Effects of Thermal Processing on Isomerization and Bio Accessibility of L Ycopene Precursors

Home , Neurosporene, Phytofluene, Retinol

EFFECTS OF THERMAL PROCESSING ON ISOMERIZATION AND BIO ACCESSIBILITY OF L YCOPENE PRECURSORS

A Thesis

Presented in Partial Fulfillment of the Requirements for

The Degree Master of Science in the

Graduate School of The Ohio State University

Marjory Renita, B.S. *****

The Ohio State University 2005

Master's Examination Committee: Approved by Dr. Steven J. Schwartz, Advisor

Dr. Joshua A. Bomser

Dr. Mark L. Failla Advisor Graduate Program in Food Science ABSTRACT

Consumption of dietary carotenoids has been associated with the prevention of several chronic age-related diseases. For example, ingestion of cooked tomato products is inversely correlated with the risk of prostate cancer. Processing to produce tomato- based products also has been shown to enhance the bioavailability of lycopene, the most abundant carotenoid in many varieties of tomatoes.

Plant breeders are developing unique varieties to deliver increased concentrations of carotenoids in food products. The characteristics and regulation of carotenoid biosynthesis in tomato fruit continues to be investigated extensively. Tangerine, one of the distinct variety of tomatoes, contains high levels of the lycopene precursors: (15Z)- phytoene, (15,9'Z)-phytofluene, (9,9'Z)-ζ -carotene, and (7,9,9'Z)-neurosporene, compared to typical red tomatoes. Either (9,9'Z)-ζ-carotene or (7,7',9,9'Z )-lycopene are also relatively abundant in tangerine tomato. These carotenoids are predominantly in the (Z)-configuration in contrast to the (E)-configuration of lycopene in red tomatoes.

Recent evidence has shown that thermal processing may increase isomerization of carotenoids such as β-carotene or lutein, whereas lycopene is relatively stable. The predominant form of these carotenoids in nature is the (E)-configuration, while thermal processing may induce (Z)-isomerization. The effect of thermal processing to the

ii tangerine tomatoes, especially on the (Z)-lycopene precursors present in this variety have not been studied.

Human clinical studies have reported significant amount of the (Z)-lycopene

isomers in plasma or serum levels and 70-80% in benign and malignant prostate tissues,

in contrast to the significant amount of (E)-lycopene in the dietary tomato products. The

(Z)-lycopene isomers have been proposed to be more bioavailable than the (E)- configuration. Greater absorption of these isomers observed in clinical trials coincide with results evaluating the bioaccesibility of (Z)-lycopene isomers using in vitro digestion coupled with Caco-2 cells. However, there is minimal reports that have been reported on the bioacessibility and bioavailability of the (Z)-lycopene precursors.

The aim of this research is to examine the effects of thermal processing on isomerization and bioaccesibility of the lycopene precursors: phytoene, phytofluene, ζ- carotene, and neurosporene, from tangerine tomatoes. Rapid analytical methods such as high performance liquid chromatography coupled with photodiode array detector

(HPLC-PDA), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and nuclear magnetic resonance (NMR) will be used for separation, identification, and quatification of the carotenoids including their isomers. In vitro digestion coupled with

Caco 2 cell cultures will be employed to determine the bioaccessibility (digestive

stability, micellarization, and cell uptake) of the lycopene precursors.

In this study, we observed that the lycopene precursors were relatively stable during thermal processing and no significant degradation was noted. Better extractability and increased isomerization towards the (E)-configuration were found. Processing was also associated with increases in the bioaccessibility of the lycopene precursors during in

iii vitro digestion and Caco-2 cell uptake. Overall, this study demonstrated that thermal processing may induce better extractability and isomerization of the lycopene precursors: phytoene, phytofluene, ζ-carotene, and neurosporene. These carotenoids and their isomers are readily digested, absorbed, and distributed in the human body. Futher clinical trials will be required to examine and confirm the bioavailabilty of the lycopene precursors and their isomers.

iv Dedicated to my beloved parents and sister

v ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Steven J. Schwartz, for his great support, encouragement, advise, patience, and faith on me, which made this research and thesis possible. I am also grateful for all of the support and guidance from my committee members, Dr. Mark Failla and Dr. Josh Bomser, both from the nutrition department.

I am very delighted being as a member of Haas Chair Lab under Dr. Schwartz's supervision. I thank all of my previous and present lab members, for their great support and teaching me in depth of using the analytical equipments. It is also a great experience to have the possibility of collaborative studies with other departments to broaden my knowledge. I also appreciate the great help and mentor from Dr. Failla and his lab for digestion and cell culture work.

Finally, I am indebted to my parents for their great support and encouragement for me to study abroad and finish my education.

vi VITA

January 27, 1982 ...... Born, Surabaya, Indonesia

June 1999 ...... Graduated from St.Ursula High School, Jakarta, Indonesia

June 2003 ...... :...... B.S. Food Science and Technology, The Ohio State University, Columbus, OH

2003-Present...... Graduate Research Associate, Food Science and Nutrition, The Ohio State University, Columbus, OH

PUBLICATIONS

Research Publications

1. Lee, J. H., Renita, M., St Martin, S. K., Schwartz, S. J., and Vodovotz, Y. "Isoflavone characterization and antioxidant activity of Ohio soybeans." Journal of Agricultural and Food Chemistry, 52, 9, 2647-2651, (2004).

Peer-reviewed Abstracts and Posters

1. Renita, M., Schwartz, S. J. "Thermal processing increases isomerization of lycopene precursors from tangerine tomatoes." IFT Annual Meeting. July 16-20, 2005. New Orleans, LO.

2. Renita, M. Failla, M. L., Schwartz, S. J. "In vitro digestion and Caco-2 cell uptake oflycopene precursors from tangerine tomatoes." International Symposium on Carotenoids. July 17-22, 2005. Edinburgh, Scotland.

FIELD OF STUDY

Major Field: Food Science and Nutrition

Vil TABLE OF CONTENTS

ABSTRACT ·------11

DEDICATION ·------v

ACKNOWLEDGMENTS ------v1

VITA ------Vll TABLE OF CONTENTS ______vm

LIST OF TABLES ------x

LIST OF FIGURES ·------Xl CHAPTER 1: REVIEW OF THE LITERATURE ______1

1.1. Carotenoids ______1 1.1.1. Structure, classifications, and nomenclature ______2 1.1.2. Carotenoids in food products ______5 1.1.3. Health benefits of carotenoids ______5

1.2 Biosynthesis of carotenoids ______8 1.2.1. Biosynthesis of carotenoids in choloroplast______8 1.2.2. Tomato as a model for the study of carotenoid biosynthesis ___ 14 1.2.3_ Recent advances of carotenoid biosynthesis studies ______16

1.3. Analytical methods ______18 1.3 .1. Isolation methods ______19 1.3.2. High performance liquid chromatography (HPLC) ______20 1.3.3. Identifications using several analytical techniques ______21

1.4. Bioaccessibility and bioavailability of carotenoids ______27 1.4_ l, Mechanisms of carotenoid bioacessibility, uptake, and absorption ·------28 1.4.2. In vitro digestion and Caco-2 cell cultures ______31 1.4.3. Methods assessing bioavailability in clinical studies ______34

Vlll 1.5. Effects of thermal processing on carotenoids ______35 1.5.1. Degradation and structural changes ______35 1.5.2. Thermal processing effects on bioavailability of carotenoids __ 37

1.6. Lycopene precursors ______39 1.6.1. Lycopene precursors in food products ______39 1.6.2. Lycopene precursors in vivo ______41 1.6.3. Lycopene precursors in cancer cells ______43

1. 7. Hypotheses and objectives ______44

1.8. List of references ______45

CHAPTER2: CHARACTERIZATION AND ISOMERIZATION OF LYCOPENE PRECURSORS DURING THERMAL PROCESSING ______58

2.1. Abstract ------59

2.2. Introduction------60

2.3. Materials and methods ------63 2.3.1. Materials, chemicals, and standards ______63 2.3.2. Thermal processing ______64 2.3.3. Carotenoid extraction______64 2.3.4. High Performance Liquid Chromatography (HPLC) analysis_64 2.3.5. Mass Spectrometry (MS) analysis ______65 2.3.6. Nuclear Magnetic Resonance (NMR) analysis ______65 2.3. 7. Data analysis------65 2.4. Results and discussion ______66 2.4.1. Characterization of the lycopene precursors ______66 2.4.2. Thermal processing effect ______72

2.5. Conclusions ______78

2. 6. List of references ______79

lX CHAPTER 3: DIGESTIVE STABILITY, MICELLARIZATION, AND UPTAKE OF L YCOPENE PRECURSORS BY IN VITRO DIGESTION AND CAC0-2 CELLS 83

3 .1. Abstract 84

3 .2. Introduction .______85 3 .3. Materials and methods ------88 3 .3 .1. Chemicals and standards------88 3 .3 .2. Sample preparation ______88 3. 3 .3. In vitro digestion ______8 8 3.3.4. Isolation of the aqueous/micellar fraction from digesta ______89 3.3.5. Caco-2 cell culture 90 3 .3.6. Cellular uptake of micellar carotenoids______91 3 .3. 7. Carotenoid extraction and analysis ______92 3 .3 .8. Data analysis ______92

3.4. Results and discussion 92 3.4.1. Composition of the processed and unprocessed tomatoes ______92 3 .4 .2. Digestive stability ______96 3 .4.3. Efficiency of micellarization______98 3 .4.4. Stability of mi cellar carotenoids ______100 3 .4 .5. Caco-2 cell uptake______101

3. 5. Conclusions ______103

3.6. List ofreferences 104

LIST OF REFERENCES 109

APPENDICES 122

APPEND IX A: Standard curves ______122 APPENDIX B: UV spectra of lycopene precursors and isomers______J24 APPENDIX C: Methods for thermal processing study ______127 APPENDIX D: NMR analysis for identification oflycopene pecursors ______128 APPENDIX E: IFT, New Orleans 2005, Abstract ______J29 APPENDIX F: International Carotenoid Symposium, Edinburgh 2005, Abstract ___ 130 APPENDIX G: Methods for in vitro digestion and Caco-2 cell study ______J31 APPENDIX H: Stability of micellar carotenoids______132

x LIST OF TABLES

1.1. Relative amount of lycopene precursors in various foods.

1.2. Amounts ofphytoene, phytofluene, ands-carotene in various human tissues.

2.1. Absorbance maxima of phytoene, phytofluene, s-carotene, and neurosporene, including their isomers present in the tomato juice used in this research.

3.1. Lycopene precursor profiles in the processed and unprocessed tangerine tomatoes before simulated digestion.

3.2. Cellular content of lycopene precursors in Caco-2 cells after 4 hr incubation.

H. Stability of micellar carotenoids diluted in 1:4 with basal DMEM (Test Media) after incubation for 4 and 20 hours in cell environment.

xi LIST OF FIGURES

1.1. Common carotenoids found in human plasma (Furr and Clark 1997).

1.2. (A). Numbering carbon atoms in acyclic (lycopene) and cyclic CP-carotene) carotenoids. (B). The major end groups of carotenoids: acyclic 'I' -group characteristic of lycopene and acyclic group p,E,y,K,cp, X· (C). Specific end groups ofxanthophylls from algae: allenic group, with two double bonds at the carbon atom 7 and acetylene group, with triple bond between carbon 7 and 8 (Ladygin 2000).

1.3. Conversion of P-carotene to retinol, retinyl esters, and retinoic acids (Parker 1996).

1.4. Phosphoglyceraldehyde-pyruvate pathway to synthesize IDP (Ladygin 2000).

1.5. Acetate-mevalonate pathway to synthesize IDP (Ladygin 2000).

1.6. The synthesis of phytoene in chloroplasts from condensation of 2 GGPP (Ladygin 2000).

1. 7. The synthesis of acyclic and cyclic carotenoids (Bramley 2002).

1.8. Synthesis ofxanthophyll carotenoids (Fraser and Bramley 2004).

1.9. Proposed biosynthesis of carotenoids in tangerine tomatoes (Lycopersicon esculentum var. Tangella) (Fraser and Bramley 2004).

1.10. UV spectra of P-carotene isomers recorded using photo diode array (PDA) (Lacker, Strohschein et al. 1999).

1.11. High performance liquid chromatography (HPLC) analysis of a mixture of different carotenoids using a C30 column coupled with atmospheric pressure chemical ionization ionization mass spectrometry (APCI-MS) with positive ionization and selective ion monitoring (SIM) mode (Lacker, Strohschein et al. 1999).

1.12. P-carotene bioacessibility, uptake, and distribution in the human body (Parker 1996).

xii 2.1. Proposed biosynthesis of carotenoids in tangerine tomatoes (Lycopersicon esculentum var. tangella) (Fraser and Bramley 2004)

2.2. Comparison of unprocessed with processed tangerine tomatoes at 250F for 30 min.

2.3. Representative mass spectrometry (MS) chromatograms of the carotenoids phytoene, phytofluene, s-carotene, and neurosporene.

2.4. Total carotenoid concentration of lycopene precursors after thermal processing at different temperature and time.

2.5. Relative percent of isomerization for lycopene precursors after thermal processing.

3 .1. Proposed biosynthesis of carotenoids in tangerine tomatoes (Lycopersicon esculentum var. tangella) (Fraser and Bramley 2004)

3.2. Representative HPLC-PDA chromatograms of lycopene precursors present in tangerine tomatoes.

3.3. Digestive stability of lycopene precursors during simulated in vitro digestion.

3.4. Micellarization efficiency oflycopene precursors during simulated in vitro digestion.

3.5. Caco-2 cellular uptake oflycopene precursors after 4 hr incubation.

A.I. Standard curves of (15Z)-phytoene and (15,9'Z)-phytofluene used for quantification of phytoene, phytofluene, and their isomers in the tomato juice, in vitro digestion and Caco-2 study.

A.2. Standard curves of (9 ,9' Z)-s-carotene and (E)-neurosporene used for quantification of s-carotene, neurosporene, and their isomers in the tomato juice, in vitro digestion and Caco-2 study.

B.1. Representative UV spectra of (15Z)-phytoene (A), (Z)-phytoene isomer (B), and (E)-phytoene (C), from tomato juice after thermally processed at 250°F for 30 mm.

B.2. Representative UV spectra of (15,9'Z)-phytofluene (A) and (E)-phytofluene (B), from tomato juice after thermally processed at 250°F for 30 min.

B. 3. Representative UV spectra of (9,9'Z)-s-carotene (A) and (9Z)-s-carotene (B), from tomato juice after thermally processed at 250°F for 30 min.

Xlll B. 4. Representative UV spectra of (7,9,9'Z)-neurosporene (A), (Z)-neurosporene isomer, and (E)-neurosporene (C), from tomato juice after thermally processed at 250°F for 30 min.

C. Schematic methods of thermal processing study described in chapter 2.

D. Proton NMR of the olefinic region of (15 Z)-phytoene (A) and (9,9'Z )-s-carotene (B).

G. Schematic methods of in vitro digestion and Caco-2 cell study described in chapter 3.

xiv CHAPTER!

REVIEW OF THE LITERATURE

1.1. Carotenoids

Carotenoids are one of the most important pigment groups in plant foods that play specific biological roles in human health. They give bright yellow, orange, and red color occurring in photosynthetic tissues. Carotenoids are ubiquitous in nature and more than 700 carotenoids have been reported, but only approximately 60 carotenoids are commonly consumed in the diet and 20 have been detected in the human body (1,2). The principal carotenoids found circulating in the blood stream and deposited in human tissues are P-carotene, a-carotene, lycopene, lutein, zeaxanthin, canthaxanthin and P cryptoxanthin (Figure 1.1 ).

In plants, most carotenoids are located in the chloroplasts among the thylakoid membranes within the photosynthetic pigment-protein complex (3,4). However, in nonphotosynthetic organisms, carotenoids are found primarily in chromoplasts. Specific carotenoids such as lycopene have its final form as crystals which are present in the chromoplast (5). P·Carotene

HO P-Cryptoxanthln Lyoopen•

H H

HO MO ZHx1nthln Lut.in

Canth1xanthln

Figure 1.1. Common carotenoids found in human plasma (2).

1.1.1. Structure, classification, and nomenclature

Carotenoids are included in the group of tetraterpenoids. Each carotenoid

contains a core, a chain of conjugated double bonds known as the chromophore in the

center of the molecules, and end groups (6). The chromophore is responsible for the light

absorption characteristics. Each of these double bonds can exist in a (Z) or (E)-

configuration, influencing the wavelength and intensity of light absorption (7). Because

of these conjugated double bonds, carotenoids have typical chemical and physical

properties such as isomerization, stability to heat, sensitivity to light, oxygen, and acids.

Most carotenoids can be described by the general formula C4oHs60n. Where n is

from 0-6. Based on the International Union of Pure and Applied Chemistry (IUPAC)

regulation, carbon atoms are numbered starting from the end groups to the core of the molecule (8). The numbering of the molecule from the end to the center is from 1 to 15

and then the additional methyl group is numbered from 16 to 20 (Figure 1.2.A). The 2 symmetrical part is numbered with a ' symbol from 1' to 20' (8). Each carotenoid is

divided into two halves and each half is designated with a Greek letter that describes the

end groups (Figure 1.2.B). The (R, S) naming is used to show conventional chirality.

Rather than to use these nomenclature regulations, trivial names are commonly used that

are derived from the isolation source, such as carotene from carrots and zeaxanthin from

Zea mays. The trivial name P-carotene is commonly used rather than p,p-carotene and

lycopene is used rather than \jl,\jl-Carotene (8-10).

Carotenoids are then divided into two groups: acyclic and cyclic carotenoids.

Acyclic carotenoids start from phytoene and continue along the biosynthetic pathway to

lycopene. The lycopene is then further cyclized to monocyclic y-carotene or bicyclic a

carotene and P-carotene. These cyclic carotenoids are further hydroxylated to the xanthophylls that contain oxygen, such as lutein, zeaxanthin, or P-cryptoxanthin. Other

classifications are the allenic group which has two double bonds at carbon atom 7 such

as neoxanthin and acetylene group which has triple bond between carbon atom 7 and 8

such as alloxanthin (Figure 1.2.C) ( 6).

3 17 ll l9 7 ll 15 16' ""' ft"q 'l\\l l'I "q 16 I 7 10 ll ~ 1'4 lO' Lyc:openc (a) 19 l7 3' 2' 2 17' 3 20'

~-Carotene

17 16 17 16 17 11 :(](. l~R ~R 4 s 11 1lA4 18 16 2 4 ' \jl ~ e (b) 16 17 16 16 16 CH2R 17~ R 17~18 11:61~ 6 1 I :Qt.. 2V4 3 18 ~ s 4 3 ~184 4 y 'IC cp x )<'.'.,•'')..... ~ (c) ~OH - HO

Figure 1.2. (A). Numbering carbon atoms in acyclic (lycopene) and cyclic ([3-carotene) carotenoids. (B).

The major end groups of carotenoids: acyclic 'I' -group characteristic of lycopene and cyclic group

[3,E,y,K,cp, X· (C) Specific end groups of xanthophylls from algae: allenic group, with two double bonds at the carbon atom 7, and acetylene group, with triple bond between carbon 7 and 8 (6).

4 1.1.2. Carotenoids in food products

Carotenoids function as accessory light-harvesting pigments, to provide energy transfer to chlorophylls, to maintain the structural integrity of the photosynthetic apparatus, and as a photoprotective compound against harmful oxygen species in plants

(11 ). Several common carotenoids in plant tissue are P-carotene and a-carotene in carrots, pumpkin, squash, and sweet potatoes; lycopene in tomatoes and watermelon; lutein and zeaxanthin in green leafy vegetables; canthaxanthin in mushroom; and capsaxanthin in pepper. Carotenoids are introduced into humans and animals through dietary intake of vegetables and fruits. Astaxanthin can be found in marine animals such as in salmon, trout, and crustaceae. In addition, supplementation of carotenoids to animal feed contributes to the color of animal tissues. For example, red carotenoids in bird's plumage Rupicola rupicola and flamingo and yellow carotenoids in egg yolks and chicken (1 ).

Carotenoids are commonly employed as colorants in the food industry (9). P-

Carotene, P-apo-8'carotenal, and canthaxanthin have been synthesized and used in the food industry as colorants in many food products such as margarine, ice cream, cheese, juices, etc. (1). Many of these carotenoids are available as dietary supplements, such as lycopene, lutein, and P-carotene, because of their promising health benefits.

1.1.3. Health benefits of carotenoids

It is widely known that carotenoids are associated with many health benefits and may play a specific biological role in the human body (12). The implications of carotenoids for human health were initiated when carotenoids were found as precursors 5 of vitamin A and retinoids. However, up until recent years, there are many other health benefits that can be obtained from the consumption of dietary carotenoids. For example, carotenoids exhibit antioxidant activity, as immunoenhancers, or inhibitors of mutagenesis and transformation of premalignant lesions. Increased dietary intake of carotenoids is associated with decreased risk of macular degeneration and cataracts, cancer, and cardiovascular diseases (9).

The main function of carotenoids in human health is the conversion to vitamin A

(retinol). If the carotenoid contains an unsubstituted P-ionone ring with a polyene side chain of at least 11 carbon atoms, it can be cleaved enzymatically to vitamin A (2). The carotenoid bioconversion is defined as the proportion of bioavailable carotene converted to retinol (9). Provitamin A carotenoids are converted to retinol by the action of 15, 15 ' - monooxygenase (Figure 1.3). Retinol is essential for vision, as it must be available in the retina as a precursor of (1 lZ)-retinal for the regeneration of rhodopsin, which is the photosensitive pigment. Approximately 50 carotenoids are found to have provitamin A activity. The major carotenoids that are widely studied for this purpose are P-carotene and a-carotene, since P-carotene is the most potent carotenoids that exhibits vitamin A activity because of its symmetrical structure yielding two molecules of retinol for each

P-carotene molecule (13,14).

6 J..,.,..r

Figure 1.3. Conversion of ~-carotene to retinol, retinyl esters, and retinoic acids (14).

Lutein and zeaxanthin have a specific biological role with their presence in the macular pigment of the human eye ( 15). The concentration of these carotenoids varies depending on the distance from the center of the eye. By preventing light-initiated oxidative damage to the retina and retinal pigment epithelium, these carotenoids may protect against age-related macular degeneration diseases (AMD) (15, 16). Khachik et al.

(17) also proposed that (3R, 3 'R, 6'R)-lutein and (3R, 3 'R)-zeaxanthin, which are the main carotenoids in the macula, may be interconverted via their oxidation products to protect the macula against bright light and prevent AMD.

Another important carotenoid is lycopene, mainly from tomatoes. It has efficient antioxidant activity that quenches highly reactive singlet oxygen ten times higher than tocopherol and twice that of ~-carotene (18). This antioxidant function is associated with 7 lowering DNA damage, inhibiting malignant transformation, and reducing biological

oxidative damage of proteins, lipids and other cell components in vitro and in vivo (5).

Lycopene from tomato products has been widely recognized as the main carotenoid associated with high anticarcinogenic activity against prostate cancer (19-22). In addition, serum level of lycopene has been inversely correlated with cancer of the oral cavity and pharynx, esophagus (5), stomach, colon, rectum, digestive tract (24), pancreas, bladder (25), and breast (23-26).

1.2. Biosynthesis of carotenoids

1.2.1. Biosynthesis of carotenoids in chloroplasts

Carotenoids and its xanthopyll derivatives are synthesized only in the chloroplasts of algae and higher plants that have two photosystems and oxygenic photosynthesis (6). In plants, carotenogenesis occurs within the plastids and by the enzymes that are nuclear encoded (27). The biosynthesis of carotenoids starts with the synthesis of isopentenyl diphosphate (IDP) from acetic acid through glucose derived from glyceraldehydes 3-phosphate and pyruvate (phosphoglyceraldehyde-pyruvate pathway) (Figure 1.4) or from mevalonic acid (acetate-mevalonate pathway) (Figure

1.5). The phosphoglyceraldehyde-pyruvate pathway synthesizes IDP as the precursor of isoprenoids in the chloroplast, while acetate-mevalonate pathway synthesizes IDP as the precursor of all isoprenoids in bacteria, higher plants, and algae in cytoplasm (6). Thus, in the absence of photosynthesis and glucose, carotenoids can also be synthesized through the acetate-mevalonate pathway. The pathway continues with several enzymatic

8 reactions which yield geranylgeranyl diphosphate (GGPP). This compound is the precursor of carotenoids and also a common precursor of the quinines, gibberellins, and phytol of chlorophylls (Figure 1.6) (28-30).

HH!O OH CH20P CHO CH3 0 HO H ---- f=o + H+OH ---- f=o H OH CH;PH CH20P COOH CH3ASC0A H OH Dihydroxyacetone Glyceraldehyde Pyruvate Acetyl-CoA CH20H phosphate 3-phosphate Glucose

CH3 TPP CH3 CH3 TPP*CH~H .) f=o ?" FTPP + HO H . HO_E~ COOH TPP OH H OH H=t=OH CH20P CH20P Pyruvate 1-Hydroxyethyl-thiamine Glyceraldehyde diphosp hate 3-phosphate D-1-deoxyxylolose- 5-phosphate

~10~ ~'°~ NADPH ~ T ox r T ox - ~O-P-P ~O-P-P 0 OH oH OH IDP DMADP 2-C Methyl-D-erythritol

Figure 1.4. Phosphoglyceraldehyde-pyruvate pathway to synthesize IDP (6).

9 ?i H3C-C-OH Acetate (C:!) /ATP, Mg++ / CoASH 0 H:JC-~- SCoA Acetyl-CoA (C:!)

H,CJ-CH,J-SCoA H3 C-~ I Acetacetyl-CoA (C4) ..):~2 / Sc;x>H ~hydroxy, ~-methylglutaryl-CoA (C6) [ Intennediates J I 2 NADPH

!IMs++ 0 0 CH3 HO-~-O-~-O-CH2-CH = C-CH3 Dimethylallyl diphosphate (C:i) I I - OH OH

Figure 1.5. Acetate-mevalonate pathway to synthesize IDP (6). 10 Isomerase ;(a) lppl,Ipp2 CytokiWns ------· ~o-P-P crtE IDP(C3) lYgpsl Monoterpeoes ------. ~O-P-P + ~o-p-p Geranyl dipbosphate (C10) crtE IDP lYgpsl Sesquiterpenes Squalene ------· ~O-P-P + ~o-P-P Sterols Farnesyl diphosphate (C1,) crtE IDP lYgpsl Diterpenes Phytol of chlorophylls ---- o-p-p QWnones Geranylgeranyl diphosphate (Cw) Gtbberellins

P-P-O (b) O-P-P H' H" Geranylgeranyl dipbospha.te (Cw) Geranylgeranyl diphosphate (Cw) ~~~Psy 0-P-P

Prephytoene dipbosphate (C4()}

H lcrtB,Psy •U :~ 'c ' ..... '"' a

a/1-trans-Phytoene (C4o)

15,15'-cis-Phytoene (C40)

Figure 1.6. The synthesis ofphytoene in chloroplast from condensation of2s GGPP (6).

11 The condensation of two GGPP molecules produces (15Z)-phytoene (Figure 1.6).

This step is catalyzed by a membrane-associated phytoene synthase (PSY) enzyme and becomes a rate-limiting step during the ripening stage of fruits, for example in tomatoes

(31 ). Phytoene is the key C40 precursors of carotenes and xanthophylls which has nine conjugated double bonds. Figure 1.7 and 1.8 shows the complete steps of carotenoid biosynthesis after the synthesis of phytoene. The first desaturation step results in phytofluene that has ten conjugated double bonds. The next acyclic carotene produced is s-carotene, which is the first to have yellow color in algae and higher plants. Further desaturation leads to neurosporene then to lycopene. With the cyclase enzymes, lycopene is then cyclized to form i::, a, ~-carotenes. Hydroxylation and epoxidation of carotenes finalize the biosynthesis to synthesize xanthophylls (6). Several components that are required during biosynthesis of carotenoids are NADP which participates in the conversion of phytoene to phytofluene, whereas FAD functions in the conversion of phytofluene to lycopene. MN++ also appears to be an absolute requirement for the conversion of phytoene to lycopene (32).

12 GGPP

l x2 Phytoene synthase

Pbytoene Phytom&e daatwra3e Phytofluene

l;.-Carotene {;,-Carotene desatvrase Neurosporene

Lycopene ~/ ~

6-c:uollme y.arotmlll /~· ~ CrtJ...b

Figure 1.7. The synthesis of acyclic and cyclic carotenoids (33).

13 Cl-llll"OlfllC ~lCRTV.

"~1Cl4')'ptoUll1'iaCRTL-11

~OH

H~- - - ~l . l . A

allldllc add l ~r uatllow OH

Figure 1.8. Synthesis ofxanthophyll carotenoids (34).

1.2.2. Tomato as a model/or the biosynthesis of carotenoids

Tomato fruits have been extensively studied as a model to reveal carotenoid biosynthesis. During ripening, the color, flavor, texture, and aroma of the fruit changes dramatically in a coordinated manner (31 ). The availability of large collections of mutant fruits is also another preference to use tomatoes for carotenoid biosynthesis study (27).

From the breaker stage to ripening of tomato fruits, there are color changes from green to orange-red because of genetic and enzymatic regulations (31 ). mRNA levels of enzymes that produce lycopene: phytoene synthase (PSY), phytoene desaturase (PDS), and s-carotene desaturase (ZDS), increase 10-20 fold during ripening. On the other hand, mRNA levels of lycopene cyclases (LCY-f3 and LCY-E) disappear. Increases in

14 Psy gene of PSY and Pds gene of PDS during the breaker stage are also controlled by transcriptional regulation (35). These genes in the tomato fruits are easily isolated, modified, and applied within other plants or other organisms.

Carotenogenesis in fruits and flowers is controlled by different regulatory mechanisms than in green tissues. Two genes are detected for PSY: Psyl encodes a fruit and flower-specific isoform and Psy2 encodes an isoform in green tissues, including mature green fruit and has no role in ripening tomato fruit (36). It is possible that the regulation of carotenoid biosynthesis in chloroplast tissues is different from that in chromoplast-containing fruit. This makes the tomato fruit as an ideal tissue to investigate carotenoid biosynthesis in relation to plastid differentiation (36). Psyl and PSY are preferred targets for gene manipulation because PSY catalyzes the first step in the carotenoid pathway (Figure 1.6). Constitutive expression of the cDNA of Psy in transgenic tomato plants has led to dwarfism, which is caused by redirecting GGPP from the gibberellin pathway into carotenoid synthesis (27).

Because of the large availability of tomatoes and that their gene regulation are easily controlled during the fruit development, unique tomato varities have been developed in order to increase the amount of carotenoid in the fruit. The genetic regulation of the enzymes plays a key role towards the development of each breed. For example, overexpression of LCY-P produces high P-carotene content, while downregulation of this enzyme produces old gold crimson (ogc) with high lycopene content. Delta mutation yields high 8-carotene variety which is affected from the overexpression of LCY-8 (27). Also available is the yellow tomato variety which is low

15 in carotenoids. The identification of the carotenoids in these breeds have been reported in previous studies (3 7).

Another interesting variety of tomato fruit is the novel tangerine tomato

(Lycopersicon esculentum var. tangella). This attention was initiated when a poly-(Z) isomer of lycopene was detected in this variety by Zechmeister in early 1940s (38). The genotype tangerine has undergone a recessive mutation at the t locus which produces a

3183 yellow-orange color tomato (39). There are two alleles of tangerine: t which carries a deletion in the 5' non-translated region and t"'ic which carries a deletion in the coding

3183 region. These two alleles contribute to a different composition of carotenoids; t contains equimolar amounts of (7,7,9',9'Z)-lycopene or prolycopene and s-carotene, while t"'ic accumulates predominantly s-carotene (40). Fruit of plants homozygous with respect to t can readily be identified by an intensification of the color of a cut surface when exposed to sunlight for a few minutes. The color change is the result of conversion of the prolycopene to isomers of a redder hue absorbing at longer wavelengths (39).

1.2.3. Recent advances of carotenoid biosynthesis studies

Recent carotenoid biosynthesis studies have used the mutant variety tangerine tomato in comparison with the regular red tomatoes because of their unique genetic regulation. Several studies have revealed that the common perception of (E) configuration synthesis from the acyclic carotenes to (E)-lycopene present in tomatoes is possibly not accurate (41,42). The carotenoids are synthesized first through the (Z) configuration and subsequently requires an enzyme isomerase to convert to (E)-lycopene

(6,7,27,42). Thus, the complete pathway requires the synthesis of (15Z)-phytoene,

16 (15,9'Z)-phytofluene, (9,9'Z)-s-carotene, (7,9,9'Z)-neurosporene, (7,7',9,9'Z)-lycopene, and isomerization to (E)-lycopene (Figure 1.9). Prolycopene transformation into cyclic carotenes cannot proceed in the dark, even in the presence of exogenous lycopene cyclase. The conversion to (E)-lycopene is necessary as a precursor for the synthesis of cyclic carotenoids ( 6).

prolycopne , JcRTISO?

Figure 1.9. Proposed biosynthesis of carotenoids in tangerine tomatoes (43).

The specific steps in carotenoid biosynthesis were discovered usmg genetic engineering application of tangerine tomato in combination with other species such as peppers, yellow daffodil flowers, C6D mutant Scenedesmus, algae Dunaliella bardawil, and Arabidopsis thaliana (6,7,27,41,42). It is known that in prokaryotes, PSY is

17 controlled by CrtB; while in eukaryotes, PSY is controlled by Psy. Both of these enzymatic reactions yield (15Z)-phytoene (6). The whole process of dehydrogenation from phytoene to lycopene in bacteria is catalyzed by a single enzyme PDS of Crtl, whereas in tomatoes, dehydrogenation is catalyzed by the two enzyme PDS of Pdsl and

ZDS of Zdsl (6). The end product of Crtl is (E)-lycopene whereas Pds and Zds yield prolycopene. PDS inserts (£)-double bonds at the 11, 11' position, where ZDS inserts

(Z)-double bonds at the 7,7' (Figure 1.9) (7). The double bond at 9,9' undergo specific

(E)-(Z) isomerization (44). When PDS and ZDS from Arabidopsis were co-expressed in

E. coli, phytoene was converted to prolycopene (27). Expression in E. coli of the tangerine gene product Crt!SO, in conjunction with Pdsl and Zdsl enabled the accumulation of (E)-lycopene, confirming that tangerine tomato encodes for a prolycopene isomerase enzyme CRTISO from Crt!SO gene (7,45). Currently, there is no clear agreement how CRTISO works but it is hypothesized to function in a complex with

ZDS (40). The presence of Crt!SO seems to be restricted to the cyanobacterial and plant lineage, which possesses a poly-(Z)-desaturation pathway mediated by both Pds and Zds.

In the absence of Crt!SO, carotenoid biosynthesis in the tangerine tomatoes is rate limiting (7).

1.3. Analytical methods

Numerous analytical methods have been reported in order to study carotenoids in different fields. The need to isolate these carotenoids from plants, animal, or human tissues would require different techniques to handle. Mainly the extraction of

18 carotenoids is based on their solubility such as non-polar organic solvents for acyclic carotenes and slightly polar solvents for xanthophylls. For raw materials or food samples, saponification is often applied to remove lipids, cholorophylls, or other impurities. Separation, identification, and quantification of each carotenoid contained in the extract generally requires chromatographic techniques.

1.3.1. Isolation methods

Isolation of carotenoids from the source fruit and vegetable are required in order to obtain standards for analytical methods. In addition, some synthetic carotenoid standards are also available. The isolation technique mainly uses open column chromatographic methods. Neutral alumina (activity III), silica, and a mixture of MgO and hyflosupercel (1: 1) with a mobile phase solvent of ether, hexane, acetone, dichloromethane, or toluene is widely used ( 46). However, other phases and mixtures can be applied as well such as CaC03, Ca(OH)2, ZnC03, sucrose, cellulose, starch, sephadex, and polyethylene. The isolated fraction can be further purified using thin layer chromatography (TLC) silica gel H plates in petroleum ether and benzene or with MgO kieselgur ( 1: 1) plates (4 7).

An alternative isolation method that results in highly purified standards uses semi-preparative HPLC columns. Generally the stationary phase for the semi-preparative column is the same material used in analytical HPLC separations but much bigger in diameter size i.e. 10-20 mm. The separation conditions are also similar by comparing the ratio in the regular column to the semi-preparative column. However, flow rate must be adjusted accordingly to achieve a similar elution order (48).

19 1.3.2. High performance liquid chromatography (HPLC)

Carotenoids are a class of molecules with diverse properties. The acyclic hydrocarbons are nonpolar, whereas xanthophylls are more polar. Molecular differences and geometrical and positional isomers are the common issues in separation of carotenoids from complex mixtures. Thus, this area has experienced a considerable research effort to optimize analytical methodology for carotenoid separations applied throughout different fields.

The application of HPLC has enabled separation of carotenoids from complex mixtures (49). The stationary phase development and the mobile phase delivery are the two subjects that receive much attention in carotenoid analysis. Sander et al. (50) have proposed three properties that are desirable for the stationary phase in carotenoid separation: enhanced shape recognition of isomers, high absolute retention, and silanol activity. The preparation of a column with these qualities requires silica substrate as well as bonding chemistry.

C1s stationary phases are most typically used for the analysis of different carotenoid structures. Different types of C1s materials have been extensively reviewed and optimized for separation prior to the identification of carotenoids in complex mixtures (51-53). However, the disadvantage of using the C1s stationary phase is the limited resolution of the geometric isomers of carotenoids (54).

The complexity of carotenoid extracts and the minor shape differences among carotenoid isomers make separation of individual species using a C18 column rather difficult (50). Normal phases such as alumina and calcium hydroxide can be used to separate the isomers of carotenoids, but these phases are tough to handle since it is

20 difficult to control the water content (54). Thus, the development of a C30 polymeric column is established. The use of a C30 polymeric surface, in conjuction with a moderate pore-size, and moderate surface area silica, results in a bonded phase with properties well suited for the separation of carotenoids ( 50).

Selectivity towards geometric isomers is a superior feature of a C30 polymeric stationary phase (54). The introduction of a C30 stationary phase for carotenoid separation offered a new type of stationary phase which is helpful for separation of polar and non-polar carotenoids geometric isomers and positional isomers such as ~-carotene

(55), a-carotene (56), lutein and zeaxanthin (57), and lycopene (54). Typical mobile phase that suits this stationary phase contains gradient of methanol, water, and methyl tert-butyl ether (MTBE) to increase the polarity (49,54). The potential application of the

C30 method with methanol, water and MTBE has been demonstrated by the separation of carotenoids from complex biological samples (58-60) and by the simultaneous metabolic profiling of several plant isoprenoids (49). This method leads to a good separation, short analysis times and results in sharp peaks, which facilitates good detector response (55).

1.3.3. Identifications ofcarotenoids using several analytical techniques

UV/Visible photodiode array detector (UV/Vis-PDA). The universal detection technique often used with HPLC is ultraviolet/visible (UVNis). Currently, it is widely used for rapid analysis of carotenoids. The PDA detector is the most developed UVNis method. It is able to measure absorption at a large number of wavelengths simultaneously giving full UV Nis spectral information. Since the observed spectrum

21 can describe the characteristic for any compound, this information can be used for identification of unknown samples (61 ).

Continuous photodiode array PDA detection assists in the identification and quantification of carotenoids after elution from the stationary phase ( 49). The spectral information that can be obtained from the PDA detector is helpful to identify some characteristics of the carotenoid geometrical isomers. It is widely known that appearance of (Z)-bond in most carotenoids leads to a hypsochromic shift of A.max compared to the

(E) (55). The (Z)-bond takes place around the center of the molecule so that the absorption maximum of the UV spectrum shifts to shorter wavelengths and an additional band appears at 340 nm. Lacker et al. (56) showed the UV spectra analysis of P-carotene geometrical isomers (Figure 1.10). The (15Z)-isomer is well characterized by the very high intensity of its (Z)-band. The intensity of (9, 13Z) has an intensity higher than that of the (9Z) isomer, but remarkably lower than that of (13Z)-P-carotene (55).

22 i---. ·--I _,I 11.0 min (15Z) I,,_,,, . 11.7 min (13Z) I : i i""""".. 1 I ; L...... 1 12.9 min (9,13Z)

I'' I ~·-·-. I '. I . I I ·-· -... 17.7 min 9(Z)

D 15.8 min (E)

300 400 500 600 700 nm

Figure 1.10. UV spectra of ~-carotene isomers recorded using photo diode array (PDA) detector (55).

Electrochemical coulometric array detector (ECD). Data collected from a PDA detector may be limited to its detection range. Thus this method may not be suitable for analysis of biological samples, which requires detection in minute amounts. The application of an electrochemical coulometric array detector (ECD) may be more preferable. This detector had been successfully applied to significantly increase sensitivity for both acyclic carotenes and oxygenated carotenoids (60,62). Small quantities of blood plasma, human prostate, cervical tissue and buccal mucosal cells had been analyzed using this methodology (60). From these studies, detection limits of ECD are determined to be 10 to 100 folds improvement relative to a conventional PDA

23 detector. However, application of this method is still limited with respect to the geometrical isomers of carotenoids because this instrument is not able to collect the UV spectra information of the carotenoids so that it is not able to distinguish the (Z)-band for the isomers.

Mass spectrometry (MS). The coupled hyphenation of chromatography to mass spectrometry (HPLC-MS) is an efficient solution for structural elucidation. But since carotenoids belong to a rather non-polar substance class and lack a site for protonation, their ionization for mass spectrometric detection is not simple. Fast atom bombardment

(63), electrospray ionization (ESI) (64), and particle beam (65) have been used in carotenoid detection, but these techniques are challenging. For ESI, good results can been obtained by the use of additional chemicals which facilitate the ionization process, such as halogen-containing eluents, silver salts, or ferrocene-based derivatives (55).

Atmospheric pressure chemical ionization (APCI) is a more prominent ionization method for mass spectrometry of carotenoids. Using this method, additional modification of the chromatographic eluent is not necessary in comparison to ESI and yields a robust method that is easily implemented (55). This method is suitable for purified solutions and also for complex samples, without extensive sample pretreatment.

The structural elucidation of (Z)/(E) isomers with mass spectrometry is not possible because of the identical fragmentation patterns. Thus, in carotenoid analysis, coupling a

PDA detector with MS is common and provides a better solution for carotenoid isomer identification.

24 Van Breeemen et al. (59) and Lacker et al. (56) have used APCI-MS and determined the detection limit for several carotenoids using either positive or negative ionization at 1-3.5 pmol. They also showed the use of RP-C30 HPLC coupled with

APCI-MS to separate and identify lycopene isomers in serum and prostate tissue. Thus, with the lower limit of detection and without the need of additional chemicals, this ionization method is more promising for carotenoids identification. Both positive and negative ionization modes of APCI have been successfully used for carotenoid identification. In addition, selected ion monitoring analysis (SIM) provides ranges of molecular weight that can be selected. This method is useful for carotenoid analysis which has ranges of molecular weights (55). Figure 1.11 shows a representative chromatogram of an HPLC C30 coupled with APCI-MS using positive ionization and

SIM mode for determining the molecular weight of carotenoids.

25 12.45 22.91 UV ':I rn I~ l 12.52 TIC 17.58 23.02 ':I ,.. 8~ A. 19.:,23 /\.. 8.72 m/z = 597 [M+H] astaxanthin ':I ~ ,,._ u <.> i 11.41 "8 i mlz =569 [M+H] zeaxanthin u ·;> ':I k. 1! 12.57 ':I m/z:::: 565 [M+H] ~ canthaxanthin 17.62

m/z = 551 [M+H] echinenone ':I ... ~ 23.02 1

:1, I ,~;,~ ~~~ ~~~~ I 1 1 1 I 1 ~~~~~~~~~' I I I :!:', I , 11 I~, 1 1 1 I , , , 1 1 1 1 1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 time(min)

Figure 1.11. HPLC analysis of a mixture of different carotenoids using a C30 column coupled with APCI·

MS with positive ionization and SIM mode (55).

Nuclear magnetic resonance (NMR). MS methods are not capable of identifying which geometrical isomers present in the samples. Thus, NMR analysis is more useful in this area. Hengartner et al. (66) showed the use of NMR for identification of lycopene isomers and some other tetrahydrolycopenes. It is difficult and expensive to couple

HPLC with NMR. Often NMR spectral analyses are conducted on isolated and purified carotenoids because of this issue. However, the use of HPLC-NMR coupling for

26 separation and identification of the isomers of P-carotene (67) and lutein-zeaxanthin (68) have been reported. The techniques used in these studies were Overhauser effect (NOE) difference experiments, double INDOR difference (DID) for simple and efficient method for the elucidation of carotenoids, homonuclear COSY, DEPT, heteronuclear 1H, 13C

COSY tuned to one and multiple bond C,H couplings, IR-double quantum 2D, and rotating-frame nuclear Overhauser 2D spectroscopy (ROESY) (66). A recent study has noted that CDCh is a rapid and gentle solvent to extract carotenoids for NMR analysis instead of using other organic solvents such as hexane-acetone (69).

1.4. Bioaccessibility and bioavailability of carotenoids

Bioavailability is defined as the fraction of an ingested nutrient that is available to the body through absorption for utilization in normal physiological functions and for metabolic processes (70). It is also defined by the US FDA as a rate and extent to which the active substances or theurapeutic moiety is absorbed from a drug product and becomes available at the site for action (71). Bioavailability of food constituents is a complex issue involving digestion, intestinal uptake and absorption, distribution and utilization by the tissues (72).

Bioavailabilty of carotenoids is one of the research area that is quite extensively studied. Poor understanding of food structure, complexity during digestion, and response variation among each individual are the main challenges that can be misinterpreted in this study (73). Understanding carotenoid bioavailability require several key issues: (A)

Biaccessibility of carotenoids, which is defined as amount of ingested carotenoids from

27 food that can be released into an absorbable form and available for absorption in the gut after digestion (73,74); and (B) Uptake, absorption, and distribution of carotenoids, which is how the carotenoids can be absorbed through the lumen and distributed to human body (73).

1.4.1. Mechanisms of carotenoid bioaccessibility, uptake, and absorption

A representative mechanistic scheme of carotenoid biaccessibility and absorption using ~-carotene is shown in Figure 1.12. Carotenoids are digested similar to cholesterol or products of triglyceride lipolysis. They are absorbed by duodenal mucosal cells by passive diffusion (75). The bioaccessibility study of carotenoids starts with digestion from the food matrix. This step requires the breakdown of the food matrix in order to release carotenoids because they are associated with protein in a variety of plant cell structures (76). The next step is the release of carotenoids into the lumen of the gastrointestinal tract (77). Gastric hydrolysis helps to partially release carotenoids and lipids from the food matrix.

28 I ~

Figure 1.12. !)-carotene bioaccessibility, uptake, absorption, and distribution in the human body (75).

The carotenoids will be dispersed into lipid droplets once they are released from the food matrix. Shearing forces from normal digestive tract motility helps to form a fine lipid emulsion consequent to the action of bile salts and pancreatic lipases. The emulsion structure consists of a triacylglycerol core surrounded by a mono-molecular layer of 29 partially digested proteins, polysaccharides, phospholipids and partially ionized fatty acids (78). The products of lipid digestion including the carotenoids are transferred from the emulsion particle to mixed bile salt micelles. Micellar capacity for carotenoids may differ by their structure or lipid composition (75). Non-digested lipid in the lumen is known to interfere with carotenoid absorption, especially to non-polar carotenoids (79).

The micelles penetrate through a series of water lamella adjacent to the microvillus surface, which form as barriers for the carotenoids to pass. Thus, the micelles serve as reservoirs for carotenoids and other lipids, which then move across the water layer down to the brush border membrane by passive diffusion (75). This step is rate limiting for absorption of lipids and carotenoids (80). The rate of diffusion is dependent on the concentration gradient between the micelle and the plasma membrane of the enterocyte (75).

Several studies have suggested that carotenoids have low capacity for micellar incorporation and intracellular translocation from the site of uptake to chylomicrons

(77). Carotenoids that are not incorporated into chylomicrons may be discarded into the lumen during normal turnover of the mucosa. Final chylomicrons in the golgi apparatus is secreted into intracellular space and by passage into the lymphatics for transport to the bloodstream (76). Structure of the carotenoid plays a key role to facilitate the incorporation of carotenoids into lymphatic lipoproteins (77). Hydrocarbon carotenoids which are located primarily in the core of the particle are transported in LDL (58-73%), whereas xanthopylls are distributed equally between HDL and LDL in human serum

(75). These distributions are not affected by high dietary intakes of carotenoids or

30 supplements because carotenoids provide a small fraction of the total mass in the lipoprotein particle (81 ).

1.4.2. In vitro digestion and Caco-2 cell cultures

In vitro digestion coupled with Caco-2 cell cultures have been extensively used as a model for studying the biaccessibility of drugs, minerals, or vitamins, such as iron

(82), phosphorus (83), and cholesterol (84). This technique is more preferred to give rapid analysis in large numbers and under controlled conditions compared to the in vivo studies (85). Recent studies have modified this technique in the field of carotenoids to examine the human intestinal carotenoid absorption. Lycopene, P-carotene, lutein, and zeaxanthin are the main carotenoids that have been analyzed using this methodology

(86-91 ). Bioaccessibility of carotenoids during in vitro digestion may be affected from its food sources including food matrix, particle size of the food, presence of fat, and low bile acid secretion (76).

The in vitro method to study the absorption of carotenoids starts with the digestion using chemical compounds simulating gastric and intestinal phase in the human body. The gastric phase includes HCl, pepsin, and bicarbonate, which are crucial for the digestion of lipophilic compounds such as carotenoids (86,92,93). Gastric acidity may help to improve absorption of several carotenoids. However, if the pH of intestine is too low, the solubilization of carotenoids into bile salt micelles may be decreased (94).

Bile and pancreatic enzymes (pancreatin and pancreatic lipase) are included during the simulated intestinal phase (75,76,86,87). According to previous studies, omission of these steps led towards the absence of carotenoids during digestion and micellarization.

31 Bile salts are also required for interaction and transport through the brush border membrane. It is added at 2.4 mg/ml which is similar to the bile salt level present in the small instestine during a fasted state (95). Studies by Garrett et al. (86) showed that elevated levels of bile beyond this concentration did not enhance carotenoid micellarization, thus the bile concentration used were already at its optimum level. They also reported that P-carotene and a-carotene micellar fractions were increased when the pancreatin level was increased; however, the enzyme did not alter the micellar fraction of lutein (87). Studies reported that 70-75% of P-carotene and a-carotene (86) and 75-

85% of lutein and zeaxanthin (91) were recovered during simulated in vitro digestion.

Many studies also include oil addition to the food that contains carotenoids. The purpose of oil addition is to aid simulated digestion. It is well-known that lipid can enhance the efficiency of solubilization of the lipophilic compounds. Lipids also help to stimulate the release of bile salts from the gall bladder, in addition to expanding the bile salt micelle size (77,78). Plasma and serum lycopene and P-carotene were enhanced with the presence of oil, suggesting that lipid may aid the absorption of these carotenoids

(96).

Micellarization of P-carotene, lutein, and zeaxanthin ranged from 15-50%, which is much higher than (E)-lycopene at 5% (86-91). The bioavailability of xanthophylls was greater than that of the acyclic carotenoids (9). It is known that the higher efficiency of micellarization of the carotenoids is due to the factors of limiting transfer from chloroplasts to oil droplets. Xanthophylls are located at the surface of oil droplets whereas the acyclic carotenes are in the deep surface, which is more difficult to facilitate transfer. Location is significant because surface components can be spontaneously 32 transferred from lipid droplets to mixed micelles, whereas the components in the core require digestion of triacylglycerol before transfer (97). The (Z)-isomers of the carotenoids are proposed to be more efficiently solubilized in lipophilic solutions, less likely to crystallize, and readily transported within cells or tissue matrix (98). This hypothesis agrees with results on the in vitro and Caco-2 study of prolycopene from tangerine tomatoes (99).

Coupling of in vitro digestion with Caco-2 human intestinal cells is a step to validate the bioavailability of carotenoids. These studies demonstrate how the carotenoids that are transferred to the micellar fraction are absorbed by the cells (87).

Caco-2 cells have similar biochemical and morphological properties of enterocytes. The cell differentiates at confluency into polarized cells with enterocyte-like characteristics, such as highly developed brush border for apical uptake and metabolism, tight junctions that minimize paracellular transport, and basolateral secretion of chylomicrons and

VLDL (100,101). The micellar fraction obtained from in vitro digestion has to be diluted before the application into the cells. Previous studies reported that digestates may be toxic to the cells. Thus, exposure of the diluted aqueous fraction can help to retain the morphology and biochemical integrity of the cells (102). The cellular content of lutein, a-carotene, and P-carotene after 6 hr accumulation in the Caco-2 were 76, 9.2, and 33 pmol/mg protein, respectively (102). Good correlations between carotenoid bioaccessibility and uptake in vitro and to that observed in vivo has made the Caco-2 technique popular for studies on structure-absorption relationships (85).

33 1.4.3. Methods assessing bioavailability in clinical studies.

Plasma carotenoids have been used predominantly as an indicator of

bioavailability in humans. It corresponds to the intake of carotenoids from a meal but

may not necessarily reflect the absorption, distribution to tissues, and excretion of

carotenoids (72). At steady state, plasma carotenoids accounted to approximately 1% of the total body content of carotenoids, whereas the highest concentration can be found in the liver (9).

To have a better estimate of bioavailability, concurrent assessment of changes in tissue and plasma concentrations of carotenoids in response to carotenoid intake has

been proposed (72). It is known that peak plasma concentration after ingestion of a

carotenoid is slower than the time required to maximize the plasma concentration of

triacyl glycerol or retinyl esters (103). Some carotenoids are also held in the enterocyte,

which are subsequently released following an additional meal. It is useful to examine the

total plasma maximum concentration because it represents the distribution of carotenoids

from chylomicron to the low-density lipoprotein (LDL) and high-density lipoprotein

(HDL). Carotenoids in the chylomicron fraction reach their maximum concentration at

about 6hr, followed by the LDL and HDL carotenoid concentration which reached a

maximum after 16-24 hr (104). Apolipoprotein B-48 in the triglyceride rich fraction

(TRL) is found to be the integral apolipoprotein in chylomicrons (105). On the other

hand, the postprandial increase of apolipoprotein B-100 was attributed to the

accumulation of hepatogenous very low density lipoprotein (VLDL) which is caused by

the saturation of lipoprotein lipase by chylomicrons (72).

34 1.5. Effects of thermal processing on carotenoids

Many food products derived from fresh fruits and vegetables have to undergo thermal processing due to commercial sterility and safety requirements. Industrial canning is the most common thermal sterilization method followed by bottling, spray drying, evaporation, etc. Such processing may alter the physical property of the fruits and vegetables. Taste and discoloration of post-processed products are also other issues to consider during processing in order to achieve consumer acceptance. The phytonutrient composition of fruits and vegetables may also be altered in this step.

Because of these concerns, many studies have been conducted to investigate the effects of thermal processing on carotenoid composition in fruits and vegetables.

1.5.1. Degradation and structural changes

Numerous studies have reported the effects of thermal processing on carotenoid content in various fruits and vegetables. There are some variations which show that each carotenoid may have different effects from one to another. Thus, separate studies would have to be conducted in order to know the effects of processing for a specific carotenoid.

Several steps such as unpeeling the skins and chopping materials before thermal processing can contribute to a better extractability of the carotenoids. There are numerous fruits and vegetables that contain carotenoids in the skins and chopping provides physical disruption of plant tissue or reducing cellular integrity ( 106). Graziani et al. (106) also reported that maximum carotenoid content such as lycopene was detected after 2hr heating for unpeeled samples at 100°C. This study showed that after moderate heat treatment, the carotenoids were more readily extracted which contributed

35 to the better extractability and higher content of carotenoids. It is also agreed in other

studies that thermal processing may help to disrupt the tissue matrix and denature the protein, favoring the dissociation between carotenoid and lipophilic protein that releases the carotenoids such that they are more readily extracted (107,108).

Another concern of thermal processing is degradation of phytonutrients. There are many conflicting studies that have been reported. This may be due to the differences in preparation or processing methodology. However, according to the more recent reports, it has been concluded that there were no significant changes of total carotenoid content during moderate thermal processing such as boiling, steaming, and microwaving.

However, degradation was noted in extensive and prolonged heating (37,106,109). The loss reported varied among each carotenoids, such as 26% for lycopene, 39% for ~ carotene, and about 50% for phytoene, whereas phytofluene was more stable compared to the other carotenoids.

The interesting issue within thermal processmg effect on carotenoids is the isomerization reactions which may alter the structural conformation of the main carotenoid from fresh materials. These changes may affect the biological properties of the carotenoids so that further understanding of these changes is important. These isomerization reactions were noted during early thermal processing studies where ~ carotene and its provitamin A activity were examined. Chandler and Schwartz (110) and

Lessin et al. (111) investigated that there were significant changes of (E)-~ -carotene to the (Z)-isomers. Other studies also reported significant isomerization of lutein during processing such as boiling, steaming, microwaving (109) and canning (57). The

36 isomerization observed ranged within 3-22% from broccoli, peas, spinach, com, and kale.

In contrast to the isomerization of P-carotene and lutein during thermal processing, there are conflicting reports on the isomerization of lycopene. Some have reported that degree of isomerization of (E) to (Z)-lycopene was correlated with the intensity and duration of heat processing and 20-30% (Z)-lycopene were formed within 1 hr when tomatoes were processed at 100°C (98,112). However, other studies reported that there was no significant thermal isomerization of lycopene found during processing

(37,109).

The difference of thermal processing effects towards each specific carotenoid was associated with the differences in molecular shape that determines hydrophobicity, crystalline state, and solubility which influence the thermal stability. Carotenoids such as lycopene are biosynthesized in a crystalline structure and posses a longer chromophore than P-carotene or lutein. Crystal lycopene also remains in the thylakoid membrane after transformation of the chloroplast to chromoplast (37). The presence of lipid that will slowly solubilize lycopene during heating and subsequently allow isomerzation reaction to occur may account for the observed difference in the extent of isomerization. Since there were variations of isomerization reactions towards different carotenoids, an in depth study of the processing effect must be conducted for each specific carotenoid.

1.5.2. Thermal processing effects on bioavailability of carotenoids

Many studies have reported that thermal processing to produce tomato-based products may enhance the bioavailability of carotenoids because of the better

37 extractability achieved. Higher total carotenoid concentration in serum was found when subjects consumed processed tomato-based products such as tomato soup and spaghetti sauce (113,114). Gartner et al. (114) also observed that tomato paste and juice were more bioavailable than fresh tomatoes. Processing with oil increased bioavailability of the carotenoids because of the better solubility of the carotenoids in the lipophilic phase

(115,116). A recommendation was proposed that greater health benefits can be attained when incorporating at least one serving per day of tomato-based product (24).

Giovannucci et al. (117) reported that intake of lycopene from tomato-products such as tomato fruit, sauce, pizza, and juice, was associated with a reduced risk of prostate cancer. Consumption of 40 mg lycopene from tomato products was found to decrease

LDL oxidation (118). Thermal processing also enhanced the nutritional value of tomatoes by increasing the total antioxidant activity (119).

The bioavailability of the geometrical isomers of carotenoids that are induced during thermal processing has become the subject of several recent investigations. For example, the (Z)-isomers of P-carotene and a-carotene were reported to be less bioavailable. These isomers were less efficienctly converted to vitamin A than the (£) form (120). It was proposed that the (Z)-P-carotene was less preferable as a substrate for the cleavage enzyme in vitamin A conversion. In contrast to the (Z)-isomers of P carotene, more than 50% of (Z)-lycopene isomers were detected in human serum and tissue (121,122). These lycopene isomers in processed tomato products were hypothesized to be better absorbed and more bioavailable than the (E)-lycopene from fresh tomatoes (98). Boileau et al. (123) showed that the (Z)-isomers of lycopene were also more bioavailable than (E)-lycopene in the mesenteric lymph of ferrets. It was 38 hypothesized that the (Z)-lycopene isomers were less prone to crystallization due to their geometric configurations, more soluble in lipophilic solutions and bile acid micelles, and thus, they are more easily incorporated into chylomicrons and tissues (124). It was also found in recent human clinical studies that (Z)-lycopene isomers from tangerine tomatoes were more efficiently absorbed in comparison to (E)-lycopene (125,126).

Accumulation of (13Z)-isomers of lutein and zeaxanthin found in plasma of macaques and squirrel monkeys relative to the isomeric composition of the diet showed that the

(Z)-isomers of these xanthophylls may be more bioavailable than (E) (127).

1.6. Lycopene precursors

Although lycopene is the predominant carotenoid pigment biosynthesized in tomatoes, a number of biosynthetic precursors are also present in the tomato tissue. Four lycopene precursors have been identified towards the biosynthesis of lycopene. These four carotenoids: (15Z)-phytoene, (15,9'Z)-phytofluene, (9,9'Z)-~-carotene, and

(7,9,9'Z)-neurosporene follow in the pathway of lycopene production (6). They are synthesized in the (Z)-configurations followed by synthesis of (7,7',9,9'Z )-lycopene

(Figure 1.9), which then converts to (E)-lycopene by the enzyme carotenoid isomerase.

These lycopene precursors are the focus of the study in this thesis.

1.6.1. Lycopene precursors in food products

Previous studies have reported the presence of lycopene precursors in various food products, mainly on phytoene, phytofluene, and ~-carotene (Table 1.1) (106,128-

39 131). However, only few reports were found that specified the amount of neurosporene present in food. This is probably due to the lack of an authentic standard required to quantify this compound and the minute amount of this carotenoid generally present in fruits and vegetables. The highest concentrations of these caroteonids reported were in red tomatoes and tomato-based products. Interestingly, the quantities of the lycopene precursors in yellow tomatoes reported in this thesis study were higher compared to red tomatoes or other various food. The composition was 9.81 mg phytoene, 1.69 mg phytofluene, l.52mg ~-carotene, and 0.57mg neurosporene per 100 gr of tomato juice

(Chapter 2).

Methods for the analysis and identification of the lycopene precursors have applied C18 HPLC-PDA and EI-MS. Thus, separations achieved were not able to distinguish between the (E) and (Z)-form of the lycopene precursors. Many of the reports stated the total concentration of both isomers (106, 128-131) or quantified either (E) or

(Z) present in food (131 ). In addition, there are some older reports which mentioned that the lycopene precursors were present in the (£)-form (128). However, recently it was revealed from the studies on carotenoid biosynthesis that these carotenoids are present in the (Z)-form (6).

40 Concentration (mg/100 gr) of carotenoid lycopene precursor in various food Food Phytoene Phytofluene !'.;-carotene Neurosporene

A. Fresh Fruits and Vei:;etables Apricot 0.06-1.050 0.025-0.45 0.038 n/a Peach 0.012-0.18 0.002-0.070 0.002-0.014 n/a Cantaloupe 0.038 0.044 0.394 n/a Pink grapefruit 0.015 0.013 0.138 0.380 Tomatoes 1.860-6.850 0.820-6.330 0.21-1.58 1.110 Nectarine 0.400 0.122 0.009 n/a Papaya 0.260-0.680 0.260-0.440 0.170-0.190 0.050 Clementine 0.070 0.070 0.050 n/a Orange 0.080 0.040 0.020 0.010 Lemon 0.010 0.002 0.003 n/a Squash 0.550 0.200 0.720 0.140

B. Tomato-based uroducts Spaghetti sauce 2.770 1.560 0.340 3.150 Tomato paste 8.360 3.630 0.840 6.950 Tomato puree 2.400 1.080 0.250 2.110 Tomato sauce 2.950 1.270 0.290 0.480 Catsup 3.390 1.540 0.330 2.630 Tomato juice 1.900 0.830 0.180 1.23 Tomato soup 1.720 0.720 0.170 n/a

Table 1.1. Relative amount of lycopene precursors in various foods (106,128-131).

1.6.2. Lycopene precursors in vivo

Interestingly, several studies have reported the levels of lycopene precursors in human tissues (Table 1.2) (132), plasma (133), buccal mucosal cells (BMC) (72), serum, and milk (72,132-134). Similar to the food products, neurosporene was able to be detected in these tissues; however, they were not quantified due to the lack of standard availability. It is important to note that phytoene has a high concentration relative to other tomato carotenoids in lung tissue, which may be due to a preferential uptake of this

41 compound from serum by this tissue. High concentration of s-carotene and phytofluene in breast tissue relative to other carotenoids was also noted (132,134).

Average concentration (ng/g) of lycopene precursors in human tissues and skin

Organs Phytoene Phytofluene l;;-carotene

Liver 168 261 150 Lung 1275 195 25 Breast 69 416 734 Cervix n/a 106 57 Prostate 45 201 187 Colon 70 116 134 Skin 65 15 13

Table 1.2. Amounts ofphytoene, phytofluene, and l;;-carotene in various human tissues (132).

In a study of carotenoid identification and quantification in human serum and breast milk by Khacik et al. (134), phytoene ands-carotene had a higher ratio of serum per L of milk than phytofluene. The concentrations in human serum were 1.9-3.3 nmol/L phytoene, 6.7-13.8 nmol/L phytofluene, and 1.1-10.8 nmol/L s-carotene. The concentrations in human milk were 0.1 nmol/L phytoene, 0.4-1.8 nmol/L phytofluene, and 0.05-0.5 nmol/L s-carotene (134).

The carotenoids phytoene, phytofluene, and s-carotene were also detected in plasma and BMC (72,133). Plasma levels of these lycopene precursors at baseline were

0.097-0.109 µmol/L phytoene, 0.269-0.302 µmol/L phytofluene, and 0.123-0.142

µmol/L s-carotene. Phytoene and phytofluene were mainly associated with LDL 65-75% while only 17-22% in VLDL and 5-13% in HDL. These distributions were altered after

42 the subjects consumed tomato-based products. The carotenoid precursors decreased in

LDL and increased in VLDL and HDL with VLDL>HDL (133). They also found that these observed that phytofluene was more efficiently absorbed than lycopene and perhaps metabolized differently than lycopene. This observation was also confirmed by

Richelle et al. (135). Similar to plasma levels, phytofluene in BMC increased after ingestion of tomato-based products at 0.98 µgig protein. In contrast, phytoene was not detected in BMC although it was detected in plasma (72). It is widely known that phytoene is unstable when purified and thus its biological activity is difficult to be examined (136,137).

1.6.3. Lycopene precursors in cancer cells

A few studies have examined the biological activity of lycopene precursors in cancer cells. Sharoni et al. (138) have reported that lycopene precursors may have a synergistic effect with other carotenoids such as lycopene to inhibit cancer cells growth.

Nara et al. (139) have observed that in human promyelocytic leukemia (HL-60) cells, s- carotene was the most effective compound in cell growth inhibition, followed by phytofluene. However, phytoene did not inhibit the growth at the concentration tested. In addition, s-carotene and phytofluene were the most susceptible to oxidation which were lost within 5 hr incubation. The oxidation mixture caused inhibition at 44.9% of s- carotene and 68.4% of phytofluene, whereas phytoene did not have any effects. The inhibition growth of phytofluene and s-carotene were enhanced by oxidation of the carotenoids before the addition of the culture medium. These two carotenoid precursors also induced apoptosis in HL-60 cells. Other studies showed that phytofluene and s- 43 carotene significantly reduced cell viabilltiy of two prostate cancer cell line to 60-70%

PC-3 and DU145 (140).

Possible oxidation products of phytoene, phytofluene, and s-carotene are geranylgeranoic acid (GGA) and (4,5)-didehydro-GGA. The later compound was demonstrated to prevent primary hepatoma in humans and apoptosis in HuH-7 human hepatoma cells (141,142). These oxidation products also were also found to have similar functions as retinoic acid in the activation of RAR and RXR nuclear hormone receptors

(143).

1.7. Hypothesis and objectives

Studies have revealed that lycopene in human plasma and tissue consists of more than 50% of the (Z)-lycopene (121,124). Extensive thermal processing especially in the presence of lipid may enhance isomerization towards the (Z)-lycopene, which is proposed to be more bioavailable than (E)-lycopene (98,124). Results indicating greater absorption of (Z)-lycopene isomers in clinical studies correlate with studies accessing their biaccessibility using in vitro digestion coupled with Caco-2 cells (99).

The tangerine tomato variety consists of higher amounts of the four lycopene precursors: phytoene, phytofluene, s-carotene, and neurosporene. These carotenoids are prevalent in the (Z)-form (6,7,27,42). The lycopene precursors have received much attention in studies of carotenoid biosynthesis and they are present in human tissues.

However, to our knowledge, there are limited studies examining the effects of thermal

44 processmg on these carotenoids, specifically on isomerization reactions and bioavailability of (Z)-lycopene precursors.

We hypothesize that lycopene precursors in tangerine tomato will isomerizes during typical thermal treatments and these carotenoids will be readily absorped.

Therefore, the objectives of this study were to investigate the effects of thermal processing on carotenoid lycopene precursors: phytoene, phytofluene, s-carotene, and neurosporene as follows;

1. To monitor if isomerization reactions and degradation occur during processing

when subjected to thermal processing temperature and time.

2. To examine the biaccessibility (digestive stability, micellarization, and cell

uptake) using in vitro digestion and Caco-2 cell cultures.

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57 CHAPTER2

CHARACTERIZATION AND ISOMERIZATION OF

LYCOPENEPRECURSORS

DURING THERMAL PROCESSING

Marjory Renita and Steven J. Schwartz

Department of Food Science and Technology, The Ohio State University, 110 Parker

Food Science and Technology Building, 2015 Fyffe Ct., Columbus, OH 43210

Acknowledgments

The authors thank Dr. Qingguo Tian for assistance with MS analysis and Stefano Tiziani for NMR analysis 2.1. Abstract

Recent evidence has shown that thermal processing may increase isomerization of carotenoids such as ~-carotene or lutein, whereas lycopene is relatively stable. The predominant form of these carotenoids in nature is the (£)-configuration, while thermal processing may induce (Z)-isomerization. Tangerine tomato variety consists of higher amounts of the four lycopene precursors: phytoene, phytofluene, ~-carotene, and neurosporene, in which these carotenoids are prevalent in the (Z)-form. The effects of thermal processing to these precursors have not been studied. Tangerine tomatoes were processed in cans at 100, 150, 200, and 250F for 30, 60, and 90 min. Each can was extracted and the carotenoids were analyzed using C30 High Performance Liquid

Chromatography (HPLC) coupled with photodiode array (PDA). Further identification of the carotenoids was achieved using their PDA-UV-spectra, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and by comparison of analytical data from previously reported studies. Temperature showed a significant correlation to the increased isomerization for all carotenoids (P<0.05). Further processing at 200F or

250F was found to have a notable effect on isomerization (P<0.01). Processing time did not show significant differences in isomerization for these precursors. However, a moderate to strong correlation of isomerization of these carotenoids was observed when processing for 60 min was plotted with temperature (P<0.05). The presence of these unique carotenoid isomers in processed tomatoes suggests that further studies to monitor these compounds in foods and biological tissues may be warranted.

59 2.2. Introduction

Consumption of dietary carotenoids from tomato products have been inversely

associated with the prevention of prostate cancer and heart diseases (1 ). The health

benefits gained from intake of tomato fruit and its processed products have been

confirmed in many clinical studies (2,3). Lycopene is the natural pigment that is responsible for the red color in tomatoes and many other fruits. It has a potential antioxidant activity to quench singlet oxygen which is helpful to prevent cardiovascular diseases (4 ).

Many studies have shown that processing to produce tomato-based products such as tomato paste, ketchup, sauces, etc. may alter the configuration of the predominant

(all-E)-lycopene from fresh tomatoes to the (Z)-configuration (5,6). (E)-lycopene usually remains stable during processing except if it is processed with the addition of oil or extreme heat that can induce isomerization reactions (7). Thermal treatment during food processing may also affect the configuration of several other carotenoids such as (E)-P carotene from carrots (8,9) or lutein and zeaxanthin from spinach and kale (10) to their

(Z)-form.

Studies have revealed that processmg may enhance the bioavailability of lycopene (6). This is due to the break down of cell walls to release lycopene from the tomato tissue matrix and perhaps because of isomerization towards the (Z)-form. (Z) lycopene is found to be better absorbed or metabolized in the body compared to the (all

E)-lycopene (6,11,12). More than 50% of the (Z)-lycopene isomers are circulated in the plasma, serum, and tissues, which may indicate that the (Z)-configuration of lycopene is the most stable form in the human body equilibrium (13). Unlu et al. (14) have observed

60 that the (7,7',9,9'Z)-lycopene (prolycopene) in tangerine tomato variety may be isomerized to (E) by extensive processing and that prolycopene was more bioavailable than the (E) configuration.

Interestingly, the unique tangerine tomato variety consists of distinct carotenoid composition compared to typical red tomatoes. This variety contains high levels of the four lycopene precursors: (15Z)-phytoene, (15,9'Z)-phytofluene, (9,9'Z)-s-carotene, and

(7,9,9'Z)-neurosporene (Figure 2.1). This tomato is also prevalent in either (7,7',9,9'Z) lycopene or (9,9'Z)-s-carotene (15, 16). All of these carotenoids are predominant in the

(Z)-form instead of (E). Studies have shown that lycopene precursors may have anticarcinogenic activity in concert with lycopene (17,18). In addition, these lycopene precursors have received much attention in studies of carotenoid biosynthesis (16,19).

Many studies have examined carotenoid biosynthesis from their genetic and enzymatic regulation, and by genetic engineering using tangerine tomatoes and other organisms such as E. coli, Arabidopsis thaliana, etc (19,20).

61 ••

prolycoptnt , ~ i<.·RTISO?

Figure 2.1. Proposed biosynthesis of carotenoids in tangerine tomatoes (21 ).

To our knowledge, there are no studies exammmg the effects of thermal processing on lycopene precursors from tangerine tomatoes. Thus, the objective of this study was to examine the effects of thermal processing on the carotenoids: phytoene, phytofluene, s-carotene, and neurosporene. Isomerization reactions and degradation as a function of processing temperature and time were also monitored in this study.

62 2.3. Materials and methods

2.3.1. Materials, chemicals, and standards

Yellow Holland tomatoes were purchased from local grocery stores. All reagents and materials were HPLC and ACS grade purchased from Sigma Chemical Co. (St.

Louis, MO) and Fisher Scientific Co. (Fairlawn, NJ USA).

Standards of (15Z)-phytoene, (15,9'Z)-phytofluene, (9,9'Z)-~-carotene, and (all

E)-neurosporene were isolated from the tomatoes used in this study using magnesium oxide and celite (1: 1) open column chromatography as described in previous reported methods (22,23) and purified using a semi-preparative C30 column 10 µm, 250 x 20 mm l.D. (Waters, Milford, MA).

2.3.2. Thermal processing

A complete scheme of the thermal processing study is shown in appendix C. The tomatoes were blended to juice using a food blender and placed into retort cans (7.5 cm in diameter and 11 cm in height). The cans were processed at The Ohio State University pilot plant using a still retort at 100, 150, and 200F with the retort lid opened. At 30 min intervals, three cans were removed from the retort and the end of processing time was 90 min. The same still retort was used but the lid was closed to achieve 250F. At this temperature, three cans of juice were processed separately for each processing time of

30, 60, and 90 min. The procedure was repeated two additional times using a fresh batch of tomatoes for each experiment.

63 2.3.3. Carotenoid extraction

The extraction method of Nguyen and Schwartz (7) was followed. All procedures were performed under subdued light to avoid carotenoid isomerization or degradation.

Tomato fruits were cut and blended to juice. Approximately 5 g of the sample was extracted with 50ml of methanol, 4 g of celite as filtering aid, and 1 g of calcium carbonate to raise the pH. The mixture was homogenized for one minute and passed through a vacuum filtration unit with #1 (top) and #42 (bottom) Whatman filter papers

(Fisher Scientific, Fairlawn, NJ). The filtrant was extracted using 50 ml 1: 1 hexane/acetone solution, homogenized, and filtered similarly as the previous step. This procedure was repeated three times or until the residual color of the filtrant was pale white. The hexane/acetone and methanol filtrate were transferred to a separatory funnel.

Water was added to induce phase separation and the hydrophilic layer was discarded.

The lipophilic layer was passed through glass wool and anhydrous sodium sulfate crystals to completely remove residual water. The extract was collected in a volumetric flask and topped off to 100 ml with hexane. Three ml of the extract was transferred to an

11 ml vial and dried under nitrogen. Samples were placed in a -20°C freezer under dark.

2.3.4. High Perfomance Liquid Chromatography (HPLC) analysis

A reverse-phase HPLC-PDA method modified from a previously reported study

(24) was used to determine the carotenoid profiles in the tomatoes. The system consists of a Waters 2695 separation module and 996 PDA (Milford, MA). Analytical polymeric

YMC-C30 column (3 µm, 250 x 4.60 mm I.D.) and Novapak-C1s guard column (4 µm,

30 x 4.6 mm ID) were used for separation (Waters, Milford, MA). Carotenoid profiles

64 were carried out using the mobile phase of methanol and MTBE for 60 min at lml/min.

An isocratic method was run at 95% methanol and 5% MTBE for 5 min and changed to a gradient run to 70% MTBE for 40 min. The last composition was switched linearly to its original condition for 15 min. The injection volume used was 50µ1. Prior to HPLC analysis, the dried extract was resolubilized in 1 ml of methyl t-butyl ether (MTBE) and

1 ml of methanol. The sample was filtered with a 0.2 µm syringe filter.

2.3.5. Mass Spectrometry (MS) analysis

The HPLC system was coupled to the triple quadrupole mass spectrometer

(Waters-Micromass, Manchester, UK). The optimum positive-ion APCI conditions for carotenoid analysis included a corona current of 10.2 µA, a cone voltage of 35 V, RF-1 of 50 V, and desolvation gas temperature of 500 °C at 3.5 L/min. Molecular cations of the carotenoid were detected using selected-ion monitoring (SIM) with a dwell time of

100 ms per ion.

2.3.6. Nuclear Magnetic Resonance (NMR) analysis

A preliminary study for identification of the lycopene precursors was conducted using NMR. Details of this study are shown in Appendix D.

2.3. 7. Data analysis

All data were analyzed using SPSS 13.0 (SPSS Inc, Chicago, IL). Descriptive statistics including mean, standard deviation, and standard error were used for total carotenoids and percent isomerization at each temperature and time. Mean values for 65 total carotenoids and percent isomerization generated from measurement at each processing temperature regardless of the processing time were compared using multiple variate analysis (MANOVA). Similarly, mean values obtained from processed samples according to processing time regardless of the temperature were compared. Differences were considered significant at P<0.05.

2.4. Results and discussion

2.4.1. Characterization of the lycopene precursors

Analytical methods such as reversed-phase C30 High Performance Liquid

Chromatography coupled with a photodiode array detector (HPLC-PDA) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) were used to achieve the separation and identification of the carotenoids including their isomers. The characterization of the lycopene precursors in this study was achieved by comparing their HPLC PDA-UV spectra to previously reported studies (25). In addition, APCI-MS was used as further identification of the lycopene precursors and isomers. Both analytical methods have been utilized as rapid methods for identification and quantification of carotenoids, especially to examine the isomers of lycopene, P-carotene, and lutein (26).

Reversed phase HPLC has been well-known as a rapid and accurate method for separation and quantification of carotenoids (26). The use of C1s or Spherisorb ODS columns has been the preferred method for rapid analysis of carotenoids (27,28).

However, after the development of the C30 column which exhibits higher selectivity than

66 C1s, a C30 method has become more preferable to separate the carotenoid isomers

(29,30). Previous studies have used C30 columns with methanol and MTBE as the mobile phase for separation of a-carotene, P-carotene, lycopene, lutein, and zeaxanthin isomers in many fruits and vegetables or from human blood serum, plasma, and tissues (2, 7).

The method used in this study was adopted from the method of Fraser et al (31 ).

The gradient time was modified in order to achieve better separation of lycopene precursor isomers. The PDA detector coupled to the HPLC system was also beneficial in this study since each lycopene precursor has different wavelength maxima. The chromatograms were monitored at wavelengths of 285nm for phytoene, 348nm for phytofluene, 400nm for ~-carotene, and 440nm for neurosporene (Figure 2.2). The absorbance maxima for each lycopene precursor are reported in Table 2.1 and the UV spectra are shown in appendix B, which corresponded to previous reported studies (25).

67 A 2

B 4

AU . --··-··· ...... c 6

i 10 D 81i Ii ,.li . .II f" .111 9 ..II

...... -···-·-···- ...... -......

0 Minutes 60

----Processed - . - . - . - . - . Unprocessed

Figure 2.2. Comparison of unprocessed tangerine tomatoes with the processed tomatoes at 250F for 30 min. A) Chromatograms at 285nm, B) 348nm, C) 400nm, D) 440nm. 1) phytoene isomer, 2) (15Z)-

phytoene, 3) (all-E)-phytoene, 4) (15,9'Z)-phytofluene, 5) (all-E)-phytofluene, 6) (9,9'Z)-s-carotene, 7)

(9Z)-s-carotene, 8) (7,9,9'Z)-neurosporene, 9) neurosporene isomer, 10) (all-E)-neurosporene. All peaks were identified using their UV, mass spectra, and by comparison to analytical data from previously reported studies (25).

68 Absorbance maxima (nm} 1 Absorbance ratio 2 Max. 1 Max.2 Max.3 Max. 3/Max. 2

(15Z )-phytoene 276 285 298 0.72 (E )-phytoene 276 285 298 0.72

(15,9'Z)-phytofluene 331 348 366 0.87 (E )-phytofluene 331 348 366 0.87

(9,9'Z)-l;-carotene 376 400 426 1.03 (9Z)-l;-carotene 376 400 426 1.03

(7,9,9'Z)-neurosporene 409 433 460 0.35 (Z)-neurosporene 415 439 469 0.69 (E )-neurosporene 415 440 468 0.96

1 Identification is based on PDA-UV spectra from HPLC, APCI-MS, and by comparison of previously reported studies (25). 2 Ratio between heights of maxima 3 and 2 using the minimum between both peaks as base-line (25).

Table 2.1. Absorbance maxima of phytoene, phytofluene, s-carotene, and neurosporene, including their isomers present in the tomato juice in this study.

In addition to the application of the C30 column as a good separation method, identification of the carotenoid isomers is another challenging problem in this field.

Many studies have coupled the use of HPLC with MS to identify carotenoid isomers of identical molecular mass. Many MS ionization techniques have been tried such as fast atom bombardment (F AB) (32) and electrospray ionization (ESI) (33). However, carotenoids are highly non-polar compounds which make the protonation step of these methods fairly difficult. APCI-MS is a more prominent method which allows for the ionization of the non-polar compounds (26).

In this study, a positive ionization mode was used to ionize the lycopene precursors (Figure 2.3). Selected ion monitoring (SIM) mode was used according to the

69 mass of each positive ionization of the carotenoids and their isomers. Phytoene was detected at m/z 545, phytofluene at m/z 543, ~-carotene at m/z 541, and neurosporene at mlz 539. This detection method correspond with the previous reported studies which used positive APCI mode to identify isomers of ~-carotene (26,34), isomers of lycopene in the human serum and prostate tissue (35) and isomers of lutein and zeaxanthin from spinach (10). Although positive ion mode gave better results for identification of the lycopene precursors, negative ion mode was also tried in this study with success and could be another technique to be used for other carotenoids (data not shown).

Although the MS method was useful to identify the carotenoid isomers, NMR analysis is more helpful to distinguish which isomers are present in the samples. It is difficult and expensive to couple HPLC with NMR. Often NMR analyses are conducted separately from HPLC because of this issue. In this study, a preliminary NMR analysis was conducted for further identification of the lycopene precursors. Details of this study are shown in appendix D.

70 285 nm 1.13e4

AP+

AP+ mlz 543 3.34e6

I I I I I I I I I 1 r Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

1001 %1

25'-r,...,~~~~~~~~~~~~~~~"-"''-,-'1-'~~~~~~~~~~~~~~~~~~

440 nm 1; rn~

AP+ m/z 539 '::'-.-,...,~~~,~~~~,~~~~, ~~~~,~~~~~~~~.;-,...,.+-~,.'h~~~~,~~~~,~~~~':~· 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Figure 2.3. Representative MS chromatograms of phytoene at 285 nm wavelength maxima and m/z 545, phytofluene at 348 nm and m/z 543, s-carotene at 400nm and m/z 541, and neurosporene at 440nm and m/z 539. 71 Using these analytical methods and by comparison to previous literature (25), there were many isomers of lycopene precursors able to be separated, identified, and quantified. For the purpose of this study, only those isomers detected at significant level were chosen as the following: Phytoene- unidentified (Z), (15Z), and (all-£);

Phytofluene- (15,9'Z) and (all-£); s-carotene- (9,9'Z) and (9Z); Neurosporene- (7,9,9'Z), unidentified (Z), and (all-£).

The carotenoid composition m the tomato juice used in this study before processing was as followed: total phytoene at 9.81 mg/lOOgr tomatoes, phytofluene at

1.69 mg/lOOgr, s-carotene at l.52mg/100gr, and neurosporene at 0.57mg/100gr.

According to this analysis, there is an indication that the tomato used in this study was not yellow tomatoes as stated. Yellow bred tomatoes have lower carotenoid content than other varieties (36). On the other hand, the tomatoes used in this study contained high concentration of the lycopene precursors, which is an indication that the variety was possibly tangerine bred. The content of the these lycopene precursors present in the tomato juice in this study was relatively high compared to the red tomato juice with 1.9 mg phytoene, 0.83 mg phytofluene, 0.18 mg s-carotene, 1.23 mg neurosporene, per 100 gr juice; and in various fruits such as peach, apricot, grapefruit, cantaloupe, or bell pepper with 0.12-0.68 mg phytoene, 0.02-0.44 mg phytofluene, and 0.02-1.58 mg s carotene, per 100 gr edible food (28,37-40). Not many studies have reported the concentration of neurosporene possibly due to the lack of available standard and minute amount of this carotenoid present in most fruits and vegetables.

72 2.4.2. Thermal processing effect

All of the processed tomato juice appeared in good condition and acceptable to be consumed. However, we did not continue to process the juice at 250F for 90 min for the second and third batch because this treatment caused discoloration and produced off note aroma and taste to the juice. Thus, the statistical analysis for this point was not included.

In this study, we found that the carotenoids phytoene, phytofluene, s-carotene, and neurosprorene were relatively stable or did not undergo degradation during thermal processing. This result conflicts with a previous study that reported the degradation of phytoene during thermal processing (3 7). Processing may also affect the extractability of the carotenoid lycopene precursors from the tomatoes. Plotting carotenoid concentration and processing temperature, a bell shape curve was noted for all carotenoids comparing processed to unprocessed samples (Figure 2.4). This observation indicated better extractability of the carotenoids when heated. Total phytoene and phytofluene increased when tomatoes were processed at 150F, while total s-carotene and neurosporene increased at 100 and 150F. After reaching the highest concentration peak at 100 or 150F, the total concentration of all lycopene precursors tended to decrease at 200 and 250F; however, not less than the unprocessed sample, confirming that little or no degradration occurred. This result indicated that once the tomatoes were processed, the carotenoid extractability was enhanced regardless of the temperature and time. Although these trends were detected for all lycopene precursors, the changes were not statistically significant. This may be due to the biological variability in carotenoid content of each batch of tomatoes used in the study.

73 Phytoene Phytofluene

14.0 2.5

13.0

Qi 12.0 2.0 u :a.. 11.0 Cl .. ------··--·~- Q 10.0 1.5 .....Q '' ~-1 Ci 9.0 .§. 8.0 1.0 c:: 50 100 150 200 250 300 0 50 100 150 200 250 300 0 :;::; I! c:: i;-carotene Neurosporene -Cl) u c:: 3.0 1.2 0 u "C 2.5 1.0 ·5 c:: 2.0 0.8 ----, ~.. '. Cll ~ ~--: .. u 1.5 ---~~ ' ...... 0.6 s0 I- 1.0 0.4

0.5 0.2 50 100 150 200 250 300 0 50 100 150 200 250 300

Temperature (F)

··················· 30 min - · · - · · 60 min 90min

Figure 2.4. Total carotenoid concentration of lycopene precursors after thermal processing at different processing temperature and time. Unprocessed samples were used as initial points at 75F 0 min. There is a trend of better extractability of the carotenoids at lOOF and 150F; however, these changes were not significant (P>0.05).

74 The results of this study were consistent with others. In general there were no significant changes of carotenoid concentration during thermal processing, especially that these carotenoids did not undergo degradation at typical food processing conditions

(28,37-40). However, these results did not correlate with the results from Graziani et al.

(37) that showed the decrease amount of phytoene after thermal processing. The difference is possibly due to the difference in the thermal processing methodology, in which they processed the tomatoes at an unusually longer heating time (100°C for 9hr).

In addition, the better extractability trend that occurred during the thermal processing also correlated with other studies (37,38,40). This result may lead to the possibility of better bioavailability of these carotenoids when thermal treatment is applied. It is known that thermal processing may help to disrupt the tomato tissue matrix, which releases the carotenoids such that they are more readily extracted and available for absorption in the body. Studies have reported that thermal processing may enhance the bioavailability of lycopene ( 6). Thus, this study also showed that thermal processing may enhance the bioavailability of lycopene precursors. However, in vitro and clinical studies will be required to confirm this hypothesis.

There were some differences notable when the HPLC chromatograms of the unprocessed samples were compared to the processed samples for each lycopene precursor (Figure 2.2). Several isomer peaks were increased after the tomatoes were processed such as (E)-phytoene (peak 3), (E)-phytofluene (peak 5), (9Z)-~-carotene

(peak 7), and (E)-neurosporene (peak 10). The isomerization trends of lycopene precursors during thermal processing were mainly towards the (E)-form in addition to other (Z)-isomers. It was anticipated that these isomerization reaction may occur because

75 the starting carotenoids were present in the (Z)-configuration and thus, isomerization may take place towards the (E)-form. This result confirms the observation that extensive thermal processing may induce isomerization towards (E)-lycopene from prolycopene

(14,41).

Percent isomerization was calculated based on the concentration changes of the increased isomers after thermal processing compared to the unprocessed samples.

Statistical analysis verified that processing temperature had a significant correlation with increased isomerization for each individual lycopene precursor (P<0.01) (Figure 2.5). In addition, processing at 200F or 250F was found to have a significant effect on isomerization (P<0.05). The isomerization of phytoene and s-carotene at 250F was different in comparison to lOOF, however, not to 150 and 200 F. Phytofluene isomerization, however, was significantly different at 200 and 250F and neurosporene was significantly different at 250F compared to other processing temperatures. These results suggested that phytoene and s-carotene may be less influenced by thermal processing towards isomerization.

76 Phytofluene 15 70

60 12 ------t-----2 ------• R = 0.71 - 50 ---;-,,.___:_ ------·--- 9

3 10

c: 0 0 ; 100 150 200 250 300 100 150 200 250 300 ('CS ·;:N Cl> E 1;-carotene Neurosporene 0 !!!. 140 250 :.e0 120 200 +------100 ------+-2 R = 0.65 - 80 150+------

40 50 ------20 -..------~~~------R2 = 0.74-

0 100 150 200 250 300 100 120 140 160 180 200 220 240 260

Temperature (F)

·················· 30 min - · · - · · 60 min ---90min

Figure 2.5. Relative percentage of isomerization after thermal processing to lycopene precursors. Percent isomerization was calculated from the concentration differences of the isomers at each processing temperature and time from the unprocessed sample. Correlation significance was determined at P

** Showed significant regression for each processing time (P>0.05)

In contrast to the processing temperature, overall processing time (regardless of the temperature) did not show any difference in isomerization for these precursors.

However, the regression plot of carotenoid concentration vs temperature for 60 min

77 processing time was significant (P<0.05) for all lycopene precursors although they were only at moderate correlations, suggesting that this processing time may be favorable for

2 2 2 the isomerization reaction (R phytoene = 0.45, R phytofluene = 0.71, R s-carotene =

2 0.49, R neurosporene = 0.65). Regression at 30 min processing time was only significant (P<0.05) for phytofluene (R2 = 0.92) and s-carotene (R2 = 0.43) while

2 regression at 90 min was only significant (P<0.05) for neurosporene (R = 0.74). These results suggested that perhaps the isomerization reactions for phytofluene ands-carotene occurred early once thermal treatment is applied, while neurosporene was isomerized at longer time and phytoene had the least effect towards isomerization. From these results, the trend observed for the isomerization reaction during thermal processing was neurosporene > s-carotene > phytofluene > phytoene. Most likely, this trend is due to the number of conjugated double bond within the carotenoid, which favors the isomerization reaction. In this study, phytofluene significantly isomerized and correlated to the extent of processing temperature and time followed by neurosporene. Further studies will be required to examine in depth the reaction kinetics of these isomerization reactions.

2.5. Conclusions

In this study, we have demonstrated the use of HPLC-PDA coupled with APCI-

MS and NMR for separation, identification, and quantification of lycopene precursors: phytoene, phytofluene, s-carotene, and neurosporene. Thermal processing induced isomerization of these carotenoids. Temperature was found to have a significant 78 correlation on isomerization reactions for all lycopene precursors. Thermal treatments at

200 and 250F had the greatest influence on isomerization. Processing time did not have a significant effect on isomerization of the carotenoids except for phytofluene. Overall, this study demonstrated that lycopene precursors: phytoene, phytofluene, s-carotene, and neurosporene are readily isomerized during thermal processing. The presence of these unique isomeric carotenoids in the processed tomatoes suggests that further studies to monitor these compounds in food and biological tissues may be warranted, given that these isomerization reactions may influence the absorption and bioavilability of these carotenoids.

2.6. List of references

1. Krinsky, N., Mayne, S. & Sies, H. (2004) Carotenoids in Health and Diseases. Marcel Dekker, New York, NY.

2. Hadley, C. W., Clinton, S. K. & Schwartz, S. J. (2003) The consumption of processed tomato products enhances plasma lycopene concentrations in association with a reduced lipoprotein sensitivity to oxidative damage. J Nutr 133: 727-732.

3. Giovannucci, E. (2002) A review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. Exp Biol Med (Maywood) 227: 852-859.

4. Agarwal, S. & Rao, A. V. (2000) Tomato lycopene and its role in human health and chronic diseases. CMAJ 163: 739-744.

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6. Stahl, W. & Sies, H. (1992) Uptake oflycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J Nutr 122: 2161- 2166.

79 7. Nguyen, M. L. & Schwartz, S. J. (1998) Lycopene stability during food processing. Proc Soc Exp Biol Med 218: 101-105.

8. Lessin, W. J., Catigani, G. L. & Schwartz, S. J. (1997) Quantification of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables. J Agric Food Chem 45: 3728-3732.

9. Chandler, L.A. & Schwartz, S. J. (1988) Isomerization and losses oftrans-b-carotene in sweet potatoes as affected by processing treatments. J Agric Food Chem 36: 129-133.

10. Updike, A. A. & Schwartz, S. J. (2003) Thermal processing of vegetables increases cis isomers oflutein and zeaxanthin. J Agric Food Chem 51: 6184-6190.

11. Gartner, C., Stahl, W. & Sies, H. (1997) Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am J Clin Nutr 66: 116-122.

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13. Clinton, S. K., Emenhiser, C., Schwartz, S. J., Bostwick, D. G., Williams, A. W., Moore, B. J. & Erdman, J. W., Jr. (1996) cis-trans lycopene isomers, carotenoids, and retinol in the human prostate. Cancer Epidemiol Biomarkers Prev 5: 823-833.

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15. Giuliano, G., Giliberto, L. & Rosati, C. (2002) Carotenoid isomerase: a tale oflight and isomers. Trends Plant Sci 7: 427-429.

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80 20. Isaacson, T., Ronen, G., Zamir, D. & Hirschberg, J. (2002) Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants. Plant Cell 14: 333-342.

21. Fraser, P. D. & Bramley, P. M. (2004) Review: The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research 43: 228-265.

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23. Rodriguez-Amaya, D. B. (2001) A guide to carotenoid analysis in food. ILSI Press, Washington, D.C. USA.

24. Fraser, P. D., Pinto, M., Holloway, D. E. & Bramley, P. M. (2000) Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant Journal 24: 551-558.

25. Breitenbach, J. & Sandmann, G. (2005) zeta-Carotene cis isomers as products and substrates in the plant poly-cis carotenoid biosynthetic pathway to lycopene. Planta 220: 785-793.

26. Lacker, T., Strohschein, S. & Albert, K. (1999) Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection. J Chromatogr A 854: 37-44.

27. Garcia-Plazaola, J. I. & Becerril, J.M. (1999) A rapid high-performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols. Phytochem Anal 10: 307- 313.

28. Muller, H. (1997) Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Z Lebensm Unters Forsch A 204: 88-94.

29. Sander, L. C., Epler-Sharpless, K., Craft, N. E. & Wise, S. A. (1994) Development of engineered stationary phases for the separation of carotenoid isomers. Anal Chem 66: 1667-1674.

30. Breitenbach, J., Braun, G., Steiger, S. & Sandmann, G. (2001) Chromatographic performance on a C30-bonded stationary phase of monohydroxycarotenoids with variable chain length or degree of desaturation and of lycopene isomers synthesized by various carotene desaturases. J Chromatogr A 936: 59-69.

81 31. Fraser, P. D., Pinto, M. E., Holloway, D. E. & Bramley, P. M. (2000) Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J 24: 551-558.

32. van Breemen, R. B., Schmitz, H. H. & Schwartz, S. J. (1993) Continuous-flow fast atom bombardment liquid chromatography/mass soectrometry of carotenoids. Anal Chem 65: 965-969.

33. van Breemen, R. B. (1995) Electrospray liquid chromatography-mass spectrometry of carotenoids. Anal Chem 67: 2004-2009.

34. Hagiwara, T., Yasuno, T., Funayama, K. & Suzuki, S. (1998) Determination of lycopene, alpha-carotene and beta-carotene in serum by liquid chromatography atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring. J Chromatogr B Biomed Sci Appl 708: 67-73.

35. van Breemen, R. B., Xu, X., Viana, M. A., Chen, L., Stacewicz-Sapuntzakis, M., Duncan, C., Bowen, P. E. & Sharifi, R. (2002) Liquid chromatography-mass spectrometry of cis- and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce. J Agric Food Chem 50: 2214-2219.

36. Nguyen, M. L., Francis, D. & Schwartz, S. J. (2001) Thermal isomerisation susceptibility of carotenoids in different tomato varieties. J Sci Food Agric 81: 910-91 7.

37. Graziani, G., Pernice, R., Lanzuise, S., Vitaglione, P., Anese, M. & Fogliano, V. (2003) Effect of peeling and heating on carotenoid content and antioxidant activity of tomato and tomato-virgin olive oil systems. Eur Food Res Tech 216: 116-121.

38. Khachik, F., Beecher, G. R. & Lusby, W.R. (1989) Separation, Identification, and Quantification of the major carotenoids in extracts of apricots, peaches, cantaloupe, and pink grapefruit by liquid chromatography. J Agric Food Chem 37: 1485-1473.

39. Takeoka, G. R., Dao, L., Flessa, S., Gillespie, D. M., Jewell, W. T., Huebner, B., Bertow, D. & Eheler, S. E. (2001) Processing effects on lycopene content and antioxidant activity of tomatoes. J Agric Food Chem 49: 3713-3717.

40. Tonucci, L. H., Holden, J., Beecher, G. R., Khachik, F., Davis, C. S. & Mulokozi, G. (1995) Carotenoid content of thermally processed tomato-based products. J Agric Food Chem 43: 579-586.

41. Ishida, B. K., Burri, B. J. & Chapman, M. H. (2005) Effects of processing on lycopene-isomer content of tangerine tomato. In: 229th ACS National Meeting, San Diego, CA, USA.

82 CHAPTER3

DIGESTIVE STABILITY, MICELLARIZATION, AND UPTAKE OF

LYCOPENE PRECURSORS BY IN VITRO DIGESTION AND CAC0-2 CELLS

1 1 2 Marjory Renita , Steven J. Schwartz , and Mark L. Failla

1 Department of Food Science and Technology, The Ohio State University, 110 Parker

Food Science and Technology Building, 2015 Fyffe Ct., Columbus, OH 43210

2 Department of Human Nutrition, The Ohio State University, 325 Campbell Hall, 1787

Neil Avenue, Columbus, OH 43210

83 3.1. Abstract

Uncooked tangerine tomatoes with added vegetable oil (3%) were subjected to in vitro digestion coupled with a Caco-2 human intestinal cell model to determine the digestive stability, micellarization, and cellular accumulation of the lycopene precursors: phytoene, phytofluene, s-carotene, and neurosporene. Tomatoes were also processed at

121°C for 30 min to monitor thermal effects on stability and bioaccessibility of these carotenoids. The extracts were analyzed usmg C30 High Performance Liquid

Chromatography coupled with Photodiode Array detector (HPLC-PDA). Thermal processing of tangerine tomatoes increased extractability of total phytoene and phytofluene. Processing enhanced the digestive stability of total s-carotenes and several isomers of phytofluene and neurosporene, but not total phytoene. Several isomerization reactions were detected during thermal processing and digestion to some lycopene precursors. Recovery of these compounds after simulated digestion exceeded 50% and

12-45% was micellarized. Processing also enhanced micellarization of total phytofluene, s-carotene, and neurosporene. Once micellarized, the carotenoids were relatively stable

(> 75% recovery) in culture medium for 4 h. Caco-2 cells accumulated 5-17% of micellarized carotenoids after incubation at 37°C for 4h. Cell accumulation of total phytoene, phytofluene, and s-carotene of the processed samples were greater than the unprocessed samples. However, some isomers of phytofluene and neurosporene were accumulated at higher level in the unprocessed samples. Results of this study suggested that these lycopene precursors are readily digested and absorbed by the human intestinal system.

84 3.2. Introduction

The health promoting activities of carotenoids have been widely investigated.

Consumption of dietary carotenoids from plant products has been associated with the prevention of several age-related chronic diseases (1). For example; ingestion of lutein zeaxanthin has been reported to prevent age-related macular degeneration disease

(AMD) (2). Consumption of tomato products which are rich in lycopene during dietary intervention trials was found to decrease the oxidative DNA damage and serum prostate specific antigen (PSA) levels in prostate cancer patients (3,4). Results also have shown that processing of tomatoes may improve the availability of lycopene for absorption

(5,6).

Apart from the studies of the carotenoids' health benefits, agricultural plant breeders also have focused on carotenoid biosynthesis and are now developing unique plant varieties in order to deliver carotenoids in food products. The regulation of carotenoid biosynthesis has been extensively evaluated in tomato fruits (7). These studies have revealed that the biosynthetic pathway of carotenoids proceeds via acyclic carotene precursors mainly in the (Z)-configurations which is further isomerized to (E) lycopene in red tomatoes and further cyclized to (£)-~-carotene and hydroxylated to (E) lutein (8,9).

The development of tangerine tomato has provided interesting insights for the biosynthetic pathway of carotenoids in higher plants (9,10). This variety contains high levels of lycopene precursors (15Z)-phytoene, (15,9'Z)-phytofluene, (9,9'Z)-~-carotene,

85 and (7,9,9'Z)-neurosporene, compared to typical red tomatoes (9) (Figure 3.1).

Tangerine tomatoes are also prevalent in either (9,9'Z)-s-carotene or (7,7',9,9'Z) lycopene (11). The (Z)-configurations of the lycopene precursors predominate in this fruit, in contrast to the (£)-configuration of lycopene in red tomatoes. These carotenoids have been detected in other fruits and vegetables in smaller quantities such as in bell peppers, apricot, peach, cantaloupe, and pink grapefruit (12,13).

Figure 3.1. Proposed biosynthesis ofcarotenoids in tangerine tomatoes (14).

We and others have observed that (Z)-lycopene isomers from tangerine tomatoes are more efficiently absorbed in comparison to (E)-lycopene (5,6,15,16). (Z)-lycopene

86 isomers are present 60-70% in plasma and 80% in prostate tissue (17). In contrast to the studies conducted on the (Z)-lycopene isomers, there are minimal reports on the bioavailability of the lycopene precursors. Several studies have examined the presence of (Z)-phytoene and (Z)-phytofluene in human plasma, serum, milk and tissues (18-20).

It has been suggested that these compounds may exhibit anticarcinogenic properties in concert with lycopene (21,22). Because these compounds are generally present in low concentration in human tissues (13-1275 ng/g), it has been difficult to examine their biological acitivities.

In agreement with the observed greater absorption of (Z)-lycopene isomers in clinical studies, they were also observed to be more bioaccessible than (E)-lycopene

(23). In vitro digestion and Caco-2 cell cultures have been extensively used as an alternative model for studying human intestinal absorption of carotenoids (24,25). Other compounds besides lycopene have been studied using this method, such as ~-carotene

(26), lutein and zeaxanthin (26-28), and chlorophylls (29). However, assessment of the bioaccesibility of the (Z)-lycopene precursors using the coupled in vitro digestion and

Caco-2 cell model has not been evaluated.

The objective of the work summarized below was to examine the bioaccesibility of the carotenoids phytoene, phytofluene, ~-carotene, and neurosporene, using the coupled in vitro digestion/Caco-2 human intestinal cell model. The stability and micellarization of these precursors of lycopene during simulated digestion of fresh and processed yellow tomatoes, as well as the uptake of the micellarized carotenoids and their stability within the cells, have been monitored in this study.

87 3.3. Materials and methods

3.3.1. Chemicals and standards

All reagents and materials were HPLC and ACS grade purchased from Sigma

Chemical Co. (St. Louis, MO) and Fisher Scientific Co. (Fairlawn, NJ USA). Standards of (15Z)-phytoene, (15, 9'Z)-phytofluene, (9, 9'Z)-~-carotene, and (E)-neurosporene were isolated from the tomatoes used in this study using magnesium oxide and hyflosupercel

(1:1) open column chromatography as described in previous reported methods (30,31) and purified using semi-preparative C30 column 10 µm, 250 x 20 mm I.D. (Waters,

Milford, MA).

3.3.2. Sample preparation

Yellow Holland tomatoes were purchased from local grocery stores. The tomatoes were blended to juice using a food blender. Olive oil (3% w/w) was added to the juice and the mixture was transferred to cans. Some of the cans were further processed in a retort at 250°F for 30 minutes and the remaining cans were not processed.

All cans were stored at -20°C for further analysis.

3.3.3. In vitro digestion

The in vitro digestion and Caco-2 cell method used in this study is outlined in

Appendix F. Canned tomato juice was thawed and mixed using a food blender. The juice was subjected to simulated gastric and small intestinal phase of digestion using procedures as described by Garrett et al. (24). Aliquots of tomato juice (3 g) were diluted

88 with 30 ml saline solution (120 mM NaCl and 150 µm BHT), homogenized (Ultra

Turrax 339619, Tekmar Company), and transferred to a 50 ml glass bottle. The pH of the meal was decreased to 2 with 1 M HCl before adding 2 ml of porcine pepsin (40 mg/ml in 0.1 M HCl). The volume was increased to 40 ml/reaction with saline solution.

The homogenate was incubated at 37°C in a shaking water bath (95 rpm) for 1 hour. The intestinal phase of digestion was initiated by increasing the pH of the chyme to 6 with 1

M sodium bicarbonate, adding 3 ml of porcine bile extract (40 mg/ml in 100 mM sodium bicarbonate) and 2 ml of pancreatin (lOmg/ml) and pancreatic lipase (5 mg/ml). Final concentrations of pancreatin and pancreatic lipase in the reaction mixture were 0.4 and

0.2 mg/ml, respectively. The pH was adjusted to 6.9 with 1 N NaOH, brought to a final volume to 50 ml with saline, and incubated at 37°C in a shaking water bath for 2 hours.

Digestion reactions were performed for five or six replicate samples for each treatment.

3.3.4. Isolation ofthe aqueouslmicellar fraction from digesta

Aliquots of the digesta (approximately 13 ml) were centrifuged at 167000 g for

35 min at 4°C using a Beckman model L7-65 ultracentrifuge (Palo Alto, CA) with 50 Ti rotor to separate the aqueous micellar phase. The aqueous fraction was collected from the centrifuge tubes using an 18 gauge needle attached to a 10 ml syringe and filtered using 0.2 micron filters to remove any contaminating aggregates of non-micellarized carotenoids.

89 3.3.5. Caco-2 cell culture

Caco-2 cells (HTB37) were obtained from the American Type Culture Collection

(Rockville, MD) and maintained as previously described by Garrett et al. (1999). The complete medium contained high glucose DMEM (D7777, Sigma), 7.5% heat inactivated fetal bovine serum (FBS), nonessential amino acids (10 ml/L medium;

M7145), L-glutamine (2 mmol/L), penicillin-streptomycin (10 ml/L), fungizone, HEPES

(15 mmol/L), and sodium bicarbonate (44 mmol/L). Cells were grown in T75 flasks

(Beckton Dickinson Labware, Franklin Lakes, NJ) in a humidified atmosphere or 95% air and 5% C02 at 37°C.

3.3.6. Cellular uptake ofmicellar carotenoids

All experiments used highly differentiated cultures of Caco-2 cells (11-14 days post-confluent) at passages 26-33. Aliquots of micellar fractions from simulated digestion were diluted 1:4 with basal DMEM. Spent medium was removed and monolayers were washed with warm basal DMEM. The cells were incubated in a humidified atmosphere of 95% air and 5% C02 at 37°C for 4 hours to examine cellular uptake of lycopene precursors. Test media were aspirated and washed with cold phosphate buffer saline (PBS) containing albumin (2g/L ), followed by two washes with cold PBS without albumin. The monolayers were scraped from the surface of the culture dish and transferred to a microcentrifuge tube. The cell suspension was centrifuged

(1800 rpm); the supernatant was discarded and the cell pellets were blanketed with nitrogen and stored at -80 °C for further analysis.

90 3.3. 7. Carotenoid extraction and analysis

Extractions were performed under subdued light to minimize isomerization and degradation of carotenoids. Digested samples were extracted using hexane:acetone (3: 1) with 100 µm BHT, vortexed for 1 min, and centrifuged for 5 min to induce phase separation. This procedure was repeated 3 times and the hexane layers were combined and dried under nitrogen. For cell pellets, the samples were resuspended in 1 ml of 35 mmol/L SDS in ethanol containing 4.5 mmol/L BHT and sonicated for 30 s on ice.

Samples were extracted as described for digesta. Prior to HPLC analysis, films were resolubilized in MTBE:Methanol (1 :3) and filtered (0.2 µm pores) (Altech, Deerfield,

IL).

A reverse-phase HPLC-PDA system was used to determine the carotenoid profiles in the tomatoes following the methods from Fraser et al. (32) with some modifications. The system consists of a Waters 2695 separation module and 996 PDA

(Milford, MA). Analytical polymeric YMC-C30 column (3 µm, 250 x 4.60 mm I.D.) and

Novapak-C1s guard column (4 µm, 30 x 4.6 mm ID) were used for separation (Waters,

Milford, MA). Carotenoids were eluted using methanol and MTBE at lml/min for 60 min. An isocratic method of 95% methanol and 5% MTBE was used for 5 min before initiating a gradient run that reached 70% MTBE for 40 min. The last composition of the mobile phase was linearly restored to its original condition for 15 min. The injection volume used in the system was 100µ1.

91 3.3.8. Data analysis

All data were analyzed using SPSS 13.0 (SPSS Inc, Chicago, IL). Descriptive statistics including mean, standard deviation, and standard error were calculated for the stability during digestion, efficiency of micellarization, cell uptake by Caco-2 cells, and stability of aqueous micelles from digestion. Means of processed and unprocessed samples were compared using multiple variate analysis (MANOV A). Differences were considered significant at P<0.05.

3.4. Results and discussion

3.4.1. Composition of the processed and unprocessed tomatoes

Carotenoids in tomatoes before simulated digestion were analyzed using HPLC

PDA (Figure 3.1). Lycopene precursors including isomers were identified by comparison with data from previous reported studies (8). Since there were numerous isomers detected using this HPLC method, only predominant isomers were monitored for this study.

92 10

2 4 i

c ___ ,.. ______- --- ~

B _ .. ~ .. ..;,---·-··-··-·····-··-·-··-...... _,,

0 Minutes 60

Figure 3.2. Representative HPLC-PDA chromatograms of lycopene precursors present in the tomato juice. A) Chromatograms at 285 nm, B) 348 nm, C) 400 nm, D) 440nm. In order of their retention times:

I) phytoene isomer, 2) (15Z)-phytoene, 3) (E)-phytoene, 4) (15,9'Z)-phytofluene, 5) (E)-phytofluene, 6)

(9,9'Z)-<;;-carotene, 7) (7,9,9'Z)-neurosporene, 8) (9Z)-<;;-carotene, 9) neurosporene isomer, 10) (E)- neurosporene. All peaks were identified using their UV spectra and by comparison to previously reported study (8).

The quantities of the lycopene precursors in processed and unprocessed samples were examined (Table 3.1). The highest concentration of lycopene precursors in this tomato was phytoene, followed by s-carotene, phytofluene, and a small quantity of neurosporene. According to this analysis, there is an indication that the tomato used in this study was not yellow tomatoes as stated. Yellow bred tomatoes have lower carotenoid content than other varieties (33). On the other hand, the tomatoes used in this study contained high concentration of the lycopene precursors, which may indicate that the variety was possibly tangerine bred. 93 Carotenoids Processed a,b Unprocessed a

Total phytoene* 18.21 16.15 (15Z)-phytoene * 15.57 14.21 Phytoene isomer* 1.49 1.95 (E)-phytoene * 1.15 n.d

Total phytofluene* 3.22 2.69 (15, 9'Z)-phytojluene 2.68 2.55 (E)-phytojluene * 0.55 0.14

Total £;-carotene 5.76 5.53 (9,9'Z)- c:;-carotene* 4.12 4.37 (9Z)- c:; -carotene* 1.62 1.16

Total neurosporene 1.08 1.10 (7,9,9'Z) neurosporene* 0.43 0.70 neurosporene isomer* 0.28 0.24 (E)-neurosporene * 0.37 0.17

•units: mg of carotenoids/l 00 gr tomatoes. bProcessed tomatoes in retort at 250°F for 30 min. *Significant between processed and unprocessed samples (P<0.05).

Table 3.1. Lycopene precursor profiles in the processed and unprocessed tomatoes before simulated digestion.

In this study, processmg enhanced the extractability of total phytoene and phytofluene (P<0.05). This result may be due to the presence of oil during heating. Oil was added to juice to facilitate the formation of mixed-micelles during simulated digestion. It is well known that lipid can enhance the efficiency of solubilization of lipophilic compounds such as carotenoids, stimulate the release of bile salts from the gall bladder, and increase the size of bile salt micelles (34 ). Plasma lycopene and P-carotene were measured by the presence of oil in a test meal, demonstrating that dietary lipid

94 facilitates absorption of carotenoids (35). However, the extractability of s-carotene and neurosporene was not altered by processing in the presence of oil.

Thermal processing also was associated with isomerization of lycopene precursors. (E)-phytoene content decreased, while (15Z)-phytoene content increased and assumed likely a (Z)-isomer of phytoene was detected. (E)-phytofluene was increased 3- fold in the cooked samples, while the (15,9'Z)-phytofluene concentration did not change.

This observation suggests that this reaction may arise from other phytofluene isomers that were not monitored in this study. The (9Z)-s-carotene increased after processing, whereas the (9,9'Z)-s-carotene decreased. The (E)-neurosporene also increased while the (7 ,9 ,9'Z)-neurosporene decreased after processing, suggesting possible isomerization reaction between these two isomers.

Severe thermal treatment has been reported to induce isomerization of (E) to (Z) isomers of lycopene (5). ~-carotene, lutein, and zeaxanthin also isomerize from (E) to

(Z)-form (36-38). Likewise, Unlu et al. (15) and Ishida et al. (16) observed that the prolycopene isomerized to the (E)-configuration after severe processing. Since these lycopene precursors were predominantly in the (Z)-configuration, partial isomerization of (Z) to (E)-isomers of the lycopene precursors occurred during thermal treatment. In contrast, analysis of s-carotene suggests possible isomerization between the (Z)-isomers.

Conversion of (Z) to (E)-isomer was not monitored in this study because we were not able to identify and quantify all isomers present after themal processing. Additional kinetic studies will be required to examine these isomerization reactions for each specific lycopene precursors.

95 3.4.2. Digestive stability

In this study, in vitro digestion was used as previously reported in other in vitro studies of carotenoids (24,26,28). This system has been shown to be a useful model to examine carotenoid bioaccessibility prior to human clinical studies. The gastric phase in this in vitro digestion included HCl and pepsin, while the small intestinal phase includes the addition of bile extract, pancreatic enzymes and biocarbonate. Previous studies have shown that bile salts and the digestive enzymes are required for the transfer of the carotenoids from the food matrix to micelles (24,34,39,40).

Stability of the lycopene precursors varied from 50-80% during in vitro digestion

(Figure 3.3). Processing did not affect the digestive stability of total phytoene and phytofluene. However, recovery of total s-carotene and neurosporene after digesta increased in the processed samples, suggesting that processing enhanced the digestive stability of these carotenoids.

96 Total phytoene (1SZ)-phytoene phytoene (E)-phytoene Total Rlytofuene (15, 9'Z)-phytofluene (E)-phytofuene isomer

100 .------~ * 80 -1------<

60 * * *

0 Total 1;-carotene (9,9'Z)-1;-carotene (9Z)-1;-carotene Total (7,9,9'Z) neurosporene (E)- neurosporene neurosporene isomer neurosporene

• Processed D Unprocessed

Figure 3.3. Digestive stability of lycopene precursors during simulated in vitro digestion. The percent of digestive stability is the amount of carotenoids in 'digesta' divided by the amount of carotenoids in 'food '.

Digesta is the product of the simulated digestion before inducing phase separation of micelles. Food is the starting material (3gr) in total of 50ml saline.

* showed significance between processed and unprocessed samples (P<0.05).

Digestive stability of the predominant isomers (15Z)-phytoene, (15,9'Z)- phytofluene, and (7,9,9 'Z)- neurosporene was similar for processed samples and unprocessed sample. However, recovery of both (9,9'Z)- s-carotene and (9Z)- s -carotene during digestion of processed samples was slightly greater than for the digested unprocessed samples (P<0.05). Recovery of the lycopene precursors after digestion was lower compared to the recovery of ~-carotene (70-75%) and lutein-zeaxanthin (75-85%)

97 usmg the similar procedure (28). Additional studies are required to examine the influence of the acid environment in the gastric phase on these compounds.

There were also some indications of isomerization reactions observed during digestion. While 96% of (E)-phytofluene was stable during digestion of the unprocessed samples and 70% of this isomer was recovered after digestion of the processed samples.

Similarly, 84% of (E)-neurosporene was recovered after digestion of the unprocessed samples compared to 74% recovery in the processed samples. It is unclear if these losses were associated with isomerization or degradation of the carotenoids.

3.4.3. Efficiency of micellarization

Formation of mixed micelles is essential for delivery of carotenoids to the brush border membrane of enterocytes. The micellarization efficiency of lycopene precursors during the small intestinal phase of digestion ranged from 14-45% (Figure 3.4).

Micellarization of total s-carotene was lower than the other carotenoid precursors.

(15Z)-phytoene was micellarized more efficiently than the (E) phytoene, whereas (E) phytofluene was more efficiently micellarized than the (15, 9'Z)-phytofluene.

Micellarization of (E) and the other (Z)-isomer of neurosporene were greater than that of

(7, 9, 9'Z)- neurosporene.

98 Total phytoene (15Z)-phytoene phytoene (E)-phytoene Total Rlytofluene (15, 9'Z)-phytofluene (E)-phytofluene isomer

20 * *

0 Total 1;-carotene (9,9'Z)-1;-carotene (9Z)-1;-carotene Total (7,9,9'Z) neurosporene (E) - neurosporene neurosporene isomer neurosporene

• Processed D Unprocessed

Figure 3.4. Micellarization efficiency oflycopene precursors during simulated in vitro digestion. The percent of micellarization is the amount of carotenoid transferred to the filtered aqueous fraction divided by the carotenoid content in food.

* Showed significance between processed and unprocessed samples (P<0.05).

Processing also increased the efficiency of micellarization of total phytofluene, s-carotene, and neurosporene, but not to total phytoene. However, (E)-phytofluene had higher micellarization in the uncooked samples. The efficiency of micellarization of these lycopene precursors during digestion was similar to that reported for P-carotene, lutein, and zeaxanthin (15-50%) in foods digested in vitro, and were much higher than for (E)-lycopene (5%) (23,28,41). This study correlates with other studies which stated that phytofluene appeared to be better absorbed than lycopene in clinical studies (20,42).

It is known that the higher efficiency of micellarization of the carotenoids is due to the 99 factors limiting transfer from chloroplasts to oil droplets (43). Xanthophylls carotenoid are localized at the surface of oil droplets whereas carotenes are localized in the core thus decreasing the rate of transfer to micelles (44 ). It is most likely that these lycopene precursors, which are categorized as acyclic carotenes, are also located near the core of the oil droplets. However, these lycopene precursors are predominantly in the (Z) configurations, that are proposed to be more efficiently solubilized in lipophilic solutions, less likely to crystallize, and readily transported within cells or tissue matrix

( 45). Such characteristic of the lycopene precursors likely contributes to their enhanced micellarization efficiency. These results also correlate with the higher efficiency of in vitro micellarization (23) and better absorption of the (Z)-lycopene isomers compared to the (E)-lycopene in humans (15,46).

3.4.4. Stability of Micellar Carotenoids

The micelles generated during simulated digestion were placed in the cell culture incubator (95% air and 5% C02 at 37°C) to determine their stability after 4 and 20 hr.

Stability for most of the micellarized carotenoids exceeded 50% after 4 hr incubation with the order of: ~-carotenes> neurosporene > phytofluene > phytoene (Appendix H).

Stability of carotenoids micellarized during digestion of processed samples (75-90%) was greater than for unprocessed samples (50-60%). Time dependent isomerization towards (E)-phytofluene and (E)-neurosporene was noted in micelles generated from unprocessed samples.

100 3.4.5. Caco-2 Cell Uptake

Caco-2 cells have been extensively used as a model to study bioaccesibility of carotenoids. These cells have similar biochemical and morphological properties of enterocytes for the apical uptake and metabolism studies (47,48). Caco-2 cells accumulated the lycopene precursors after incubation at 37°C for 4h. The cellular content of these lycopene precursors are shown in Table 3.2.

Carotenoids Processed a,b Unprocessed a

Total Phytoene* 185.66 147.06 (15Z)-phytoene * 141.54 125.01 (E)-phytoene * 22.06 22.06 phytoene isomer* 20.22 n.d

Total Phytojluene* 25.83 16.60 (15, 9'Z)-phytofluene * 19.19 12.91 (E)-phytofluene * 5.54 3.69

Total (-carotene* 55.18 43.33 (9, 9 'Z)- (-carotene* 34.07 21.48 (9Z)- (-carotene 21.48 18.15

Total Neurosporene * 6.09 3.28 (7,9,9'Z) neurosporene 1.78 1.56 neurosporene isomer* 1.67 0.76 all-(E)-neurosporene * 2.63 0.94

•units: pmol of carotenoids/mg protein. bProcessed tomatoes in retort at 250°F for 30 min. *Significant between processed and unprocessed samples (P<0.05).

Table 3 .2. Cellular content of lycopene precursors in Caco-2 cells after 4 hr incubation.

101 Cells accumulated 14-17% of total s-carotene from medium. Accumulation of phytoene > phytofluene > neurosporene and represented 4-8% of that present in medoium (Figure 3.4). Uptake by Caco-2 cells did not alter the composition of the carotenoids and no isomerization reactions were detected. Accumulation by Caco-2 cells of these lycopene precursors, except for s-carotene, appeared to be lower than that reported for P-carotene and xanthophylls (23,24,26,28).

!!!. Qi u 10 6 "'u 0"' 5 >. .0 al 1il 0 3 E Total phytoene ( 152)-phytoene phytoene (E)-phytoene Total Phytofluene (1 5, 9'Z)-phytofluene (E)-phytofluene ::J is omer u u "'(/) 20 "O '6 c * Q) e 15 "'u "O Q) N 'C 10 .!!! Qi u ~ 5 ;fl

0 Total c-carotene (9,9'Z)-c-carotene (9Z)-c-carotene Total (7,9,9'Z) neurosporene (E) - neurosporene neurosporene isomer neurosporene

• Processed D Unprocessed

Figure 3.5. Caco-2 cellular uptake oflycopene precursors. The cell uptake is the amount of carotenoids in the cell divided by the carotenoid content in the test media. Test media is a 1 :4 dilution of aqueous micelles from simulated digestion.

* showed significance between processed and unprocessed samples (P<0.05).

102 Cell accumulation of (15Z)-phytoene and (15,9'Z)-phytofluene from micelles generated during digestion of thermally processed samples (7%) was significantly greater (P<0.05) than the unprocessed tomato (4.5-6%). In contrast, cells accumulated greater (E)-phytofluene isomer from the digested non-thermally processed tomato than from unprocessed tomato. Cell accumulation of (9,9'Z)-s-carotene from micelles generated by digestion of the processed samples (15%) was greater than that from micelles produced during digestion of unprocessed samples (11 %), while accumulation of (9Z)-s-carotene was independent of processing of test food. In addition, processing slightly decreased uptake of the (7,9,9'Z)- and (E)-neurosporene, while the cell uptake of the unknown isomer was enhanced. After these carotenoids were accumulated by the cells during the 4 hr exposure, the qualitative and quantitative profiles remained stable during overnight incubation.

3.5. Conclusions

This study demonstrated that the carotenoids phytoene, phytofluene, s-carotene, and neurosporene were transferred from food to micelles during simulated digestion and were delivered to Caco-2 human intestinal cells. Use of the coupled in vitro digestion/Caco-2 cell system revealed that thermal processing was generally associated with increases in the bioaccessibility of lycopene precursors. Isomerization of some carotenoids occurred during processing and digestion. The efficiency of micellarization

103 and uptake by Caco-2 cells varied for some of the geometric isomers of the carotenoids, where (Z)-isomers were generally better micellarized than (E).

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121 APPENDIX A

STANDARD CURVES

Phytoene

3500000

Cll 3000000 I!! 2500000 < ...: 2000000 Cll a.Cll 1500000 (.) 1000000 ....I a. 500000 ::c 0 -500000 05 concentration (mol/L)

Phytofluene

2500000 Cll 2000000 I!! < ...: 1500000 Cll a.Cll 1000000 (.) ....I 500000 a. ::c 0 -500000 006 Concentration (mol/I)

Figure A. 1. Standard curves of(15Z)-phytoene and (15,9'Z)-phytofluene used for quantification of phytoene, phytofluene, and their isomers in the tomato juice, in vitro digestion and Caco-2 study.

122 ~-Carotene

3500000 ca I!! 3000000 < 2500000 .II::ca 2000000 QI c.. 1500000 0 ..J 1000000 c.. ::c 500000 0 0 5E-07 1 E-06 2E-06 2E-06 3E-06 3E-06 4E-06 4E-06 5E-06 concentration (mol/I)

Neurosporene

800000 ca I!! < 600000 .II::ca QI 400000 c.. 0 ..J c.. 200000 ::c

0.0000002 0.0000004 0.0000006 0.0000008 0.000001 0.0000012 concentration (mol/I)

Figure A. 2. Standard curves of (9,9'Z)-s-carotene and (E)-neurosporene used for quantification of s carotene, neurosporene, and their isomers in the tomato juice, in vitro digestion and Caco-2 study.

123 APPENDIXB

UV SPECTRA OF L YCOPENE PRECURSORS AND ISOMERS

2852

334.9

200 400 nm

Figure B. 1. Representative UV spectra of (l 5Z)-phytoene (A), (Z)-phytoene isomer (B), and (E)-phytoene (C), from tomato juice after thermally processed at 250°F for 30 min.

124 A .6

200 400 nm

Figure B. 2. Representative UV spectra of (I 5,9'Z)-phytofluene (A) and (E)-phytofluene (B), from tomato juice after thermally processed at 250°F for 30 min.

A 425.5 400.3

376.3

200 500 nm

Figure B. 3. Representative UV spectra of(9,9'Z)-s-carotene (A) and (9Z)-s -carotene (B), from tomato juice after thermally processed at 250°F for 30 min.

125 432.7

67.7

200 600 nm

Figure B. 4. Representative UV spectra of(7,9,9'Z)-neurosporene (A), (Z)-neurosporene isomer, and (E) neurosporene (C), from tomato juice after thermally processed at 250°F for 30 min.

126 APPENDIXC

METHODS FOR THERMAL PROCESSING STUDY

c§ ... Bl~ded to l"'~ """9 food bl'"du 1 CaMe:d

Unprocased / e:l'!'lp~ 75F, ....------+-----. time 0 min Tha-.1 Procasi11f

We erba h Re: oM process

(o~d still ~ . tort) (c:lose:d s 111 ~tort) l OOF l '°F 200F 2'° F D Each proee.s$e.d at proce.sse:d at 30 and 30, 60, 90 l'!'lin 60 min ~ ...._ __.. Hexou: A~tone: (1:1) e:>Ctrac ion, colle:ct Huane laye:r l tfl.C - PpA YMC C30 Carcte:,noid column

R.evtt~d Phase. Me:OH:MTBE g~die:nt Time: 60 min, Flow Ro e:; lml/min

APCI-MS

• riple: quadrupole: ma$S s~c l'O~te:r. • Ca!"ono curr-e:nt of 10.2 ,uJ, , a cone voltage. of 35 V, RF-1 of 50 V, and desolva ion gas te:,mperature: of 500 °C at 3.5 Llmin. · Se:le:cte:d-ion monitoring (SIM) with o dwe:ll time: of 100 ms pe:r ion.

Figure C. Schematic methods of thermal processing study described in chapter 2.

127 APPENDIXD

NMR ANALYSIS FOR IDENTIFICATION OF LYCOPENE PRECURSORS

Standards (15Z)-phytoene, (15,9'Z)-phytofluene, (9,9'Z)-s-carotene, and (all-E) neurosporene were dissolved in chloroform and analyzed using a Bruker 800 MHz spectrometer with a 5 mm QXI probe. The following NMR condition was achieved: lH

30° flip angle of 5.033 ms, 65K data points, lK number of scans, spectral width 11.5

KHz, and relaxation delay 2s. Only (15Z)-phytoene and (9,9'Z)-s-carotene (Figure 3) were able to be identified since the other standards were too low in concentration. It was also difficult to isolate an adequate quantity of individual isomers such that an NMR spectra could be obtained.

A B

6.7 6.6 6.5 6.4 6.3 6.2 6.I 6.0 5.9 s.s ppm 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.o 5.9 s.s ppm

Figure D. Proton NMR of the olefinic region of (15 Z)-phytoene (A) and (9,9'Z )-s-carotene (B).

128 APPENDIXE

ABSTRACT 2005 IFT ANNUAL MEETING New Orleans, LO, July 15-20 Session 36E-45, Fruit & Vegetable Products: General

Thermal processing increases isomerization of lycopene precursors from yellow tomatoes M. RENITA and S. J. Schwartz. Dept. of Food Science and Technology, Ohio State Univ., 2015 Fyffe Ct., Columbus, OH 43221

Recent evidence has shown that thermal processing may increase isomerization of carotenoids such as ~-carotene or lutein. The predominant form of these carotenoids in nature is the all trans configuration, while thermal processing may induce cis isomerization. The yellow tomato variety consists of higher amounts of four lycopene precursors: phytoene, phytofluene, ~-carotene, and neurosporene, in which these carotenoids are prevalent in the cis form. The effects of thermal processing on isomerization reactions of these precursors have not been studied. The objective of this study is to determine the effects of thermal processing on carotenoid lycopene precursors. Yellow tomatoes were processed in cans at 100, 150, 200, and 250° F for 30, 60, and 90 min. Each can was extracted and analyzed using reversed phase C30 High Performance Liquid Chromatography coupled with a Photo Diode Array detector (HPLC-PDA). Temperature showed a significant correlation to the increase of isomerization for all carotenoids (P<0.01). Further processing at 200F and 250F was found to have a notable effect on isomerization (P<0.05). Processing time did not show any difference in isomerization for these precursors. However, only phytofluene behaved differently from the group and correlated significantly with the extent of processing time. We also observed that during mild heat treatments, these carotenoids tended to be more easily extracted from the thermally processed tomato matrix. However, these changes were not significant. Results of this study suggested that temperature affected isomerization of lycopene precursors, whereas processing time only increased isomerization to phytofluene. Further investigation will be required in order to determine the structures of these isomers, whether they were converted to trans or other cis configurations.

129 APPENDIXF

ABSTRACT 2005 INTERNATIONAL CAROTENOID SYMPOSIUM Edinburgh, UK, July 17-20

IN-VITRO DIGESTION AND CAC0-2 CELL UPTAKE OF PHYTOENE AND PHYTOFLUENE FROM TANGERINE TOMATOES aMarjory Renita, bMark L. Failla, and aSteven J. Schwartz aFood Science and Technology, The Ohio State University, 110 Parker Food Science Building, 2015 Fyffe Road, Columbus, OH 43210 (email: [email protected], [email protected]) , bHuman Nutrition, The Ohio State University, 325 Campbell Hall, 1787 Neil Avenue, Columbus, OH 43210 (email:[email protected])

Dietary carotenoids from consumption of plant products have been associated with the prevention of several chronic age-related diseases [1]. Plant breeders have studied carotenoid biosynthesis and are now developing unique plant varieties in order to deliver carotenoids in food products. Tomato fruits have been extensively evaluated to regulate and control biosynthesis of carotenoids [2]. The tangerine tomato variety contains high levels of lycopene precursors: 15-(Z)-phytoene, 15,9'-(Z)-phytofluene, 9,9'-(Z)-s-carotene, and 7,9,9'-(Z) neurosporene, compared to typical red tomatoes [3]. Tangerine tomato is also prevalent in either 9,9'-(Z)-s-carotene or 7,7',9,9'-(Z)-lycopene [4]. These carotenoids are predominant in the (Z) configuration in contrast to the (E)-configuration of lycopene in red tomatoes. We have observed in human clinical studies that (Z)-lycopene isomers are more efficiently absorbed in comparison to (E)-lycopene [5]. These results also correlate with observations using simulated in-vitro digestion to evaluate the bioaccesibility of (Z)-lycopene isomers [6]. However, the bioaccesibility of the (Z)-lycopene precursors (phytoene, phytofluene, s-carotene, and neurosporene) have not been evaluated.

In this study, uncooked tangerine tomatoes with added vegetable oil (3%) was subjected to in vitro digestion coupled with the Caco-2 human intestinal cell model [7] to determine the digestive stability, micellarization, and cellular accumulation of the lycopene precursors with focus on 15-(Z)-phytoene and 15,9'-(Z)-phytofluene. Tomatoes also were processed at 121°C for 30 min to monitor thermal effects on stability and bioacessibility of these carotenoids. The extracts were analyzed using C30 HPLC-PDA.

Thermal processing of tangerine tomatoes increased extractability and induced isomerization of 15-(Z)-phytoene and 15,9'-(Z)-phytofluene (P<0.05). Recovery of these compounds exceeded 50%, and 20-30% were micellarized during simulated digestion. Processing did not affect the digestive stability of these carotenoids. However, thermal treatment increased micellarization of 130 15,9' -(Z)-phytofluene (P<0.05).Some isomerization of 15,9' -(Z)-phytofluene to (E)-phytofluene may have occurred during digestion of the uncooked tomato. Once micellarized, the carotenoids were relatively stable(> 85% recovery) in culture medium for 4 h.

Caco-2 cells accumulated 4-8% of micellarized carotenoids after incubation at 3 7°C for 4h. Cell accumulation of 15-(Z)-phytoene and 15,9' -(Z)-phytofluene from digested thermally processed samples was significantly (P<0.05) greater than from uncooked digested samples. Unlike the predominant 15,9' -(Z)-phytofluene, the (E)-phytofluene isomer showed higher cell uptake from the non-thermally processed tomatoes (P<0.05).

Results of this study suggested that 15-(Z)-phytoene and 15,9'-(Z)-phytofluene in tangerine tomatoes were isomerized during thermal processing. 15,9' -(Z)-Phytofluene also was partially isomerized during digestion. Although thermal processing did not affect digestive stability of either lycopene precursors, it enhanced the efficiency of micellarization of 15,9' -(Z)-phytofluene and intestinal cell uptake of both 15-(Z)-phytoene and 15,9'-(Z)-phytofluene.

[l] N. Krinsky, S. Mayne, H. Sies, Carotenoids in Health and Disease. Marcel Dekker, New York (2004). [2] J. Hirschberg, Curr. Opin. Plant Biol 4, 210-218 (2001). [3] V. Ladygin, Biochemistry 65, 1113-28 (2000). [4] G, Giuliano, L. Giliberto, Rosati, Trends Plant Sci. 7, 427-429 (2002). [5] Z. Unlu, S. Clinton., D. Francis, S. Schwartz, Proceeding: Pigments in Food, more than colours ... , 295 (2004). [6] M. Pusateri, C. Chitchumroonchokchai, S. Schwartz, M. Failla, FASEB J. 17, A696, 425 (2003). [7]. Garrett, M. Failla, R. Sarama, J Agric. Food Chem. 47, 4301-4309 (1999).

131 APPENDIXG

METHODS FOR IN VITRO DIGESTION AND CAC0-2 CELL STUDY

Homogttiiud Start ,,,Digestion ...... Add 3"1. oil ..... l pH • 2 with 1M HCI 2 ml Porcine Pcpsift: 40 mg/ml in 0.1 M HCI Total solution with sohne : 40 ml Gastric Phase l Incubation: 1 hour. src. 95 """' l pH • 6 with 1M NaHCO, 3 ml Bile ExtNct : 40 mg/ml in 0.1 M NaHCO, !'-.-tin= 0.4 mg/ml in 0.1 M NaHCO, P-..atlcUpua: 0.2 ma/ml in 0.1 M NaHCO· Intestinal Phase l pH • 6.9 with IN NaOH Total solution with 1Glinc: !50 oil l Incubation: "Digcsta" ...... 41. Digc.stive .rto.b1lity.: 2 hours. Jrc. 95 """' l dige.stQ/food x 1007. "Aqueous" MIGellar Formation ...... 7. M icell<1rizot ion = aqueous/food x lOO'X. l Test Media: "Miccllor Stability" Aqueous micelles d ilution ...... after 4 hr Cl1ld 20 kr =1:4 with Be.sol DMEM incubation in cell l environment HT837 m T75 flasks "Cell Uptake" Cac;o-2 Celle 4rir 951c oir 5t. CO, ...... • GmoW'lt inc&ll/test Retcrit10n 20hr media x 100"

Figure G. Schematic methods of in vitro digestion and Caco-2 cell study described in chapter 3.

132 APPENDIXH

STABILITY OF MICELLAR CAROTENOIDS

Test Media Stability 4 hr Stability 20 hr

Carotenoids Processed•,h Unprocessed" Processed"'b Unprocessed" Processed•,h Unprocessed"

Total phytoene 1.261 1.350 0.910 0.678 * 0.795 0.690 (l 5Z)-phytoene 0.991 1.167 0.764 0.590 * 0.644 0.602 (E)-phytoene 0.143 0.183 0.078 0.089 0.076 0.087 phytoene isomer 0.127 not detected 0.068 0.059 0.007 0.065

Total phytotluene 0.188 0.185 0.163 0.089 * 0.100 0.069 * (15, 9'Z)-phytojluene 0.145 0.160 0.125 0.073 * 0.059 0.043 * (E)-phytojluene 0.043 0.024 0.039 0.015 * 0.040 0.026 *

Total l;-carotene 0.188 0.180 0.235 0.168 * 0,193 0.168 * (9, 9'Z)- (-carotene 0.121 0.122 0.150 0.107 * 0.101 0.080 * (9Z)- (-carotene 0.067 0.058 0.084 0.061 * 0.093 0.071 *

Total neurosporene 0.048 0.025 O.o38 * 0.028 * 0.023 O.ot8 (7,9,9'Z) neurosporene 0.014 0.012 0.011 0.010 0.004 * 0.008 * neurosporene isomer 0.014 0.007 0.010 * 0.005 * 0.003 0.002 a/1-(E)-neurosporene 0.007 0.006 0.017 * 0.006 * 0.016 * 0.007 *

'Units: µg of carotenoids/ml !>processed tomatoes in retort at 250°F for 30 min. *Significant between processed and unprocessed samples (P<0.05).

Table H. Stability ofmicellar carotenoids diluted in I :4 with basal DMEM (Test Media) after incubation for 4 and 20 hours in cell environment. 133