1. Carotenoids, Health Benefits and Bioavailability Steven J. Schwartz, Ph.D. Food Science & Interdisciplinary Graduate Program in Nutrition The Ohio State University Presented at Phytochemicals in Fruit and Vegetable to Improve Human Health November27, 2007

5. Phytoene Phytofluene  -Carotene Neurosporene Lycopene Lycopene Biosynthesis

6. Lycopene  -Carotene  -Carotene  -Carotene  -Carotene  -Carotene Adapted from Britton, 1983 and Gross, 1991. Biosynthesis of common β and ε cyclic carotenes

7. Tomatoes Varieties with Unique Carotenoid Profile REDOR1OR2YELGRE

8. Common Carotenoids HydrocarbonsXanthophyllsLuteinZeaxanthin  -Cryptoxanthin  - Carotene  - Carotene Lycopene

9. Biological Functions of Carotenoids  Provitamin A Activity  Non-provitamin A Activity: Singlet Oxygen Quenching Activity Singlet Oxygen Quenching Activity Antioxidant Activity (Trap Free Radicals) Antioxidant Activity (Trap Free Radicals) Enhancement of Immune Response Enhancement of Immune Response Potential Chemopreventive Properties Potential Chemopreventive Properties

10. Conversion to Vitamin A  -carotene O 2 15,15’-monooxygenase H+ reductase retinol OH retinol OH

11. Carotenoids and Health Benefits  Epidemiological  Cell culture  Animal (experimental)  Human (clinical)

12. Dietary carotenoids, vitamin-A, vitamin-C, and vitamin-E, and advanced Age-related Macular Degeneration Seddon et al. J. Am. Med. Assoc. 272: (18) 1413-1420, 1994 “Conclusion.-Increasing the consumption of foods rich in certain carotenoids, in particular dark green, leafy vegetables, may decrease the risk of developing advanced or exudative AMD, the most visually disabling form of macular degeneration among older people.”

13. Lutein & Zeaxanthin in the Macula Macula is the Region Directly Behind the Lens, Receiving the Most Light.Macula is the Region Directly Behind the Lens, Receiving the Most Light. Lutein & Zeaxanthin are Deposited Specifically.Lutein & Zeaxanthin are Deposited Specifically. The Role Is To Prevent Photo-Oxidation.The Role Is To Prevent Photo-Oxidation. Macula Lens Lutein & Zeaxanthin

15. Epidemiological Studies Investigating Lutein and Cataract Risk StudyComparisonOutcome spinach intake 1  5 times/week vs.  1 time/month  risk 39% carotenoid intake 2 13.7 vs. 1.1 mg/day of lutein  risk 22% carotenoid intake 3 7.0 vs. 1.3 mg/day of lutein  risk 19% antioxidant intake 4 1.3 vs. 0.3 mg/day of lutein  risk 50% serum carotenoids 5 0.4 vs. 0.18  mol/L of lutein  risk 30% 1 Handelman, et al., 1988 2 Chasan-Taber, et al., 1999 3 Brown, et al., 1999 4 Lyle, Mares-Perlman, Klein, Klein, Greger, 1999 5 Lyle, Mares-Perlman, Klein, Klein, Palta, Bowen Greger, 1999

17. Vegetables, fruit, and cancer prevention: A review Steinmetz KA, Potter JD Journal of the American Dietetic Association 96: (10) 1027-1039, 1996 “The types of vegetables or fruit that most often appear to be protective against cancer are raw vegetables, followed by allium vegetables, carrots, green vegetables, cruciferous vegetables, and tomatoes.”

18. Bioavailability is the fraction of an ingested nutrient that is available for utilization in normal physiological functions, metabolism and/or storage. In the context of this discussion, bioavailability of carotenoids is defined as the amount of these micronutrients that are absorbed by the intestinal enterocytes and transported in the bloodstream. Absorption and Bioavailability

19. “Physical inaccessibility of carotenoids in plant tissues may reduce their bioavailability. It may be difficult to free beta-carotene in dark- green leafy vegetables from its matrix. Our findings do not support the long-standing assumption that vitamin A deficiency can be combated by increasing the intake of dark-green leafy vegetables. “ Lack of Improvement in Vitamin A Status with Increased Consumption of Dark-Green Leafy Vegetables dee Pee, S., West, C.E., Muhilal, Karyadi, D. and Hautvast, J.G.A.J. Lancet 346: 75-81. (1995)

20. Food Matrix of Spinach Is a Limiting Factor in Determining the Bioavailability of  -Carotene and to a Lesser Extent of Lutein in Humans (Castenmiller et al. J. Nutr. 129: 349-355, 1999) Lutein 51%  -Carotene 7%

21. CarotenoidsCleavageOxidationIsomerizationMetabolites Carotenoids LYMPH PORTAL MICELLES Chylomicron Carotenoid Absorption & Metabolism Adapted from Erdman et al. Ann. N.Y. Acad. Sci. 691:76-85 (1993)

22. Plasma  -Carotene Response after Meals Supplemented with Dietary Pectin Plasma  -Carotene Response after Meals Supplemented with Dietary Pectin (Rock and Swendseid, Am. J. Clin Nutr. 1992)

23. Food Processing and Preparation  Physical and thermal treatment of foods causes degradation of plant cell structural constituents. Physical and thermal treatment of foods causes degradation of plant cell structural constituents. Thermal processing brings about disruption of the carotenoid protein complexes. Thermal processing brings about disruption of the carotenoid protein complexes. Inactivation of oxidizing enzymes results in less degradation and greater stability of carotenoids. Inactivation of oxidizing enzymes results in less degradation and greater stability of carotenoids. These changes result in enhanced uptake and efficiency of carotenoid absorption. These changes result in enhanced uptake and efficiency of carotenoid absorption.

24. Percentage Change Altered “Bioavailability” By Food Processing  All-trans Rock, C., Lovalvo, J., Emenhiser, C., Ruffin, M., Flatt, S., Schwartz, S., J. Nutr., 128: 913-916, 1998.

25. Intake of carotenoids and retinol in relation to risk of prostate cancer Giovannucci et al. Journal of the National Cancer Institute, 87(23): 1767-76, 1995 “Combined intake of tomatoes, tomato sauce, and pizza (which accounted for 82% of lycopene intake) was inversely associated with risk of prostate cancer.”

27. Epidemiological Data: Relative Risks TotalOralEsophagus*StomachColonRectumPancreas*Lung*ProstateBladderBreastCervixMesothelioma Relative Risk: 0 0.5 1.0 1.5 Relative Risk: 0 0.5 1.0 1.5 Statistically Significant  Not Statistically Significant Giovannucci, 1999

28. Carotenoids as Antioxidants Endogenous and Exogenous Reactive Oxygen Species (ROS) Antioxidants Antioxidants Lipids, Proteins, DNA Damage Chronic Disease

29. Lycopene as a Singlet Oxygen Quencher Lycopene is the most efficient singlet oxygen quencher and phenoxyl radical scavenger among the naturally occurring carotenoids. Stahl and Sies, 1996. Lycopene: a biologically important carotenoid for humans? Arch. Biochem. Biophys. 336:1-9. Mortensen and Skibsted, 1997. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 45:2970-2977.Lycopene is the most efficient singlet oxygen quencher and phenoxyl radical scavenger among the naturally occurring carotenoids. Stahl and Sies, 1996. Lycopene: a biologically important carotenoid for humans? Arch. Biochem. Biophys. 336:1-9. Mortensen and Skibsted, 1997. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 45:2970-2977.

30. Other Hypothesized Mechanism of Action  Stimulate enzymes that detoxify toxins/carcinogens  Enhance DNA repair mechanisms  Increase apoptosis of cancer cells  Decrease cell proliferation  Possess anti-angiogenesis activity  Restore gap-junction communication

32. PDA Chromatogram of Tomato Carotenoids all-trans Lycopene cis Lycopene all-trans  -Carotene

33. Selected Geometrical Isomers of Lycopene  all-trans lycopene  5-cis lycopene  9-cis lycopene  13-cis lycopene  7,9,9’,7’-cis lycopene

34. nm 250 300 350 400 450 500 360 (cis peak) C. 464 A. 472 B. 435 A. All-trans lycopene B. Tetra-cis lycopene C. 13-cis lycopeneLycopene

35. C 30 Analysis of Lycopene Geometrical Isomers

36. Am J Clin Nutr 1997; 66:116-22 Lycopene is more bioavailable from tomato paste than from fresh tomatoes Christine Gärtner, Wilhem Stahl and Helmut Sies ABSTRACTLycopene bioavailability, from a single dose of fresh tomatoes or tomato paste (23 mg lycopene) ingested together with 15 g corn oil was compared by analyzing carotenoid concentrations in the chylomichron fraction. The lycopene isomer pattern was the same in both fresh tomatoes and tomato paste. The triacylglycerol response in chylomicrons was not significantly different after both treatments. Ingestion of tomato paste was found to yield 2.5-fold higher total and all-trans-lycopene peak concentrations (P<0.05 and P<0.005, respectively) and 3.8-fold higher area under the curve (AUC) responses (P<0.001) than ingestion of fresh tomatoes. The same was calculated for lycopene cis-isomers, but only the AUC response for the cis-isomers was significantly higher than ingestion of tomato paste (P<0.005). No difference was observed in the  - and  -carotene response. Thus, in humans, the bioavailability of lycopene is greater from tomato paste than from fresh tomatoes.

37. Electron Microscopy Electron micrograph of lycopene crystalloids in mature red tomato chromoplast. A. Lycopene crystalloids. B. Plastoglobulin-type sacs in which  -carotene reportedly accumulates. 3.5 m m B A

38. Tomato Product Consumption – Correlation with Plasma Lycopene Tomato Saucep < 0.0001 Tomatoesp < 0.056 Lower Prostate Cancer Risk in Men with Elevated Plasma Lycopene Levels: Results of a Prospective Analysis (Gann et al., Cancer Research, 1999, 59: 1225-1230)

39. Commercial Products Study – Experimental Design Vegetable Juice Wash-out Meals Analysis ( 14 days) (28 days) Tomato Soup 36 subjects 18 M, 18F Tomato Sauce

40. Plasma Lycopene Response 0.000 1.000 2.000 3.000 Baseline Week 1Week 2Week 3 Week 4 Week 5Week 6 umoles/L saucesoupVeg washout feeding Allen et al. J. Nutr. 2003

41. Total Lycopene Levels in Buccal Cells 0.0 1.5 3.0 4.5 012345678 Juice Soup Sauce Week Allen et al. J. Nutr. 2003

42. Conjugated Diene Formation (nm) Lipoprotein Oxidation Lag Period (min, mean ± SE) CS Baseline 64.7±2.4 Treatment 70.1±4.0 Significance p<0.05 RTS 64.7±2.468.3±2.4 p<0.05 V8 64.7±2.471.7±4.0 p<0.01

43. Two Stage In Vitro Digestion Garrett et al. (1999) J. Agric. Food Chem. 47:4301 Centrifugation TestMeal*GastricDigesta GastricPhase IntestinalDigesta IntestinalPhase * Aqueous Micellar Fraction *

44. Caco-2 Human Intestinal Cells            

45. Caco-2 Cells Test Media DMEM + Micellar Fraction Apical Basolateral Harvest Cells Analyze for Pigments Collect Basolateral Media Analyze for Pigments 4 hours 0-10 hours Uptake Transport

46. Massive doses of a carotene-rich vegetable to the basic diet over a long period did not greatly increase serum carotene and vitamin A levels and that most carotene was excreted. Carotene Balances on Boys in Rwanda Where Vitamin A Deficiency is Prevalent Roels, Trout and Dujacquier. J. Nutr. 1958

47. Absorption of β-Carotene and Vitamin A with Different Levels of Fat Absorption of β-Carotene and Vitamin A with Different Levels of Fat ( adapted from Roels et al. J. Nutr. 1958 ) 200 g carrots (n=5) 200 g carrots + 20 ml olive oil (n=5) dayβ-carotene (µg/100 ml serum) Vitamin A (µg/100 ml serum) β-carotene (µg/100 ml serum) Vitamin A (µg/100 ml serum) 043.036.647.831.0 3180.651.0297.850.4 difference37.614.4250.0*19.4 * significantly different from β-carotene serum levels without fat addition (unpaired Student t-test, p<0.005)

48. Addition of fats to the diet may contribute to the relief of vitamin A deficiency in this region. Roels, Trout and Dujacquier. J. Nutr. 1958

49. CarotenoidsCleavageOxidationIsomerizationMetabolites Carotenoids LYMPH PORTAL MICELLES Chylomicron Carotenoid Absorption & Metabolism Adapted from Erdman et al. Ann. N.Y. Acad. Sci. 691:76-85 (1993)

50.  a L 4 hour L   6 hour   L 9 hour b a L 2 hour L   0 hour Post-prandial Response 440 mV Applied Potential 20.0 nA Full Scale

51. Brown et al. Am. J. Clin Nutr. In press (2004) Carotenoid Response at Three Different Lipid Levels Fat Free 0 g Fat Free 0 g Reduced Fat 6 g Reduced Fat 6 g Full Fat 28 g Full Fat 28 g Full Fat Reduced Fat Fat Free

52. Lettuce: 40g, Avocado: 75g, Carrots: 100g, Spinach:80 g

53. A) Salad B) Salad with 150g of Avocado C) Salad with 24g of Avocado Oil obtained from the same subject at 5th hour HPLC Chromatograms of TRL Fraction after Consumption of; lutein β- carotene α -carotene

54. Values are based upon mean AUC ± SEM

55. Tomato Varieties

56. Postprandial Absorption of Tangerine vs High Lycopene (Pasta Sauce)

57.  Accumulating evidence (epidemiological, cell-culture, animal and human) continues to associate health benefits with fruit and vegetable consumption.  Carotenoid pigments unique to fruits and vegetables, through their antioxidant activity, may be important components with beneficial physiological properties.  “Bioavailability” of carotenoids from specific food products is influenced by a number of dietary factors including food processing treatments and product formulation (i.e. lipid content) and should be determined to more accurately assess delivery of these phytonutrients from the diet. Summary

58.  Consumption of carotenoid containing F&V’s may have a protective effect against oxidative stress in vivo by decreasing the susceptibility of lipoproteins to oxidation.  In vitro assays can be employed to mimic digestion and absorption processes resulting in estimates of in vivo bioavailability for carotenoids.  Care must be taken not to make the assumption that all the health benefits brought about by fruit and vegetable consumption are attributed to a single class of compounds such as carotenoids. Summary

59. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Steven K. Clinton, M.D., Ph.D.Internal Medicine, OSUSteven K. Clinton, M.D., Ph.D.Internal Medicine, OSU Dave Francis, Ph.D.Horticulture &Dave Francis, Ph.D.Horticulture & Crop Science, OSU Mark Failla, Ph.D.Human Nutrition, OSUMark Failla, Ph.D.Human Nutrition, OSU Cheryl Rock, Ph.D.UC - San DiegoCheryl Rock, Ph.D.UC - San Diego Wendy White, Ph.D.Iowa State UniversityWendy White, Ph.D.Iowa State University Minhthy Nguyen, Ph.D.Minhthy Nguyen, Ph.D. Charlotte M. Allen, Ph.D.Charlotte M. Allen, Ph.D. Mario Ferruzzi, Ph.D.Mario Ferruzzi, Ph.D. Nuray Unlu, Ph.D.Nuray Unlu, Ph.D. Torsten Bohn, Ph.D.Torsten Bohn, Ph.D. Craig Hadley, Ph.D.Craig Hadley, Ph.D. Elena Vittadini, Ph.D.Elena Vittadini, Ph.D.

60. Questions