyanins in juice or extracts are thermally unstable (63, 64), but studies have not previously considered thermal stability of anthocyanins in whole foods. When cooked and uncooked carrots were administered to study volunteers at matched carrot dose sizes, cooking did not appear to affect total anthocyanin mass in blood or urine after the dose. However, since the cooked carrots showed a trend (though not statistically significant) toward having slightly reduced anthocyanin contents, expressing the blood and urine appearance of anthocyanins as fractional recovery suggests cooking may be important. When anthocyanin appearance in urine is expressed as fractional recovery (anthocyanin mass in urine / anthocyanin mass in treatment), urinary recovery of anthocyanins from cooked carrots was found to be greater than that from the same size dose of raw carrots (44). Previous studies have shown that cooking and processing influence carotenoid bioavailability (65, 66). Thermal processing may disrupt cell walls, making compounds more accessible for absorption. Since it has been suggested that anthocyanin absorption is mediated by a carrier in the gastrointestinal epithelium, absorption may be prone to saturation. To investigate this possibility, purple carrots were administered to study volunteers in two dose sizes. Plasma and urine appearances of anthocyanins were not different after ingestion of 250 g cooked carrots compared to 500 g (44). The 250 g carrot treatment contained approximately 350 umol anthocyanins. This suggests that anthocyanin absorption may be saturated at levels of 350 umol (or lower, which cannot be determined since our lowest dose was 350 umol), though it should be noted that specific saturation levels would likely be compound specific. Saturation of an absorption mechanism has been observed previously for carotenoids (55). The apparent saturation supports carrier involvement in anthocyanin absorption. Additionally, the large size and the polarity of these compounds makes passive transport less likely since they would not partition into a lipid bilayer. Carrots, like many other anthocyanin-rich vegetables, contain a substantial fraction of acylated anthocyanins. Both acylated and non-acylated anthocyanins have been recovered in blood and urine of volunteers after consumption of purple carrots (44). Other recent studies have also shown that acylated anthocyanins can cross the gastrointestinal tract intact (67-69). In human studies with purple carrots, acylated anthocyanins were recovered at much lower levels in blood and urine than non-acylated anthocyanins. Similarly, Wu et al (69)

161 reported that recovery of the single acylated anthocyanin from freeze-dried marionberry powder was lower than that for the three non-acylated anthocyanins. And Mazza et al (70) were unable to detect acylated anthocyanins in serum of subjects after consumption of lowbush blueberries, while many non-acylated anthocyanins in serum were measurable. One possible explanation for this is that acylation of anthocyanins reduces bioavailability. Alternatively, it is possible that acyl groups are cleaved prior to appearance in blood and urine.

Consumer Evaluation and Promotion Before growers embrace carrots of various colors, a market needs to be established. Consumer sensory evaluation showed that the high-p-carotene dark orange and white carrots were favored over the yellow, red and purple carrots in both blind and non-blind treatments (26). However, all the carrots were wellaccepted by the consumer panel and therefore growers should be encouraged to cultivate specialty carrots to provide dietary sources of vitamin A and phytochemicals. Outreach activities with community gardens resulted in positive attitudes toward all the carrot colors. Seed companies and growers ranging from small-scale to large are beginning to direct their attention and resources to this wide array of carrot colors as this small niche market begins to grow.

Future Directions for Research Genetic research has provided a useful basis for improving the nutrient composition, of carrot, but many questions remain. Will use of molecular markers improve selection efficiency for desired pigments? Will changes in pigment composition alter color to reduce visual appeal for consumers? Can we select for one specific pigment, say a - carotene, to the exclusion of other carotene isomers? Can we breed for higher bioavailability of all pigments? Can we breed crops for those consumers who have specific nutrient needs? Can we determine the molecular basis of the mutations that resulted in high pigment accumulation? Can we take that knowledge and apply it to genetic improvement of nutrient composition in other crops? How far can we apply naturallyoccurring crop variation, and what more could transgenic approaches bring? More research is needed on vegetables' contribution to alleviating vitamin A deficiency globally. The carrot could play a major role in this regard as it can be grown in many areas of the world. Determining whether smaller amounts of dark-orange carrot with higher carotenoid concentrations can result in similar vitamin A status to a larger amount of typical orange carrot is an important next step in both human and animal models. Studies that compare the lycopene-rich

162 red carrot to tomato in disease prevention models are also needed. The influence of anthocyanin presence in the purple carrot on carotenoid bioavailability is also needed in humans to ascertain phytochemical interactions from this unique vegetable. Many interesting questions also remain to be answered with respect to nutritional aspects of anthocyanins and how carrots can provide anthocyaninrelated health benefits. The mechanisms for anthocyanin absorption across the gastrointestinal tract are currently unknown. The effect of increasing dose size on anthocyanin absorption efficiency will be important for determining effective intake patterns. Dietary factors that increase or decrease absorption must be understood for the development of dietary recommendations. And the active forms of the compounds (parent anthocyanins or metabolites) must be elucidated. As the importance of anthocyanins to health continues to unfold, it is expected that consumer demand for anthocyanins will increase, and carrots will likely play an important role in providing dietary anthocyanins. Carrot color has generated much interest in the past. As crop specialists collaborate with nutritionists to study the pigments that confer these distinctive colors, a broad new array of research questions arise, and consumers will ultimately benefit from those collaborations.

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