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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. STABILITY OF CAROTENOIDS IN RED PALM OIL AND ITS EFFECTS ON THEIR BIOAVAILABILITY, PROVITAMIN A ACTIVITY AND TOXICITY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Ni Luh Puspitasari-Nienaber, M.S.

* * * * *

The Ohio State University 2002

Dissertation Committee: Approved by Dr. Steven J. Schwartz, Adviser

Dr. Mark L. Failla

Dr. James W. Harper Adviser Food Science and Technc Dr. Polly D. Courtney

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Red palm oil (RPO) is a mildly refined crude palm oil containing at least 500 |.ig/g

carotenoids and 800 pg/g tocopherols and tocotrienols. The major carotenoids found in

RPO are the provitamin A a- and p-carotene. Provitamin A carotenoids in RPO are

more efficiently converted to vitamin A than provitamin A carotenoids in other plant

sources. Short-term supplementation with RPO was shown to improve the vitamin A

status of both mother and infant. Therefore, RPO has been suggested to be an effective

food-based strategy to combat vitamin A deficiency. Although these findings were

promising, information on RPO stability during storage and food processing is limited.

This information is critical in determining the shelf life, proper storage and processing

conditions for RPO. Furthermore, the characteristics of carotenoid degradation products

formed during storage merit investigation. The present study was designed to examine

the oxidative and thermal stability of RPO, and secondly to assess the in vitro

bioavailability, cellular uptake, conversion to vitamin A and potential mutagenicity of p-

carotene oxidation products formed during storage.

RPO carotenoids were stable during storage in the dark at room temperature.

The storage time for the carotenoids to deplete 50% (t

and carotenoid degradation followed zero order kinetics. When tocopherols were

removed from RPO, the ty2 value was only 18 h and the degradation kinetics changed to

first order. In RPO, tocopherols degraded faster during storage than when carotenoids

were removed. RPO was found not suitable for frying. Heating of RPO for 15 min

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. degraded more than 90% of the carotenoid. These results suggest that it is important

to keep RPO from light, oxygen and excessive heat treatment.

Epoxy p-carotene was found as the main carotenoid degradation product in RPO

during storage. The subsequent studies were designed to investigate the bioavailabilty.

vitamin A conversion and mutagenicity of epoxy p-carotene. Epoxy p-carotene was

subjected to a simulated gastric and small intestinal digestion. The micellarized

compounds were then fed to Caco-2 cells to determine cellular accumulation and

cleavage to vitamin A. p-Carotene epoxides were readily accessible for absorption but

the degree of micellarization depend on the presence of other carotenoids and food

matrix. Cellular accumulation of these compounds by Caco-2 cells indicates their

availability for absorption. However, the relative accumulation of p-carotene epoxides

was lower than p-carotene. Epoxy p-carotene was converted to vitamin A less efficiently

than p-carotene. The bacterial reverse mutation assay demonstrated that epoxy p-

carotene was mutagenic in S. typhimurium TA102 but not in TA98, TA100 and TA97.

This study indicated that RPO was stable during storage, however RPO was not

suitable for frying. Proper storage conditions were important to preserve the carotenoids

in RPO. Oxidation products of carotenoids, mainly p-carotene epoxides, accumulated

during storage. Although these compounds are accessible, bioavailable and exhibited

partial provitamin A activity, consumption of p-carotene epoxide is not recommended

given their potential toxicity as mutagens. Information acquired from these studies has

provided the basic information on the potential of RPO as a food-based source of

provitamin A in areas with high prevalence of vitamin A deficiency. These data can be

used to aid for future studies in investigating the relationship between consumption of

RPO and modulation of vitamin A deficiency.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This work is dedicated to the children of Indonesia.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

I would like to especially thank my adviser, Dr. Steven Schwartz, for introducing

me to this fascinating research area, for the endless opportunity to explore and for the

challenges he presented. I am truly grateful of his continuous support and

understanding of my circumstances throughout my graduate program. I would like to

extend a special appreciation to Dr. Mark Failla for providing an invaluable learning

experience, enthusiasm and encouragement. Drs. David Min, Polly Courtney and Jim

Harper are acknowledged for their advise and support.

I am deeply grateful for the assistance and friendship of Mario Ferruzzi, Charlotte

Allen, Chureeporn Chitchumroonchokchai, Corey Scott, Keith Harris, Carla Bailey,

Minhthy Nguyen, Maureen Hackett, Christine Clinger, Elizabeth Altstaetter, Igor

Milosevic, Nuray Unlu, Ashley Updike, Yu Chu Zhang, Mario Pusateri, Julie Jenkins,

Claudia Harris, Hye Won Yeom, Sudarat Jiamyangyeun and Benjawan Thumtanaruk. I

thank my dance partners Judy Wilaputra and Becky Lydon for their friendship and for

nurturing the other side of me. Ruth Roberts-Kohno, who showed me that a mother

could do this too, was an inspiration.

I would also like to thank my colleagues at the Department of Food Technology

and Human Nutrition, Bogor Agricultural University for allowing me to take this

opportunity. Drs. Aman Wirakartakusumah, Dedi Fardiaz and Deddy Muchtady are

acknowledged for their support. Special thanks also go to Drs. Lilts Nuraida, Nuri

Andarwulan, Ratih Dewanti-Hariyadi, Purwiyatno Hariyadi and Slamet Budiyanto.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Finally, I am truthfully indebted of my family: my parents Bondan and Merdah

Hermanislamet, and my brothers Dias Pradadimara and Syah Inderaprana, for their love

and prayer throughout my education. I am most grateful of my husband, Uwe, for his

love, support and encouragement during my study. My daughter, Maya Rakitri, has

been a source of motivation the entire way.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA

August 16, 1963 ...... Born - Bandung, Indonesia 1985 ...... B.S. Agricultural Technology, Gadjah Mada University 1990...... M.S. Food Science, University of Wisconsin - Madison 1986 - 1997 ...... Researcher, Inter-University Center for Food and Nutrition, Bogor Agricultural University, Indonesia 1986 - 1997 ...... Lecturer, Department of Food Technology and Human Nutrition, Bogor Agricultural University, Indonesia 1992...... Visiting scientist, Department of Food Science, North Carolina State University 1992 - 1993...... Visiting scientist. Federal Center for Cereal, Potato and Lipid Research, Munster, Germany 1997 - present ...... Graduate Research Associate. The Ohio State University

PUBLICATIONS

Puspitasari-Nienaber, N.L., Ferruzzi, M.G. and Schwartz, S.J. 2002. Simultaneous detection of tocopherols, carotenoids and chlorophylls in vegetable oils by direct injection on RP-HPLC with coulometric electrochemical array detection. J. Am. Oil Chem. Soc. 79:633-640. Bohm, V., Puspitasari-Nienaber, N.L., Ferruzzi, M.G. and Schwartz. S.J. 2002. Trolox equivalent antioxidant activity of different geometrical isomers of a-carotene, [3-carotene, lycopene and zeaxanthin. J. Agric. Food Chem. 50:221-226.

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Henry, L.K., Puspitasari-Nienaber, N.L., Jaren-Galan, M., van Breemen, R.B., Catignani, G.L. and Schwartz, S.J. 2000. Effects of ozone and oxygen on the degradation of carotenoids in an aqueous model system. J. Agric. Food Chem. 48:5008- 5013. Apriyantono, A.; Husain, H.; Lie, L.; Judoamidjojo, M.; Puspitasari-Nienaber, N. L. 1999. Flavor characteristics of Indonesian soy sauce (kecap manis). Flavor Chem. Ethn. Foods, (Proc. Meet. 5th Chem. Congr. North Am.], 15-31. Apriyantono, A.; Wiratma, E.; Husain, H.; Nurhayati; Lie, L.; Judoamidjojo, M.; Puspitasari-Nienaber, N.L.; Budiyanto, S.; Sumaryanto, H. 1996. Analysis of volatiles of kecap manis (a typical Indonesian soy sauce). Spec. Publ. - R. Soc. Chem. 197 (Flavour Science), 62-65. Puspitasari, N.L., Greger, J.L. and Lee, K. 1991. Calcium fortification of cottage cheese with hydrocolloid control of bitter flavor defects. J. Dairy Sci. 71:1-7.

PUBLISHED ABSTRACTS

Puspitasari-Nienaber, N.L., Failla, M.L. and Schwartz, S.J. 2002. Accumulation, retention and vitamin A conversion of micellar p-carotene epoxides by Caco-2 human intestinal cells. Experimental Biology Meeting, New Orleans, LO. Schwartz, S.J. and Puspitasari-Nienaber, N.L. 2002. Analysis and bioavailability of lycopene. AOCS Annual Meeting, Montreal, Canada Puspitasari-Nienaber, N.L., Failla, M.L. and Schwartz, S.J. 2001. Micellarization of beta-carotene oxidation products by an in vitro digestion. IFT Annual Meeting, New Orleans, LO. Puspitasari-Nienaber, N.L. and Schwartz, S.J. 2001. Stability of oil-in-water emulsions from red palm oil: Role of indigenous carotenoids and tocopherols. AOCS Annual Meeting, Minneapolis, MN. Bohm, V., Puspitasari-Nienaber, N.L., Ferruzzi, M.G. and Schwartz, S.J. 2001. Antioxidant activity of carotenoid geometrical isomers. International Congress of Nutrition, Vienna, Austria. Puspitasari-Nienaber, N.L., Ferruzzi, M.G. and Schwartz, S.J. 2000. Simultaneous detection of tocopherols, carotenoids and chlorophylls in vegetable oils by direct injection on RP-HPLC with coulometric electrochemical array detection. IFT Annual Meeting, Dallas, TX. Puspitasari-Nienaber, N.L., Min, D.B. and Schwartz, S.J. 2000. High concentration of a- and p-carotene increase oxidation and tocopherol degradation in red palm oil. AOCS Annual Meeting, San Diego, CA. Moxley, C.L., Ferruzzi, M.G., Nguyen, M L., Puspitasari-Nienaber, N.L., Francis, D., Clinton, S. and Schwartz, S.J. 1999. Diets rich in cis lycopene increase circulating c ij isomers in human. Experimental Biology, Washington DC.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Puspitasari-Nienaber, N.L., Priata, A., Hastinah, T. 1997. Thermal degradation of p- carotene in a lipid model system. AOCS Annual Meeting, Seattle, WA. Puspitasari-Nienaber, N.L. and Aitzetmuller, K. 1993. Analytical investigation on Pangium edule seed oil. Joint Congress of 2nd EUROLIPID and 5m German Society for Fat Science, Munster, Germany Puspitasari-Nienaber, N.L. and Aitzetmuller, K. 1993. Development of an improved method for the quantification of vitamin A esters in margarine using HPLC with an internal standard. Joint Congress of 2nd EUROLIPID and 5m German Society for Fat Science, Munster, Germany Puspitasari-Nienaber, N.L. and Schwartz, S.J. 1992. Carotenoid composition of several unique Indonesian plant foods. IFT Annual Meeting, New Orleans, LA.

FIELDS OF STUDY

Major Field: Food Science and Nutrition

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

Page

A bstract ...... ii

Dedication ...... iv

Acknowledgements ...... v

V ita ...... vii

List of Tables ...... xii

List of Figures ...... xiii

Chapters:

1. Introduction ...... 1

2. Review of literature ...... 4

2.1. Palm O il ...... 4 2.2. Red Palm O il ...... 8 2.3. Carotenoids ...... 8 2.4. Quenching of Singlet Oxygen by Carotenoids ...... 9 2.5. Autoxidation of Carotenoids ...... 9 2.6. Reaction of Carotenoids with Free Radicals ...... 12 2.7. Carotenoids and Lipid Oxidation ...... 18 2.8. Thermal Degradation of Carotenoids ...... 21 2.9. Products of Carotenoid Degradation ...... 22 2.10. Carotenoid Digestion, Intestinal Absorption and Metabolism to Vitamin A ...... 22 2.11. Chemopreventive Activity of Carotenoids ...... 24

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Simultaneous detection of tocopherols, carotenoids and chlorophylls in 25 vegetable oils by direct injection C3o RP-HPLC with coulometric electrochemical array detection ......

3.1. Abstract ...... 26 3.2. Introduction ...... 27 3.3. Experimental Procedures ...... 29 3.4. Results and Disccusion ...... 31 3.5. Conclusions ...... 37 3.6. References ...... 45

4. Effects of carotenoids and tocopherols on the oxidative and thermal 49 stability of red palm o il ......

4.1. Abstract ...... 50 4.2. Introduction ...... 51 4.3. Experimental Procedures ...... 52 4.4. Results and Disccusion ...... 55 4.5. Conclusions ...... 62 4.6. References ...... 72

5. (3-Carotene epoxides. In vitro digestive stability, and their uptake and 77 conversion to vitamin A by human Caco-2 human intestinal cells ......

5.1. Abstract ...... 78 5.2. Introduction ...... 80 5.3. Experimental Procedures ...... 83 5.4. Results and Disccusion ...... 91 5.5. Conclusions ...... 105 5.6. References ...... 123

6. Mutagenicity of epoxy p-carotene in Salmonella typhimurium...... 131

6.1. Abstract ...... 132 6.2. Introduction ...... 133 6.3. Experimental Procedures ...... 130 6.4. Results and Discussion ...... 134 6.5. Conclusion ...... 138 6.6. References...... 141 Dissertation Summary ...... 145

Bibliography ...... 148

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

Table Page

3.1 Linear ranges and detection limits of carotenoids, tocopherols and 44 chlorophyll a analyzed on a C3o RP-HPLC with electrochemical array detection. 4.1 Kinetic parameters for a-carotene, p-carotene and total carotene 63 degradation in red palm oil during storage. 4.2 Kinetic parameters for total tocopherol degradation in palm oil during ... 64 storage. 5.1 Conversion efficiency of p-carotene epoxides in the TC7 clone of 122 Caco-2 cells. Conversion efficiency was calculated as the ratio of (1/2 retinol) to (1/2 retinol + carotene) in the cells 48 h after incubation. Values are mean ± SEM (for n = 3-5). Different letters indicate statistically significance difference (p < 0.0001). n.d. = not detected. 6.1 Mutagenicity of epoxy p-carotene to S. typhimurium TA98, TA100 ...... 139 TA97 and TA102 without activation. Values are mean ± SEM (n=3-7). 6.2 Mutagenicity of epoxy p-carotene to S. typhimurium TA98, TA100 ...... 140 TA97 and TA102 with activation. Values are mean ± SEM (n=3-7).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

Figure Page

2.1 Structures of the most common carotenoids, tocopherols and ...... 7 tocotrienols in crude palm oil 2.2 Mechanism of p-carotene autoxidation as proposed by Mordi (1993).... 11 2.3 Reaction pathways of p-carotene oxidation via free radical addition 13 (Liebler, 1993) 2.4 Structures of p-carotene geometrical isomers, carbonyl and epoxy 14 compounds 2.5 Competitive reaction of p-carotene with phenoxyl radical to yield 16 a) p-carotene radical cation and b) phenoxyl adduct (Mortensen and Skibsted, 1996) 2.6 Reaction of p-carotene (PC) and lipid peroxyl radical (LOO*) as 20 proposed by Tsuchihashi et al. (1995) 3.1 Structures of carotenoids, tocopherols and tocotrienols ...... 38 3.2 C30 HPLC separation of tocopherols, tocotrienols and carotenoid 39 geometrical isomers in red palm oil 3.3 C30 HPLC separation of tocopherols, carotenoids and chlorophylls in .... 40 A) virgin olive oil and B) virgin olive oil spiked with spinach leaves 3.4 Current-voltage curves of A) p-carotene and its geometrical isomers .... 41 and B) a-carotene and its geometrical isomers 3.5 Current-voltage curves of a-tocopherol, a-tocotrienol, 5-tocopherol 42 and y-tocopherol 3.6 Current-voltage curves of chlorophyll derivatives ...... 42 3.7 C30 HPLC separation of tocopherols in A) wheat germ oil, B) soybean 43 oil and C) mixture of wheat germ + soybean oil (2:1) 4.1 Separation of major RPO carotenoids and tocopherols on a C30 ...... 65 RP-HPLC with coulometric electrochemical array detection. xiii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2 A) Degradation of carotenoids in RPO with indigenous tocopherols 66 during storage at 25, 37 and 60 °C. B) Degradation of carotenoids in RPO without indigenous tocopherols during storage at 60 °C. 4.3 A) Degradation of tocopherols in RPO during storage at 25, 37 and 67 60 °C. B) Degradation of individual tocopherol in RPO during storage at 37 °C. 4.4 Peroxide values (meq/kg) of A) RPO and B) control oil during ...... 68 storage at 25, 37 and 60 °C. 4.5 A) Degradation of carotenoids in RPO during come-up time and 69 frying of french fries. B) Formation and degradation of carotene isomers during come-up time and frying of french fries. 4.6 Degradation of tocopherols in A) RPO and B) control oil during ...... 70 come-up time and frying of french fries. 4.7 Formation and degradation of A) p-carotene and B) a-carotene 71 Isomers in RPO during storage at 37 °C 5.1 Structures of all-trans p-carotene and its epoxy derivatives ...... 106 5.2 Separation of p-carotene isomers and p-carotene epoxides o n ...... 107 a C30 RP-HPLC with coulometric electrochemical array detection 5.3 Separation of retinol, retinyl palmitate and p-carotene o f ...... 108 Caco-2 cell extracts on a Ci8 RP-HPLC. Cell extracts were A) not saponified (325 nm), B) saponified (325 nm) and C) saponified (450 nm) 5.4 Concentration of A) p-carotene, B) 5,6-epoxy p-carotene and ...... 109 C) 5,8-epoxy p-carotene in applesauce, fresh palm oil and used palm oil test meals 10 min after preparation. The horizontal broken line represents the amount of test compounds added to the test meal. Preparation of test meal, digestion and analyses were described in Materials and Methods. Data represent mean ± SEM (n=3-6).

x/V

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.5 A) Recovery of p-carotene and its epoxy derivatives in the ...... 111 digesta following in vitro digestion of applesauce, fresh palm oil and used palm oil. Data represent the percentage of carotenoids recovered from the digested to the starting test meal which is arbitrarily set to 100%. B) Generation of diepoxy p-carotene in the digesta following in vitro digestion of applesauce, fresh palm oil and used palm oil. 5.6 Micellarization of p-carotene and its epoxy derivatives from ...... 112 applesauce test meals. A) Micellarization of p-carotene in the presence of different p-carotene epoxides. B) Micellarization of various p-carotene epoxides in the presence of p-carotene at 1.1 ratio. Applesauce test meals contain 10% (w/w) oil and various p-carotene derivatives (25-35 mmol/g). Data represent mean ± SEM (n=6). Different letters above the bars indicate statistically significance different (p < 0.05) between samples. 5.7 Micellarization of p-carotene and its epoxides in applesauce (AS), ...... 113 fresh palm oil (FPO) and used palm oil (UPO) test meals following in vitro digestion. Apple sauce test meal contained 10% corn oil with the test compounds. All test meals contained 21-37 mmol/g of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene. Data represent mean ± SEM (n=3-6). Different letter above the bars indicate statistically significance different (p <0.05) between test meals. 5.8 Effect of pH on the conversion of 5,6-epoxy p-carotene to ...... 114 5,8-epoxy p-carotene during the gastric phase of in vitro digestion procedure. Applesauce test meal contained 10% com, 5,6-epoxy p-carotene and p-carotene. Gastric pH was adjusted with 1.0 mol/L HCI or 1.0 mol/L NaOH before addition of pepsin. Samples were incubated at 37 °C for 1 h, and neutralized before analysis. Data represent mean ± SEM (n=3-6). The asterisk (*) indicate that concentration of these carotenoids differed significantly (p < 0.05) from the food

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.9 Effect of bile acid content on the transfer of p-carotene epoxides 115 from the test meal to the aqueous micellar fraction. Preparation of raw material, digestion and analyses were described in Materials and Methods. Data represent mean ± SEM (n=3-6). The asterisk (*) indicate that the concentration of these carotenoids differed significantly (p < 0.05) from the concentration when bile acid concentration was 0 mg/mL. 5.10 Stability of micellarized p-carotene and its epoxides in cell culture 116 environment. The concentration of each test compound in the medium at 0 h was 60 nmol/L. Data represent the percentage of carotenoids relative to the raw material (arbitrarily set to 100%). Data represent mean ± SEM (n=3-6). Different letters above the bar indicate statistically significance different (p < 0.05) within time points. The asterisk (*) indicate that the remaining (%) concentration of these carotenoids differed significantly (p < 0.05) from the previous time points. 5.11 Cellular accumulation and retention of micellarized epoxy ...... 117 p-carotenes in differentiated cultures of Caco-2 HTB37 cells. Each test compound was solubilized in com oil and digested in vitro. Aqueous fractions were diluted (1:4) with DMEM before addition to cell monolayers. A) Cellular accumulation after various periods of incubation (0-6 h). Initial concentrations of 5,6-epoxy and 5,8-epoxy p-carotene was 60 nmol/L. B) Cellular accumulation after 4h incubation in medium with different concentrations of epoxy p-carotenes. C) Retention of cells loaded with test compound during 4h of incubation and subsequently incubated in medium free of test compounds for 18 h. Data represent mean ± SEM (n=3).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.12 Cellular accumulation and retention of micellarized (3-carotene a n d ...... 119 epoxy p-carotenes in the TC7 clone of the Caco-2 cells. Each test compound was solubilized in corn oil and digested in vitro. Aqueous fractions were diluted (1:4) with DMEM before addition to cell monolayers. A) Cellular accumulation after various periods of incubation (0-6 h). Initial concentrations of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene was 20 nmol/L. B) Cellular accumulation after 4h incubation in medium with different concentrations of epoxy p- carotenes. C) Retention by cells pre-loaded with test compound during 4h of incubation and subsequently incubated in medium free of test compounds for 18 h. Data represent mean ± SEM (n=3). Different letters above the bars indicate statistically significance different (p < 0.05) between samples. 5.13 Recovery of p-carotene and its epoxides from differentiated ...... 121 monolayers of the TC7 clone of Caco-2 grown on inserts. Cells were loaded by addition of DMEM containing individual test compound as Tween micelles to the apical compartment for 4 h. Cell monolayer was washed and incubated for 20 h in fresh DMEM. Cells, apical medium and basolateral medium were collected and analyzed as described in Material and Methods. Data represent mean ± SEM (n=3).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

INTRODUCTION

The traditional biological function of carotenoids, mainly p-carotene, is its

provitamin A activity. Plant carotenoids are the main supply of provitamin A in

developing countries where consumption of diets rich in vitamin A is low and vitamin A

deficiency is a problem. Bioconversion efficiency of plant carotenoids to vitamin A,

however, is very low. Interestingly, crude palm oil (CPO) which contains very high levels

(500-800 ng/g) of provitamin A carotenoids (Goh et al., 1985; Tan et al., 1986; Tan.

1989) in a more bioavailable form (Solomon, 1998) and produced mainly in these

countries has not been optimally utilized to eliminate the problem.

a- And p-carotenes are the major components (together they constitute more

than 90% of the total carotenoids) of CPO carotenoids, with y-carotene, lycopene and

xanthophylls present in mtch lower levels (Tan et al., 1986). CPO also contains high

amount (about 1000 pg/g) of both tocopherols and tocotrienols. Unlike other vegetable

oils, it is unusually high in y-tocotrienols (Tan, 1989). The current practice for palm oil

production involves fractionation, refining, bleaching and deodorizing to produce light

yellow palm oil known as refined bleached deodorized (RBD) palm oil. These steps

destroy all carotenoids, about half of the tocopherols and tocotrienols, and represent a

loss of a potential source of provitamin A and natural antioxidants (Tan et al., 1986).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since the late 1960s CPO has been introduced as a source of dietary provitamin

A for preschool children in Indonesia, India and West-African countries (Oey et al., 1967;

Rajagopal and Mudambi, 1978; Villard and Bates, 1984), but was not popular because

of its non-pleasant taste and deep red color. Recent findings on the efficient

bioconversion of p-carotene to vitamin A from palm oil (Mahapatra and Manorama,

1997; Solomon, 1998) and the suggested cancer preventive activity of carotenoids (Peto

et al., 1981; Block et al., 1992), tocopherols and tocotrienols (Guthrie et al., 1997 a and

b; 1999) have led to the commercialization of a refined CPO known as red palm oil

(RPO) in Malaysia and Indonesia. Because provitamin A does not produce

hypervitaminosis A, RPO, once again, was suggested to be an effective food-based

strategy to combat vitamin A deficiency (Solomon, 1998 and 1999).

RPO is produced using a process employing short-path distillation to CPO and

contains not less than 500 mg/kg carotenoids and 800 mg/kg of tocopherols and

tocotrienols (Choo et al., 1998). Little is known, however, about the oxidative and

thermal stability of RPO. Studies have shown the prooxidant effect of p-carotene and

other carotenoids such as lycopene and lutein when added to vegetable oils at high

levels (Haila and Heinonen, 1994; Haila et al., 1996; Henry et al., 1998 a and b).

Recently, Steenson and Min (2000) discovered that thermal degradation products of p-

carotene, but not lycopene, act as prooxidants in soybean oil.

Findings from two human intervention studies reported an increased risk for lung

cancer among smokers taking high dose of p-carotene supplements (ATBC Study

Group, 1994; Omenn et al., 1994). Several subsequent reports provided useful

information regarding what may cause the harmful effect of p-carotene in those studies,

and pointed out that oxidative metabolites of p-carotene may be one factor. Salgo et al.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1999) showed that p-carotene oxidation products increased the binding of

benzo[a]pyrene metabolites to DNA in an in vitro experiment using calf thymus DNA.

Another study using BALB/c 3T3 cells showed that in the presence of p-carotene

metabolites, p-carotene induced cell transformation by benzo[a]pyrene (Perocco et al.,

1999). In general, these studies demonstrated that p-carotene oxidation products, but

not p-carotene itself, may facilitate carcinogenesis.

Information on RPO stability is critical in determining its shelf life, storage

conditions and suitable use. The characteristics of carotenoid degradation products

merit further study, including their effects on oxidative and thermal stability of the oil. on

bioavailability as well as on bioconversion to vitamin A. Only with this knowledge can we

be certain that RPO is a potential food-based source of provitamin A in areas with high

prevalence of vitamin A deficiency.

The specific objectives of this study are:

1. To investigate the oxidative and thermal stability of RPO, by understanding the role

of carotenoids, tocopherols as well as their degradation products.

2. To investigate the in vitro bioavailability, cellular uptake and conversion to vitamin A

of a) palm carotenes in the presence of their oxidation products and b) the oxidation

products.

3. To screen for potential mutagenicity of carotene oxidation products of RPO.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2

REVIEW OF LITERATURE

2.1. Palm Oil

Palm oil, together with soybean oil, is a major contributor to the total oil and fat

production in the world. Approximately 90% of all palm oil is used as cooking oil,

margarine, shortenings and confectionery fats (Tan, 1989; Ong et al., 1995). Global

production of palm oil in 1999-2000 was forecasted to reach 20.1 million metric tons

(MMT) and the major producers and exporters are Malaysia and Indonesia. It has been

predicted that palm oil supply will exceed soybean oil and that Indonesia will produce

more palm oil than Malaysia by 2012 (Gunstone, 1999).

Red colored and carotenoid-containing CPO is typically fractionated, refined,

bleached and deodorized to produce light yellow RBO palm oil. These steps not only

remove all undesired impurities, but also destroy all carotenoids, about half of the

tocopherols and tocotrienols, and represent a loss of a potential source of natural

provitamin A and antioxidants (Tan, 1989). Minor components of CPO constitute of

carotenoids (500-700 (ig/g), tocopherols and tocotrienols (600-1200 pg/g), sterols (250-

620 (ig/g), phospho- and glyco-lipids, terpenic and aliphatic hydrocarbons and other

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. trace impurities (Cottrell, 1991; Ong et al., 1995; Choo et al., 1996). The major

carotenoids, tocopherols and tocotrienols present in CPO are presented in Figure 2.1.

Carotenes impart a distinctive red color to CPO, where a-carotene and p-

carotene combined make up about 90% of the total, in a 1:2 ratio, respectively. CPO is

the only major source of a-carotene besides carrot. Other carotenoids found in CPO are

phytoene, phytofluene, 8-carotene, y-carotene, ^-carotene, neurosporene, p-

zeacarotene, a-zeacarotene and lycopene (Ng and Tan, 1988; Ong et al., 1995; Schierte

et al., 1997). Depending on the palm variety, carotenoid concentrations in CPO can be

as high as 3000 ug/g (Yap et al., 1991).

p-Carotene from RPO is much more efficiently bioconverted to vitamin A than

that from other plant sources. Two parts of p-carotene from RPO yields one RE of

vitamin A (Solomon, 1998) while 12 parts from orange fruits (de Pee et al., 1998) and 26

parts from green vegetables (de Pee et al., 1995) are required to produce the same

effect. Supplementation with palm carotenoids (15 mg/d) was reported to significantly

increase plasma concentration of p- and a-carotenes in humans (Faulks et al., 1998).

Canfield et al. (1999) reported that palm carotenoids in palm oil improved vitamin A

status of both mother and infant after a short-term supplementation. The presence of a -

carotene did not affect p-carotene bioavailability and both were delivered by the

supplement (van het Hof et al., 1999). a-Carotene was suggested to be more effective

in reducing the risk of skin (Murakoshi et al., 1992) and lung (Murakoshi et al., 1992;

Ziegler et al., 1996) cancer than p-carotene.

Palm carotenoids were proven to have no mutagenic activity in vitro and in vivo

(Masuda et al., 1995). In addition, palm carotenoids exhibited anticarcinogenic and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. detoxifying activities by suppressing cytochrome P45o-enzyme activity in benzo (a)

pyrene metabolism in rats (Tan and Chu, 1991). Okuzumi et al. (1992) reported that

palm carotenoids inhibit intestinal carcinogenesis.

Palm oil is a good source of tocopherols and tocotrienols, which are present in a

1:4 ratio, respectively (Eitenmiller, 1997; Belitz and Grosch, 1999). The major

homologous are a-tocopherol (200 mg/kg), a-tocotrienol (390 mg/kg), p-tocotrienol (25

mg/kg), y-tocotrienol (430 mg/kg), 8-tocopherol (26 mg/kg) and 8-tocotrienol (100 mg/kg)

(Belitz and Grosch, 1999). This composition is unique, since palm oil is the only

tocotrienol-containing oil among the four major oils (with soybean, rapeseed/canola and

sunflower oils) consumed in the world (Eitenmiller, 1997).

It is generally accepted that the differentiation between the biological effects of

tocopherols as vitamin E and antioxidant is difficult, if not impossible. Tocopherols have

been reported to prevent the development of cardiovascular disease and to reduce the

risk for several cancers, which are linked to their antioxidant activities (Cottrell, 1991).

Palm tocotrienols were effective inhibitors of protein oxidation and lipid peroxidation in

rat liver microsomes (Kamat et al., 1997). Guthrie et al. (1997a and b, 1999) reported

that palm tocotrienols inhibited human breast cancer cells more effectively than a -

tocopherol, with y-tocotrienol being the most effective.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p-carotene

a-carotene

y-carotene

lycopene

a-tocopherol a-tocotrienol

p-tocopherol p-tocotrienol u 7 -tocopherol y-tocotrienol

C 5-tocopherol 5-tocotrienol

Figure 2.1. Structures of the most common carotenoids, tocopherols and tocotrienols in crude palm oil 7

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2. Red Palm Oil fRPOl

RPO is produced using a process employing short-path distillation to CPO and

contains not less than 500 pg/g carotenoids and 800 ^ig/g of tocopherols and

tocotrienols (Choo et al., 1993; 1998). CPO is degummed by phosphoric acid treatment,

followed by deodorization and deacidification by molecular distillation under low

temperature and pressure. (3-Carotene from RPO has been shown to be bioavailable

and RPO was suggested to be a good source of provitamin A for combating vitamin A

deficiency (Rukmini, 1994). When fed to rats, RPO did not induce phase I but induced

phase II enzymes (Manorama et al., 1993a).

2.3. Carotenoids

Carotenoids are long-chain, conjugated polyenes synthesized in plants and

found in animals (Pfander, 1992). Epidemiological as well as animal data have been

accumulated about the possible role of carotenoids in decreasing risk of some type of

cancers and cardiovascular disease (Peto et al., 1981; Ziegler, 1989; Mayne, 1996;

Giovannucci, 1999). The evidence supports the involvement of lipid peroxidation or

oxidative stress in the development of such diseases, and carotenoids - with (3-carotene

and more recently lycopene being the most studied - were believed to act as

antioxidants in vivo. The chemical reactivity of carotenoids towards oxidizing agents and

free radicals present in foods and biological systems, thus their role as antioxidants, is

mainly due to the polyene chain in the mid-portion of the molecule (Burton and Ingold,

1984; Palozza and Krinksky, 1992; Liebler, 1993; Mortensen and Skibsted, 1997,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Woodall et al., 1997a). In addition, carotenoids are involved in energy transfer reactions

such as cis-trans isomerization (Mordi et al., 1991; Finkelshtein and Krasnokutskaya,

1996) or quenching of singlet oxygen (Stahl and Sies, 1993; Yang and Min, 1994).

2.4. Quenching of Singlet Oxvaen bv Carotenoids

Carotenoids are the most effective singlet oxygen quenchers known (Foote and

Denny, 1968) and they act via two distinct mechanisms (Krinsky, 1989; Stahl and Sies,

1993). The predominant mechanism is a physical quenching reaction, in which

carotenoids absorb excited state energy and convert singlet oxygen to triplet oxygen.

The triplet carotenoids are converted back to their ground state by releasing a small

amount of heat to the environment. One (3-carotene molecule is capable of quenchinn

up to 1000 singlet oxygen molecules, before they react chemically. The chemical

reaction eventually destroys the carotenoids and generates specific marker products not

found in free radical oxidation such as (3-carotene-5,8-endoperoxide (Stratton et al..

1993). Endoperoxide was produced via singlet oxygen 1,4-diene addition to the 13-

carotene molecule.

2.5. Autoxidation of Carotenoids

The fact that carotenoids undergo self-initiated autoxidation with molecular

oxygen has been recognized for a long time (Zechmeister et al., 1944; Budowski and

Bondi, 1960; El-Tinay and Chichester, 1970; Burton and Ingold, 1984). In 1958, Friend

proposed that the initial step in (3-carotene autoxidation is isomerization of all -trans to c/s

isomers that has been recently confirmed by Mordi et al. (1991); Finkelshtein and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Krasnokutskaya (1996). Isomerization led to shorter conjugated double bonds on either

side of the cis bond where the overlap in the 71-electron fields became weaker. Glover

(1960) postulated a mechanism for the degradation by eccentric cleavage mechanism,

derived from the identification of apocarotenals as the major degradation products, p-

Carotene is cleaved from one end of the polyene chain by a p-mechanism producing a

series of apocarotenals. When the central 15,15' double bond is reached, the C-13

methyl group that is at a p position from the central double bond blocked the reaction.

It is known that products of p-carotene oxidation with molecular oxygen are very

similar to the products obtained from singlet oxygen oxidation. More recently, Henry et

al. (2000) they are also very similar to the products of ozonation. Then, one would

expect that one reactant in the first reaction is activated. Handelmann and co-workers

(1991) showed the involvement of peroxyl radical intermediate in p-carotene

autoxidation, because a-tocopherol was able to suppress the reaction. Mordi (1993)

proposed the involvement of a singlet diradical of p-carotene, formed after all -trans cis

isomerization. He reasoned that in c/s-form oxygen attack on either side of the cis bond

is enhanced. To date, however, p-carotene diradicals have never been identified. The

resulting p-carotenyl peroxyl radicals react further as shown in Figure 2.2. The exact

point of attack is still a question and studies on autoxidation products point more to a

random rather than specific attack on the conjugated polyene of carotenoids (Mordi et

al., 1991). Using attenuated total reflection IR spectroscopy, Finkelshtein et al. (1999)

recently demonstrated that polyperoxides are the primary oxidation products and that

they are gradually transformed into epoxy and then carbonyl compounds.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All-frans p-carotene 15,15’-c/s p-carotene \

oo \ / epoxides + RO* dioxetanes

apocarotenals

Figure 2.2. Mechanism of p-carotene autoxidation as proposed by Mordi (1993)

Budowski and Bondi (1960) found that lower p-carotene concentrations

increased the length of induction period for p-carotene oxidation and the rate of oxidation

following the induction penod. They called this phenomenon as dilution effect, but no

further explanation was offered. The activation energy for p-carotene autoxidation was 11

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. found to be 10.2 kcal/mol, 5 kcal lower than that of linoleic acid (El-Tinay and

Chichester, 1970).

2.6. Reaction of Carotenoids with Free Radicals

The mechanism of carotenoid reaction with free radicals is less well understood.

Burton and Ingold (1984) postulated that p-carotene inhibited lipid oxidation by trapping

peroxyl radicals and thus a chain-breaking antioxidant. The mechanism is different from

the conventional chain-breaking antioxidant such as tocopherols, in that p-carotene does

not react with peroxyl radical via hydrogen atom abstraction (Burton and Ingold, 1984).

Several co-workers showed that addition to the 5,6 double bond followed by

elimination of an alkoxyl radical produced p-carotene epoxides as shown in Figure 2.3.

(Samokyszyn and Mamett, 1987; Kennedy and Liebler, 1991; Liebler, 1993; Lieblerand

McClure, 1996). Zechmeister et al. (1944) suggested that the central double bonds ir. p-

carotene molecule were more stable because of resonance, and therefore were less

susceptible to oxidation than the terminal bonds. In 1970, El-Tinay and Chichester

proposed that 5,6-p-carotene epoxide was the initial product formed during autoxidation

of p-carotene because the terminal double bonds were sites of the highest electron

density. 5,6-p-Carotene epoxide was then subsequently decomposed to other oxidation

products. Handelmann et al. (1991), Mordi et al. (1993) and Yamauchi et al. (1993)

showed that a variety of polar degradation products (Figure 2.4.) were produced at

approximately similar rates, suggesting the multiple sites of the initial radical attack or p-

carotene.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. P-carotene

ROO ROO»

ROO»

p-carotene epoxide 1. homolytic cleavage (- 2RO* ) 2. heterolytic cleavage (- ROOR)

p-apo-carotenals

Figure 2.3. Reaction pathways of p-carotene oxidation via free radical addition (Liebler. 1993)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. all-frans p-carotene

15-c/s p-carotene

13-c/s p-carotene

9-cis p-carotene

p-apo-10’-carotenal

5,6-epoxy p-carotene p-apo-12’-carotenal

5,6,5',6-diepoxy p-carotene p-apo-14'-carotenal

5,8-epoxy p-carotene p-apo-12-carotenal

p-apo-13-carotenone 5,8,5',8'-diepoxy [1-carotene

Figure 2.4. Structures of (3-carotene geometrical isomers, carbonyl and epoxy compounds

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It should be noted that the epoxide-forming reaction of peroxyl radical and p-

carotene also releases an alkoxyl radical, thus is not expected to produce an antioxidant

effect. Whether the carbonyl-forming reaction produces an antioxidant effect or not, it

depends on how the bis-peroxyl adducts decompose (Liebler, 1993). Homolytic

cleavage would release two alkoxyl radicals, resulting in no net radical consumption. On

the contrary, heterolytic cleavage produces nonradical products, thus an antioxidant

effect. The conditions when the reaction will lead to the homolytic or heterolytic

cleavage were not mentioned.

Another mechanism of peroxyl radical reaction with p-carotene was proposed

(Grant et al., 1988; Jovanovic et al., 1992). Peroxyl radicals are strong oxidants and

they are capable of removing an electron from the electron-rich p-carotene polyene. The

p-carotene cation radicals exhibit strong near-infrared absorption at 955 nm (Grant et al..

1988) and specific resonance raman spectra (Jeevarajan et al., 1996). Using laser flash

photolysis, Mortensen and Skibsted (1996) showed that phenoxyl radicals react with p-

carotene to yield phenoxyl radical adduct and p-carotene radical cation, indicating tha.

the two reactions proceed simultaneously (Figure 2.5 ).

In the presence of electron donors such as a-tocopherol, p-carotene cation

radicals are able to accept electrons (Simic, 1992), but in their absence the cation

radicals decay bimolecularly (Mortensen and Skibsted, 1996). Based on pulse

radiolysis, Boehm et al. (1997) claimed that the reaction with tocopherol can be

reversed, in which a-tocopherol radical can be regenerated to a-tocopherol by p-

carotene. Valgimigli et al. (1997), however, could not reproduce the same effect using

EPR spectroscopy, which can monitor the presence of tocopherol radicals. They

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pointed out the pitfall of drawing mechanistic conclusions based only on the UV, visible

or near IR spectra of transient intermediates.

Figure 2.5. Competitive reaction of p-carotene with phenoxyl radical to yield a) p- carotene radical cation and b) phenoxyl adduct (Mortensen and Skibsted, 1996)

Burton and Ingold (1984) suggested that p-carotene’s antioxidant activity was

greatest at low oxygen pressure such as found in most tissues under physiological

conditions, therefore complements that of tocopherols which are effective at higher

oxygen concentration. They also found that the rate of oxygen consumption at

atmospheric oxygen pressure was 30 fold higher than that could be explained by initiator

decomposition alone, indicating that p-carotene undergo chain-propagated autoxidation

analogous to autoxidation of polyunsaturated fatty acids. Kennedy and Liebler (1992),

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. however, found that p-carotene provided similar antioxidant protection at both high and

low oxygen pressures. They also observed that p-carotene was consumed faster at high

oxygen pressure, again, suggesting a concomitant autoxidation reaction of p-carotene.

Liebler (1993) implied that at atmospheric oxygen concentrations, oxygen react

reversibly with p-carotene peroxyl radical to form a new radical. The new radical then

oxidizes a new p-carotene molecule, thus a prooxidant, as reported earlier by Burton

and Ingold (1984). At low oxygen concentration, the p-carotene peroxyl radical traps a

second peroxyl radical to form bis-peroxyl radical (Liebler, 1993). As in p-carotene

autoxidation, p-carotene is more reactive towards free radical at lower concentration

(Burton and Ingold, 1984).

Different carotenoids showed a marked difference in reactivity towards free

radicals and this was partly influenced by the structure of individual carotenoids (Miller et

al., 1996; Mortensen and Skibsted, 1997a; Woodall et al., 1997a and b). The

decreasing number of coplanar conjugated double bonds and the presence of hydroxyl

and keto groups in carotenoid molecules have been shown to decrease their reactivity in

radical-scavenging reactions such as with radical cation (Miller et al., 1996), phenoxyl

(Mortensen and Skibsted, 1996 and 1997b), peroxyl as well as non specific (Fenton)

radicals (Woodall et al., 1997a and b). The relative rate was shown to be carotenes >

hydroxycarotenes > ketocarotenes. Lycopene was the most active carotenoid, owing to

its 11 conjugated double bonds. Although p-carotene also has 11 conjugated double

bonds, the two in p-ionone rings are not coplanar with the polyene chain and its

reactivity followed lycopene closely. In contrary to the most popular hypothesis, which is

free radical addition to the polyene chain and not hydrogen abstraction, Woodall et al.

(1997a and b) noted that hydrogen abstraction from positions allylic to the polyene chain

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (C-4 of p-carotene and lycopene) must be considered. This co-worker found that

isozeaxanthin, canthaxanthin and astaxanthin (in which the C-4 and C-4’ positions are

occupied by functional groups) reacted more slowly than p-carotene and zeaxanthin

(those positions are free) because the possibility of hydrogen abstraction from these

positions was reduced.

2.7. Carotenoids and Lioid Oxidation

Cooxidation of carotenoids and fatty acids has been recognized for years in

soybean flour as a result of lipoxygenase activity and observed as bleaching of p-

carotene (Klein et al., 1985). Subsequently, cooxidation involving lipoxygenase activity

has been studied in model system (Jaren-Galan and Minguez-Mosquera, 1997), potato

(Aziz et al., 1999), pepper (Jaren-Galan and Minguez-Mosquera, 1999) and pea (Wu et

al., 1999). Lipoxygenases are also found ubiquitously in mammalian cells, especially

cells of the immune system (Canfield and Valenzuela, 1993). There have been reports

of the positive effect of carotenoids in immune response and inhibition of tumor

promotion. The mechanism, however, is very complex and the details are not known.

Lipids are the major source of free radicals in foods and biological systems.

Cooxidation between lipids and carotenoids without lipoxygenase action has been

reported in lipid containing solutions (Holman, 1949; Palozza et al.. 1995; El Oualja et

al., 1995; Tsuchihashi et al., 1995) and bulk oils (Warner and Frankel, 1984; Haila and

Heinonen, 1994; Haila et al., 1996; Henry et al., 1998).

The reaction mechanism of lipid and carotenoid cooxidation is very similar to that

of free radical oxidation of carotenoids. Tsuchihashi et al. (1995) proposed that lipid

peroxyl radical adds to a double bond in p-carotene molecule to give a resonance-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stabilized, strongly delocalized carbon-centered radical, LOOpC* (Figure 2.6 ). This

radical may undergo several competing reactions. LOOpO can react with another

radical (X*) to give a stable product and lead to antioxidation reaction. In the presence

of oxygen, LOOpC* reacts further with oxygen to yield LOOpCOO. At higher p-

carotene concentration, this radical will attack another p-carotene molecule and leadii.g

to autoxidation and eventually giving polymeric products. Polymeric autoxidation

products of p-carotene have been observed and confirmed by Tsuchihashi and co

workers (1995). LOOpCOO* may also attack lipid to generate lipid radical and induce

chain oxidation reaction, or be scavenged by a-tocopherol. At lower oxygen

concentration, the formation of p-carotene epoxide is more important than the polymer

formation. Tocopherols, when present in high amount, may also scavenge LOOpCOO.

In general, carotenoids can act as lipid antioxidants but they are less potent than

tocopherols. p-Carotene is 32 times less reactive towards peroxyl radical than a -

tocopherol, an effective hydrogen donor and about as reactive as BHT in benzene

solution (Tsuchihashi et al., 1995). In addition, in the presence of oxygen p-carotene

oxidation adducts can promote further lipid oxidation and consume other antioxidants

present in the system. Since the rate of lipid oxidation increases as the degree of

unsaturation increases, p-carotene oxidation also increased as reported by Palozza et

al., 1995.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LOOpCOOX LOOpCX

Autoxidation of BC

LO O + PC —» LOOpC* <— > LOOBOO* o2 \ LH LOOpCOOH + LO* ox. \ LOOpCOOH + aT* epoxide (PCO) + LO* ------> oxidation

Figure 2.6. Reaction of p-carotene (PC) and lipid peroxyl radical (LOO*) as proposed

by Tsuchihashi et al. (1995)

The interaction between carotenoids and other antioxidants in lipid oxidation has

been recognized as an important factor. However, there are some contradictory results

regarding their effects. The presence of tocopherol was reported to enhance the

protective effect of p-carotene on singlet oxygen initiated photooxidation of methyl

linoleate (Terao et al., 1980). The antioxidant action of p-carotene and a-tocopherol was

reported to be additive in hexane solution, but it was synergistic in microsomal

membranes (Palozza and Krinsky, 1991 and 1992) and linoleic acid in fert-butyl alcohol

(Li et al., 1995). Neither synergistic nor cooperative effect between p-carotene and a-

tocopherol, however, was observed by Henry et al. (1998b) in safflower seed oil.

Combinations of p-carotene and y-tocopherol (Haila and Heinonen, 1994) and lutein and

y-tocopherol (Haila et al., 1996) were more efficient in inhibiting lipid oxidation than y-

tocopherol alone. They suggested that y-tocopherol was able to retard the formation of

carotenoid degradation products, which have prooxidant effects.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The prooxidant effect of p-carotene oxidation products on lipid oxidation was not

observed in photooxidation but autoxidation of methyl linoleate (Suzuki et al., 1989) and

ethyl linoleate (El Oualja et al., 1995). This effect was reported to be concentration

dependent, with higher prooxidation at higher p-carotene concentrations. Prooxidant

action of p-carotene was also noted when oxidation of oil had proceeded (Suzuki et al.,

1989). More recently, Steenson and Min (2000) found that p-carotene thermal

degradation products can act as prooxidants in soybean oil. In contrast, lycopene

thermal degradation products were found to have antioxidant activity in the same oil.

2.8. Thermal Degradation of Carotenoids

Heat causes trans - cis isomerization of carotenoids. The type of isomer formed

after heat treatment depends on the temperature, length of heating and presence of

other constituents during heating (Chandler and Schwartz, 1988; Marty and Berset,

1990; Henry et al., 1998). Reflux heating resulted in greater isomerization and

degradation of p-carotene than oven heating (Chen and Huang, 1998). The presence of

food constituents such as water and starch, combined with mechanical mixing leads to a

much higher loss of p-carotene than prolonged heating at 180°C (Marty and Berset,

1990).

The major cis forms of p-carotene include 9-c/s and 13-c/s, while the 15-c/s and

the di-c/s isomers present in minor amount (Chandler and Schwartz, 1988; Chen and

Huang, 1998; Lessin et al., 1997; Henry et al., 1998a). Pesek et al. (1990) observed

that the 9-c/s p-carotene isomer can be converted to 13-c/s isomer only after its

conversion to the all-trans isomer, implying that isomerization is a reversible process and

direct conversion between the cis isomers was not possible.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. After prolonged heating, volatile and non-volatile thermal degradation products

are formed. Reported volatile decomposition products of (3-carotene include toluene,

xylene, [3-ionone, 5,6-epoxy-p-ionone, [3-cyclocitral, 2,6-dimethylnaphtalene and

dihydroactinidiolide (Schrier et al., 1979; Kanasawud and Crouzet, 1990). Non-volaltile

thermal degradation products of (3-carotene include compounds with mass units of 378

(C2aH4 2) and 444 (C 33H48) by the loss of dimethylcyclodecapentaene and toluene from

the polyene chain of p-carotene (Onyewu et al., 1982). Non-volatile oxidized derivatives

of carotenoids such as epoxy and hydroxy carotenoids, apocarotenals and

apocarotenones have also been reported (Ouyang et al.. 1980; Marty and Berset, 1990).

2.9. Products of Carotenoid Degradation

Degradation products of carotenoids have been reported as isomers, epoxy,

carbonyl and alcohol compounds (Chandler and Schwartz, 1988; Handelmann et al.;

1991; Mordi, 1993; Yamauchi et al., 1993; Henry et al., 2000). Yeum et al. (1995) noted

that degradation products of carotenoids were similar whether formed by human gastric

mucosal homogenates, lipoxygenase, or linoleic acid hydroperoxide. Predominant p-

carotene degradation products are presented in Figure 2.4.

2.10. Carotenoid Digestion. Intestinal Absorption and Metabolism to Vitamin A

Carotenoid digestion and absorption involves disruption of food matrix via

mechanical and chemical means, release and dispersion of carotenoids in lipid emulsion

particles, solubilization into mixed bile salt micelles, movement across the unstirred

water layer and the brush border membrane, uptake by the enterocyte and incorporation

into lipoproteins (Bowen et al., 1993; Erdman et al., 1993; Furr and Clark, 1997). The

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. presence and type of lipids during carotenoid digestion is an important factor. Fine lipid

emulsion, containing triacylglycerol core surrounded by partially digested proteins,

polysaccharides and phospholipids, was formed in the stomach. Carotenes are

incorporated in the triacylglycerol core, whereas the more polar xanthophylls are

distributed at the emulsion surface, thus facilitating their transfer to the mixed micelles.

Digestion of the triacylglycerol core by pancreatic colipase-dependent lipase is required

before carotenes can be transferred to the mixed micelles.

Although carotenoid solubilization into mixed micelles is a well-known event, its

exact mechanism is not fully understood. However, the presence of bile salts is known

to be essential for carotenoid absorption, where they serve to solubilize carotenoids in

the small intestine and may be required for interaction with and transport through the

brush border membrane. The mixed micelles must pass two barriers, i.e. the unstirred

water layer and the microvillus membrane, with the former being the rate limiting step for

absorption of lipids and lipid soluble vitamins. Passage across the brush border

membrane is a passive diffusion and occurs very rapid. The content of mixed micelles

are subsequently transferred to the intestinal cells, packaged in chylomicrons, secreted

into the lymph, and transported into the liver.

In the intestinal cells, carotenoids with an unsubstituted p-ionone ring and a

polyene side-chain with at least 11 carbon atoms are metabolized enzymatically to

vitamin A. The enzyme p-carotene 15,15-dioxygenase - which cleaves p-carotene into

retinal, the parent compound of retinol (vitamin A) and retinoic acid - was partially

isolated and characterized as a cytosolic enzyme in the mid-60's (Olson and Hayaishi,

1965; Goodman et al., 1966). Recently, During et al. (1998) characterized this enzyme

in TC7 clone of human intestinal cell line Caco-2.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The mechanism of carotenoid cleavage is still controversial. Originally, the

central cleavage pathway that yields two molecules of all -trans retinal from all -trans p-

carotene was proposed (Olson and Hayaishi, 1965; Goodman et al., 1966) and more

recently reconfirmed (Devery and Milborrow, 1994; Nagao et al., 1996). Wang et al.

(1991) and Tang et al. (1991) demonstrated an alternative pathway involving an

eccentric cleavage with the formation of apocarotenals as intermediates. Ferreira et pi.

(2001) reported both central and eccentric cleavage of lycopene in the rat intestinal

mucosa.

2.11. Chemoprevantive Activity of Carotenoids

In a number of epidemiological studies, consumption of dietary carotenoids has

been linked to a decreased risk of some types of cancer (Peto et al., 1981; Block et al.,

1992; Giovannucci, 1999). The mechanism by which carotenoids act as cancer

chemopreventive agents, however, is not fully understood. The view that carotenoids

are active because of their conversion to retinoids, which are known for their

chemopreventive activity, restricts the activity to a limited number of carotenoids. A

number of studies on laboratory animals and cell cultures show that activity is

independent of such conversion.

The proposed mechanisms on how carotenoids exert their chemopreventive

effects include; 1) act as an antioxidant; 2) induce cytochrome P450 enzymes; 3)

enhance gap-junctional communication and 4) metabolism to retinoic acid and

subsequent activation of retinoic acid receptors (RARs) (Lotan, 1999).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3

Simultaneous Detection of Tocopherols, Carotenoids and Chlorophylls in

Vegetable Oils by Direct InjectionCjo RP-HPLC with

Coulometric Electrochemical Array Detection

Ni Luh Puspitasari-Nienaber, Mario G. Ferruzziand Steven J. Schwartz

Department of Food Science and Technology,

The Ohio State University

2015 Fyffe Road, Columbus, OH 43210-1007

Keywords: C30 column, carotenoids, chlorophylls, electrochemical detection. HPLC,

tocopherols, tocotrienols

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1. ABSTRACT

Tocopherols, carotenoids, and chlorophylls in vegetable oil have been used to

aid their authentication. Their importance in influencing the oxidative stability of

vegetable oils and their possible health benefits have been shown in numerous studies.

Therefore, the need for a rapid and reliable analysis method has become increasingly

important. This study demonstrates the application of C30 RP-HPLC with

electrochemical detection for the simultaneous analysis of tocopherols, carotenoids and

chlorophylls in vegetable oils. Aliquots of vegetable oils were dissolved in appropriate

solvents and injected directly without saponification, thus preventing sample loss or

component degradation. Effective separation of tocopherols, carotenoidss and

chlorophylls was achieved. Detection was performed using a coulometric

electrochemical array detector set between 200 and 620 mV. For a 25 pL injection, the

respective detection limits for carotenoids, tocopherols and chlorophylls were 1 fmol,

0.15 pmol and 0.5 pmol, and representing 1000, 25 and 5 fold enhancement over the

UV-vis methodology. The detector response was linear between 0.01 and 2.00 pg/mL

for all compounds studied. Within-day variations (CV) were between 2.0 and 6.3%,

while between-day variations were between 2.7 and 7.4%. This method can be applied

for rapid and sensitive analysis in the study of oil quality and adulteration.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2. INTRODUCTION

Minor constituents of vegetable oils such as tocopherols, carotenoids, sterols and

chlorophylls have been widely utilized as authentication aids. Olive oil adulteration can

be traced based on its tocopherols and tocotrienols content (Dionisi et al., 1995; Aparicio

et al., 2000) as well as chlorophyll and carotenoid composition (Gandul-Rojas et al..

2000). Carotenoids, tocopherols and tocotrienols are abundant and characteristic of red

palm oil (Ong and Tee, 1992). The presence of tocotrienols in olive oil can be attributed

to contamination or adulteration with palm oil (Dionisi et al., 1995). More recently, the

importance of these minor constituents on the oxidative stability of vegetable oils

(Psomiadou and Tsimidou, 1998; Haila et al., 1996) and their possible health benefits

(Guthrie et al., 1997; Solomon, 1998) have prompted the need for rapid and reliable

analytical methods.

Typically, analysis of minor compounds of vegetable oils involves their

enrichment into the unsaponifiable fraction followed by separate analyses with gas

chromatography (GC) and/or high performance liquid chromatography (HPLC) utilizing

various modes of detection. Saponification is often employed to remove lipids and

chlorophylls, and to release esterified xanthophylls (Lietz and Henry, 1997). However,

this step has been reported to result in a significant loss of carotenoids, especially the

epoxycarotenoids and xanthophylls (Khachik et al., 1986; Kimura et al., 1990). When

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. applied to red palm oil, saponification significantly decreased the amount of a- and 13-

carotene and a-tocopherol extracted and facilitated isomerization of the carotenoids

(Lietz and Henry, 1997).

HPLC with electrochemical detection (ECD) has been used in the analysis of

tocopherols in olive oil (Dionisi et al., 1995), vegetable oils (Sanchez-Perez et al., 2000)

and neonatal plasma samples (Finckh et al., 1995), tocopherols and carotenoids in

human blood plasma and various biological microsamples (Motchnik et al., 1994;

Ferruzzi et al., 1998 and 2001). The coulometric array detector employed in the latter 2

studies offers several advantages, including high sensitivity, selective detection and

ease of implementation. Limit of detection for y-tocotrienol analyzed by RP-HPLC with

amperometric detection was reported to be 10 times lower compared to NP-HPLC with

fluorometric detection (Dionisi et al., 1995). Detection limits for all -trans 3-carotene and

lycopene were reported to be 10 and 50 fmol, respectively, which was 10 to 100 times

lower than the conventional UV-vis detection (Ferruzzi et al., 1998 and 2001). The

application of a multichannel electrochemical detector allowed for enhanced selectivity

because interfering compounds can be eliminated prior to the detection of the compound

of interest. In addition, current-voltage curves (CVC’s) may be constructed based on the

analyte's response across the electrochemical array and used as an aid in peak

identification (Ferruzzi et al., 1998 and 2001).

In this study, tocopherols, tocotrienols, carotenoids and chlorophylls (Figure 3.1.)

were simultaneously determined in vegetable oils by C3Q RP-HPLC with coulometric

electrochemical array detection. Oil samples were dissolved in the mobile phase and

directly injected without exctraction and saponification. Limit of detection, linearity range

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and reproducibility of the method were tested. This method was then applied to red

palm oil, virgin olive oil and various vegetable oil mixtures to illustrate the potential

usefulness in the detection of oil adulteration.

3.3. EXPERIMENTAL PROCEDURES

Materials. HPLC grade solvents were used throughout the experiments.

Tocopherols (a, 5 and y), chlorophyll a, all -trans p-carotene and lycopene standards

were purchased from Sigma-Aldrich (St. Louis, MO). All -trans a-carotene was obtained

from Fluka Chemie AG (Buchs, Switzerland). Red palm oil, virgin olive oil and soybean

oil were purchased from a local grocery store, while wheat germ oil was from a local

health food store.

Standard Preparation. Stock solutions of carotenoids, tocopherols and

chlorophyll a were prepared in hexane, methanol and diethyl ether, respectively. Their

concentrations were determined spectrophotometrically using published extinction

coefficients (AOCS, 1998; Britton, 1995; Schwartz et al., 1981). Working solutions were

made between 0.005 and 0.5 i^g/mL by serial dilutions using methyl-tert-butyl ether

(MTBE) + methanol (1+1) followed by filtration (0.2 pm nylon filter) prior to injection.

Sample Preparation. Vegetable oils (10-20 mg) were solubilized in 6 mL of

MTBE + methanol (1+1), filtered through a 0.2 pm nylon filter, and analyzed

immediately.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Instrumentation and Chromatography. An analytical (4.6 mm i d. x 250 mm, 5

urn) polymeric YMC™ C30 column (Waters, Milford, MA) was used and protected by a

Ci8 guard (4.6 mm i.d. x 50 mm) column (Vydac, Hisperia, CA) and a pre-column filter.

Column temperature was held constant at 25 °C to control for temperature fluctuations in

the laboratory at different times. The HPLC system consisted of a Hewlett Packard

model 1050 (Santa Clara, CA) solvent delivery system. Injection volume was 25 pL.

An eight channel 5600 Coularray™ electrochemical detector (ESA, Chelmsford,

MA) with potentials set between 200 and 620 mV in 60 mV increments from channel 1 to

8, respectively. At the end of each day, the analytical cells were cleaned by applying

oxidation and reduction potentials of 800 mV and -600 mV, respectively for 60 seconds.

This step aids in elimination of deposited compounds present on the cell surface that are

unreactive at the applied analytical potentials. Data collection and integration were

performed using the ESA Coularray™ version 1.01 software and data management

system.

The method of Ferruzzi et al. (1998) was modified to achieve baseline separation

of tocopherols and tocotrienols. 1.0 M ammonium acetate (pH 4.6) was prepared with

Cis Sep-Pak® (Waters, Milford, MA) solid phase extraction purified HPLC-grade water.

Solvent A consisted of methanol:MTBE:ammonium acetate water (88:5:5:2), while

solvent B consisted of methanol:MTBE: ammonium acetate (20:78:2). The following

linear gradient was used: 0 to 5 min, 100% solvent A; 5 to 45 min, 15% solvent A, 85%

solvent B; 45 to 50 min 100% solvent B.

Identification. When available, authentic standards were used for the

identification of carotenoids, tocopherols and chlorophylls. Carotenoid cis isomers

identification was aided by comparison to previously reported C30 UV-vis and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. electrochemical methods (Ferruzzi et al., 1998; Ferruzzi et al., 2001; Emenhiser et al.,

1995; Emenhiser et al., 1996). Tocotrienols were identified by co-chromatography using

tocotrienol containing oils and comparison to reported data (Damoko et al., 2000;

Drotleff and Tesoriere, 1999).

Detection limit.We defined the detection limit when the signal-to-noise ratio

equals 3. Standard solutions were diluted in 5-fold series and analyzed until the

detection limits were reached.

3.4. RESULTS AND DISCUSSION

HPLC separation. Figure 3.2. depicts a typical separation of major red palm oil

carotenoids, tocopherols and tocotrienols. The C3o stationary phase allows for efficient

separation of a-carotene (ail-frans, 13-c/s, 13’-c/s and 9-c/s isomers), p-carotene (all-

trans, 9-c/s and 13-c/s isomers) and all -trans lycopene. The 9 -c/s isomer of a-carotene

coeluted with all -trans p-carotene and further modification of the gradient elution was not

able to completely resolve the two peaks while maintaining a reasonable analysis time.

Lessin et al. (1997) faced the same problem when analyzing thermally processed carrot

using the same stationary phase. The amount of 9 -c/s isomer typically is comparable to

the corresponding 9-c/s a-carotene (Emenhiser et al., 1996; Lesin et al., 1997). In the

case of red palm oil, where p-carotene comprises more than 60 % of the total

carotenoids (Ong and Tee, 1992), the quantity of 9 -c/s a-carotene is minor compared to

the all -trans p-carotene and most likely does not affect the quantification of all -trans 15-

carotene. In addition to the carotenoids, baseline separation of a-, 5-, and y-tocopherols,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and a- and 5-tocotrienols present in red palm oil was achieved. Using a C30 column

coupled with a diode array detector, Darnoko et al. (2000) achieved a similar baseline

separation of tocopherols and carotenoids in red palm oil.

A typical electrochemical chromatogram of virgin olive oil is presented in Figure

3.3.A. Major tocopherols in olive oil were well resolved, although baseline separation of

(3- and y-tocopherol was not achieved. The two tocopherols differ only in the position of

the two methyl groups in their structures and most normal as well as reversed phase

HPLC methods (Dionisi et al., 1995; Psomiadou and Tsimidou, 1998; AOCS, 1998)

failed to completely separate this pair. Balz et al. (1992) used a diol column to achieve

baseline separation of (3- and -/-tocopherols and (3- and y-tocotrienols. The only

detectable chlorophyll derivative present in our virgin olive oil sample was pheophytin a.

To further test our method, we spiked the oil with fresh spinach leaves (Figure 3.3.B)

and were able to detect chlorophyll a and b in addition to pheophytin a. Gandul-Rojas et

al. (2000) detected chlorophylls and pheophytins in their virgin olive oil samples. The

discrepancy most likely comes from the age of the oil. Gandul-Rojas et al. (2000)

analyzed freshly extracted virgin olive oil, while our sample was purchased from a local

store and may have been exposed to light for a period of time. Studying the pigments

present in virgin olive oil, Minguez-Mosquera et al. (1990) found a decrease in

chlorophyll concentration during storage and pheophytin a was found to be the major

chlorophyll derivative after 8 weeks of storage.

In 1998, Psomiadou and Tsimidou developed a C18 HPLC method coupled with a

photodiode array detector, which was able to simultaneously detect tocopherols,

carotenoids and chlorophylls in virgin olive oil. Their method, however, was not able to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. separate the c/s-isomers of carotenoids. Using a C30 stationary phase and gradient

elution, analysis of tocopherols, geometrical isomers of carotenoids and chlorophylls in

vegetable oil was completed within 50 min. For the study of major palm oil carotenoids

(i.e. a- and (3-carotene), virgin olive oil or samples devoid of lycopene, analysis can be

terminated after the elution of 9-c/s (3-carotene in 37 min. Furthermore, analysis

specifically applied for tocopherols and tocotrienols can be completed in 20 min.

Electrochemical behavior. The use of a coulometric electrochemical array

detector enabled us to construct the characteristic CVC’s of carotenoids (Figure 3.4.A

and B), tocopherols (Figure 3.5.) and chlorophylls (Figure 3.6 ). Dominant responses for

(3-carotene and a-carotene were detected at the applied potentials of 380 and 440 mV,

respectively and similar to an earlier report (Ferruzzi et al., 1998). Using static systems.

Liu et al. (2000) demonstrated that the oxidation potential of p-carotene was lower than

a-carotene, indicating that the latter is more difficult to oxidize. The observed results

most likely were caused by the difference in the double bond position between p- and a-

carotene in their p-ionone rings. As a consequence, p-carotene has 11 conjugated

double bond systems, one more than a-carotene. Extension of the conjugated double

bond system, and not the oxygenation in the carotenoid structures as with xanthophylls,

was found to affect their electrochemical behavior. The observed dominant oxidation

potentials for zeaxanthin and lutein (dihydroxy p- and a-carotene, respectively) were

very similar to p- and a-carotene (Ferruzzi et al., 1998; Liu et al., 2000). Our data also

indicated that geometrical isomers of carotenoids oxidized at a higher potential than their

parent compounds, which was in agreement with previous studies (Ferruzzi et al.. 1998

and 2001). Therefore, in addition to their structures, molecular configuration appeared

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to be an important aspect of carotenoid electrochemical behavior. 5,6-Epoxy p-

carotene, an oxidation product of p-carotene, displayed a slightly higher maximum

response oxidation potential than its parent compound and similar to the 9-c/s and 13-c/s

P-carotene isomers.

The dominant response oxidation potentials of tocopherols are lower than

carotenoids (Figure 3.5 ). a-Tocopherol exhibited a dominant response oxidation

potential at around 260 mV, y-tocopherol and 6-tocopherol at around 320 and 380 mV,

respectively. A similar observation was made by Finckh et al. (1995) and Litescu and

Radu (2000). a-Tocotrienol behaved similar to its corresponding tocopherol, indicating

that the presence of three double bonds in its structure did not affect its electrochemical

properties. This observation reaffirms that electrochemical behavior is dependent on the

oxidation and reduction properties of a compound (Ferruzzi et al., 1998).

Chlorophylls and pheophytins oxidized at much higher potentials than

tocopherols and carotenoids (Figure 3.6 ), showing their dominant oxidation potentials

between 560 and 620 mV. The response factors of pheophytins were notably lower than

those of chlorophylls (data not shown), and this may affect the detection of pheophytins

when present in low concentrations in vegetable oil.

Sample preparation. Our C30 RP-HPLC-ECD methodology allowed for direct

injection of diluted oil samples (20-30 ng oil), therefore eliminating saponification

procedure and reducing the time for sample preparation. Psomiadou and Tsimidou

(1998) injected more than 20 times the amount of oil sample for their NP-HPLC with UV-

vis detection in order to obtain acceptable detector response. Furthermore,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. saponification is known to adversely affect carotenoids (Lietz and Henry, 1997; Khachik

et al., 1986; Kimura et al., 1990) and tocopherols (Lietz and Henry, 1997). This step has

been reported to induce carotenoid isomerization and degradation (Lietz and Henry,

1997). The same authors developed an enzymatic method, which hydrolyzes

triglycerides in red palm oil as an alternative to saponification. No significant loss of

carotenoids and tocopherols occurred. However, a 4 h sample preparation time was

required for the enzymatic method. Sanchez-Perez et al. (2000) employed a continuous

tocopherol extraction from vegetable oil by a silicone non-porous membrane coupled in

line with an RP-HPLC and a coulometric detector. Although the system was fully

automated, a washing step was required between successive analyses and a surfactant

was required to aid sample solubilization in injection solvents.

Detection limit, linearity range and reproducibility.Detection limits for

carotenoids, tocopherols and chlorophylls were determined by serial dilutions of (3-

carotene, a-tocopherol and chlorophyll a standards, respectively and the results are

shown in Table 3.1. The detection limits for carotenoids, tocopherols and chlorophylls

were found to be 1 fmol, 0.15 pmol and 0.5 pmol, respectively for a 25 faL injection, and

represent 1000, 25 and 5 fold enhancements over the reported UV-vis data (Psomiadou

and Tsimidou, 1998). Values for (3-carotene and a-tocopherol were similar to those

found in previous studies (Finckh et al., 1995; Ferruzzi et al., 1998; Ferruzzi et al., 2001).

The detector response was linear between 0.01 and 2.00 pg/mL for all the compounds

studied, as indicated by the correlation coefficients (Table 3.1.). Response factors for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. carotenoids (a-carotene, p-carotene and lycopene) were in similar ranges, while

tocopherols showed slightly lower responses by comparison. Chlorophyll a maintained a

response factor between that of carotenoids and tocopherols.

The reproducibility of the method was tested using samples representative of red

palm oil. Within-day variations (CV, n=5) were 4.5% for a-tocopherol, 2.3% for a-

tocotrienol, 6.3% for y-tocopherol, 4.0% for a-carotene and 2.0% for p-carotene. These

values are lower than previous reports for similar compounds (Finckh et al., 1995).

Between-day variations were similar for the tocopherols (3.2 % for a-tocopherol, 2.7%

for a-tocotrienol, 7.2% for y-tocopherol) and slightly higher for the carotenoids (5.2% for

a-carotene and 7.4% for p-carotene). Finckh et al. (1995) found the between-day

variations for both tocopherols and carotenoids were significantly higher than the within-

day variations. These differences can be attributed to sample preparation and baseline

noise from the electrochemical detector. Motchnik et al. (1994) and Ferruzzi et al.

(1998) suggested the use of a pulse dampener and/or low pulse pumps to reduce

background noise. Our solvent delivery system has a built-in pulse dampener. Although

Damoko et al. (2000) were able to achieve similar separations of red palm oil

tocopherols and carotenoids, their method exhibited larger variations (up to 25%) than

the current methodology. They noted larger variations for compounds with low

concentrations. This is most likely the result of harsh saponification conditions applied,

where red palm oil was heated at 100 °C for 30 min (Damoko et al., 2000). The ability of

our methodology to inject oil samples directly onto the HPLC system without introducing

error and loss from extraction, saponification and solvent removal may explain our lower

variations.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Application. The current methodology was applied to detect vegetable oil

adulteration based on their tocopherol patterns. Figures 3.7.A and B illustrate the

distinct tocopherol patterns of wheat germ and soybean oils. Figure 3.7.C demonstrates

the change in the tocopherol pattern when wheat germ oil was adulterated with soybean

oil in 2:1 ratio. In addition, this method has been successfully applied in studying the

oxidative stability of red palm oil (Puspitasari-Nienaber et al., 2000). Tocopherols,

tocotrienols, carotenoids (including their geometrical isomers) were quantified

simultaneously. This method also allowed for the identification of p-carotene oxidation

products (i.e. epoxy p-carotene) not previously identified because of their loss during

saponification.

3.5. CONCLUSIONS

We demonstrated here the use of a C3o RP-HPLC coupled with coulometric

electrochemical array detection to simultaneously separate and detect tocopherols,

tocotrienols, carotenoids and chlorophylls in vegetable oils. The detection limits for

carotenoids, tocopherols and chlorophylls were 5-1000 times better than the

conventional UV-vis detector. The detector’s sensitivity allowed direct injection of diluted

oil samples, eliminating sample loss during saponification, reducing time for sample

preparation and avoiding column overloading and fouling. This method can be applied

for rapid and sensitive analysis of oil quality and adulteration.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p-carotene 13-c/s p-carotene

a-carotene 9-c/s P-carotene

lycopene

a-tocopherol a-tocotrienol

OH OH U o y-tocopherol 5-tocopherol

Figure 3.1. Structures of carotenoids, tocopherols and tocotrienols

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All-frans ()-carotene

a-tocotrienol i All-frar?s a-carotene )

13-c/s p-carotene 13 -c/s a-carotene \ jj 9-c/s a-carotene 13-c/s u-carotene \ \

a-tocopherol i!n 9-c/s p-carotene

lycopene

•A: 500 mV 440 m v 380 mV

320 mV

- 260 mV

10.0 20.0 30.0 40.0 50.0 Retention time (minutes)

Figure 3.2. C30 HPLC separation of tocopherols, tocotrienols and carotenoid geometrical isomers in red palm oil.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) VIRGIN OLIVE OIL

j I a-Tocopherol

li! Pheophytin a , ,1 * I «r„e 620 mV lj\\K----- jl1V - /> , — P ft 'C ' />A'-— KI >->• 480 mV v>..:z:J c c i ’ _z." 200 mV

B) OLIVE OIL * SPINACH LEAVES

n Chlorophyll a

! a-Tocopherol i V I i i

I ’ Chlorophyll b I i!

• »ij, Lutein .I , Pheophytin o a (i-Carotene

620 mV 480 mV 200 mV 'j I i r

15 25 35 Retention time (minute)

Figure 3.3. C30 HPLC separation of tocopherols, carotenoids and chlorophylls in A) virgin olive oil and B) virgin olive oil spiked with spinach leaves.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Normalized Area (nC)

1200 • A) - A - all-trans-(i-Car 900 { -13 cis-li-Car A 9 cis-p-Car • epoxy (1-Car 600 j

300 j

200 260 320 380 440 500 560 620

1200 - A - all-trans-u-Car • 9 cis-a-Car 900 -A - 9' cis-a-Car -A -13 cis-a-Car -K- 13'cis-u-Car 600 • epoxy u-Car

300

380 440 500 560 Applied Potential (mV)

Figure 3.4. Current-voltage curves of A) p-carotene and its geometrical isomers and B) a-carotene and its geometrical isomers

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Normalized Area (nC) 1200

♦ — a -TOC ■ - a -Tot 900 A 0 -Toe • v -Toe 600 •

200 260 320 380 440 500 560 620 Applied Potential (mV)

Figure 3.5. Current-voltage curves of a-tocopherol, a-tocotrienol, 5-tocopherol and y-tocopherol

Normalized Area (nC) 1200 - ♦ Chi a • Chib 900 A - Phe a • Phe b 600

300 A

200 260 320 380 440 500 560 620 Applied Potential (mV)

Figure 3.6. Current-voltage curves of chlorophyll derivatives

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. !] A) WHEAT GERM OIL

j I a-Tocopherol

(i-Tocopheroi A

cx-Tocotrienol 420 m v nil\ \ MO m v -J \ J 320 m v ' 260 mV

y-tocopherol f, B) SOYBEAN OIL

o-Tocopherol i.: ' i: 1 t 1 j 1 «-Tocopherol I,'.1. i V 420 m v .. 360 m v - -• * e*' - 320 m v 260 m v

u-Tocopherol y-Tocopherol

(i-Tocopherol C) WHEAT GERM OIL ivTocopherol \ ^ + SOYBEAN OIL

n-Tocotrienol ^ /A 1 I..!i > ,1 1 [■ 1 « ° mv V - . . . 380 m v - - - - - ' - - 330 mv ' 280 mv

4 8 12 16 Retention time (minute)

Figure 3.7. C30 HPLC separation of tocopherols in A) wheat germ oil, B) soybean oil and C) mixture of wheat germ + soybean oil (2:1)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Linear range R2 y = a + b x a Detection (pg/mL) limit*3

a-Carotene 0.01 -2.00 0.9993 y = 3.3 + 4316.6 x 1 fmol

(3-Carotene 0.01 - 2.00 0.9953 y = 154.9 + 4271.0 x 1 fmol

Lycopene 0.01 -2.00 0.9988 y = - 42.4 + 3877.4 x 1 fmol a-Tocopherol 0.01 -2.00 0.9989 y = - 18.5 +2891.5 x 0.15 pmol 5-Tocopherol 0.01 -2.00 0.9988 y = - 19.3 + 1261.0 x 0.15 pmol y-Tocopherol 0.01-2.00 0.9959 y = 30.4 + 1020.9 x 0.15 pmol Chlorophyll a 0.01 -2.00 0.9981 y = - 59.5 +3130.3 x 0.5 pmol a y = peak area (nC) and x = concentration (pg/mL) b For 25 pL injections

Table 3.1. Linear ranges and detection limits of carotenoids, tocopherols and chlorophyll a analyzed on a C30 RP-HPLC with electrochemical array detection

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.6. REFERENCES

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Monitoring Of Ubiquinol-10, Ubiquinone-10, Carotenoids, And Tocopherols In

Neonatal Plasma Microsamples Using High-Performance Liquid Chromatography

With Coulometric Electrochemical Detection. Anal.Biochem. 232:210-216 (1995).

Gandul-Rojas, B., M. R. L. Cepero, and M. I. Minguez-Mosquera, Use Of Chlorophyll

And Carotenoid Pigment Composition To Determine Authenticity Of Virgin Olive

Oil. J.Am.Oil Chem.Soc. 77:853-858 (2000).

Guthrie, N., A. Gapor, A. F. Chambers, and K. K. Carroll, Inhibition Of Proliferation Of

Estrogen Receptor-Negative MDA-MB-435 And - Positive MCF-7 Human Breast

Cancer Cells By Palm Oil Tocotrienols And Tamoxifen, Alone And In

Combination. J.Nutr. 127:544S-548S (1997).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Haila, K. M., S. M. Lievonen, and M. I. Heinonen, Effects Of Lutein, Lycopene, Annatto,

And Gamma-Tocopherol On Autoxidation Of Triglycerides. Ibid. 44:2096-2100

(1996).

Khachik, F., G. R. Beecher, and F. Whittaker, Separation, Identification, And

Quantification Of The Major Carotenoid And Chlorophyll Constituents In Extracts

Of Several Green Vegetables By Liquid Chromatography. J.Agric.Food Chem.

34:603-616 (1986).

Kimura, M., D. B. Rodriguez-Amaya, and H. T. Godoy, Assessment Of The

Saponification Step In The Quantitative Determination Of Carotenoids And

Provitamin A. Food Chem. 35:187-195 (1990).

Lessin, W. J., G. L. Catignani, and S. J. Schwartz, Quantification Of Cis-Trans Isomers

Of Provitamin A Carotenoids In Fresh And Processed Fruits And Vegetables.

J.Agric.Food Chem. 45:3728-3732 (1997).

Lietz, G. and C. Henry, A Modified Method To Minimise Losses Of Carotenoids And

Tocopherols During HPLC Analysis Of Red Palm Oil. Food Chem. 60:109-117

(1997).

Litescu, S.-C. and G.-L. Radu, Estimation Of The Antioxidative Properties Of

Tocopherols - An Electrochemical Approach. Eur.Food Res. Technol. 211:218-

221 (2000).

Liu, D., Y. Gao, and L. D. Kispert, Electrochemical Properties Of Natural Carotenoids.

J.EIectroanal.Chem. 488:140-150 (2000).

Minguez-Mosquera, M. I., B. Gandul-Rojas, J. Garrido-Fernandez, and M. L. Gallardo-

Guerrero, Pigments Present In Virgin Olive Oil. J.Am.Oil Chem.Soc. 67:192-196

(1990).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Motchnik, P. A., B. Frei, and B. N. Ames, Measurement Of Antioxidants In Human

Blood-Plasma. Methods Enzymol. 234:269-279 (1994).

Ong, A. S. H. and E. S. Tee, Natural Sources Of Carotenoids From Plants And Oils.

Methods Enzymol. 213:142-167 (1992).

Psomiadou, E. and M. Tsimidou, Simultaneous HPLC Determination Of Tocopherols,

Carotenoids, And Chlorophylls For Monitoring Their Effect On Virgin Olive Oil

Oxidation. J.Agric.Food Chem. 46:5132-5138 (1998).

Puspitasari-Nienaber, N. L., D. B. Min, and S. J. Schwartz, High Concentration Of a -

And p-Carotenes Increase Oxidation And Tocopherol Degradation In Red Palm

Oil. Inform 11: S121 (2000).

Sanchez-Perez, A., M. M. Delgado-Zamarrefio, M. Bustamante-Rangel, and J.

Hemandez-Mendez, Automated Analysis Of Vitamin E Isomers In Vegetable Oils

By Continuous Membrane Extraction And Liquid Chromatography-

Electrochemical Detection. Ibid. 881:229-241 (2000).

Schwartz, S. J., S. L. Woo, and J. H. von Elbe, High-Performance Liquid

Chromatography Of Chlorophylls And Their Derivatives In Fresh And Processed

Spinach. J.Agric.Food Chem. 29:533-537 (1981).

Solomon, N. W., Plant Sources Of Vitamin A And Human Nutriture: Red Palm Oil Does

The Job. Nutr.Rev. 56:309-311 (1998).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4

Effects of Carotenoids on the Oxidative and Thermal Stability of Red Palm Oil

Ni Luh Puspitasari-Nienaber, David B. Min and Steven J. Schwartz*

Department of Food Science and Technology,

The Ohio State University

2015 Fyffe Road, Columbus, OH 43210-1007

Keywords: carotenoid, oxidative stability, thermal stability, palm oil, red palm oil.

tocopherol, tocotrienol

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1. ABSTRACT

The effects of carotenoids (500 f^g/g) and tocopherols (900 pg/g) on the oxidative

and thermal stability of red palm oil (RPO) were investigated. RPO was stored at

different temperatures in the dark for 12 weeks. Ten batches of frozen French fries were

fried per day for three consecutive days at 180 °C. Refined bleached and deodorized

palm oil (RBDPO) was the control oil. Oxidative degradation of carotenoids in RPO

followed zero-order reaction kinetics. Activation energies for the degradation of a- and

p-carotene in RPO were 8.10 and 9.12 kcal/mole. Storage time for the carotenoids to

deplete 50% (t1/2) was calculated as 12, 4 and 1.3 months at 25, 37 and 60 °C,

respectively. Tocopherols degraded 1.3 to 3.2 times faster in RPO than control.

Peroxide values of RPO increased during storage and peaked after 4, 3 and 1 week at

25, 37 and 60 °C, respectively. When used for frying, > 90% carotenoids were degraded

at the end of the come-up time (15 min). Tocopherol also degraded faster in the

presence of carotenoids, especially after French fries were added to the hot oil. These

observations suggest that RPO is stable during storage but not suitable for frying. In

addition, tocopherol played an important role in maintaining the oxidative and thermal

stability of RPO.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. INTRODUCTION

The traditional biological function of carotenoids, mainly (3-carotene, is its

provitamin A activity. Plant carotenoids are the main supply of provitamin A in

developing countries where consumption of diets rich in vitamin A is low and vitamin A

deficiency is a problem. The bioconversion efficiency of plant carotenoids to vitamin A is

low (de Pee et al., 1998). However, crude palm oil (CPO) which contains high amount

(500-800 pg/g) of provitamin A carotenoids (Goh et al., 1985) in a more bioavailable

form (Solomon, 1998, van het Hof et al., 1999) has not been optimally utilized to

eliminate vitamin A deficiency.

CPO was introduced in the late 1960s as a source of daily dietary provitamin A

for preschool children in Indonesia (Oey et al., 1967). CPO decreased xerophthalmia

significantly, but the oil was not popular among the children because of its non-pleasant

taste. Rukmini, 1994 and Manorama et al., 1996 reported the effectiveness of [3-

carotene from red palm oil in restoring and maintaining the vitamin A status in school

children. Red palm oil was also reported to improve the vitamin A status of lactating

women and their infants (Canfield and Kaminsky, 2000; Lietz, et al., 2000). Solomon

(1998) suggested that RPO is an effective food-based strategy to combat vitamin A

deficiency. Red palm oil (RPO) is a refined CPO that contains more than 80% of the

original carotenoids and tocopherols and is now available on the market (Ong and Choo,

1997).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Palm oil is rich not only in p-carotene but also in a-carotene, another provitamin

A carotenoids, in the ratio 2:1 (Goh et al., 1985). The structure of a-carotene is very

similar to p-carotene, except the double bond located on the second p-ionone ring is in a

different location and therefore not part of the chromophore. The presence of a-

carotene was shown not to affect the bioavailability of p-carotene from palm oil (van het

Hof et al., 1999). Information on the carotenoids stability in RPO during storage,

however, is not available. The present study investigated the effects of a- and p-

carotene on the oxidative and thermal stability of RPO during storage and frying.

4.3. EXPERIMENTAL PROCEDURES

Materials. Refined bleached deodorized palm oil (RBDPO) was purchased from

Arista Industries Inc. (Darien, CT). RBDPO was purified prior to use by passing it

through a silica gel 60 (Fluka Chemie AG, Switzerland) open column chromatography

Tocopherol stripped palm oil was prepared following the method of Lampi et al. (1992).

Palm carotene was a gift from Quest International (Hoffman Estates, IL).

Chromatography standards (P-carotene, a-, 5- and -/-tocopherols) were from Sigma

Chemical Co. (St. Louis, MO) while a-carotene was from Fluka, Buchs, Switzerland.

HPLC grade solvents were used throughout the experiment.

Three different oils were tested: 1) RPO (RBDPO + palm carotene, containing

470 ^g/g total carotenoid and 935 pg/g total tocopherols), 2) RPO without the indigenous

tocopherols (tocopherol stripped RBDPO + palm carotene, containing 500 p.g/g total

carotenoid and 3) RBDPO as the control oil.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Oxidation conditions. Oil (1 mL) was stored at 25, 37 and 60 °C in 2 mL vials

in the dark for 12 weeks. Sampling was done every week and samples were stored at -

80 °C until analyzed. RBDPO was used as the control oil.

Frying conditions. The frying experiment was designed to simulate typical

frying practices. One liter of RPO was used to fry frozen French fries (100 g/frying). Ten

frying experiments (at 180 °C, 3 min/frying) were conducted each day for 3 consecutive

days. The time needed to raise the oil temperature to 180 °C (come-up time) was 15

min, and therefore, the total heating time each day was 45 min. After the tenth frying,

used frying oil was filtered and stored in a capped brown bottle at room temperature.

Before the first frying on the next day, used frying oil was mixed with fresh oil in 1 :1 ratio

to replace oil losses due to absorption into foods and sampling.

Sampling of oil was performed after the 1st to the 10th frying at day 1, 2 and 3.

Additional samples were collected during the come up time (1, 2, 3, 5, 10 minutes). Hot

oil was cooled down as quickly as possible in an ice bath, placed into small vials ( 2 vials

per sample) and stored at -80 °C until analyzed. Samples were analyzed for

carotenoids, tocopherols, their degradation products and total polar compounds.

Peroxide value and total polar compound analysis. Peroxide values were

analyzed using the AOCS Official Method #Cd 8-53 (AOCS, 1998). Total polar

compound measurements were according to Walking and Wessels (1981).

Carotenoid and tocopherol analysis. Carotenoids (a-carotene, p-carotene and

their isomers) and tocopherols (a, 5, y-tocopherols and a-tocothenol) were analyzed

simultaneously with C 30 HPLC equipped with an electrochemical detector (Puspitasari-

Nienaber et al., 2002).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sample and standard preparation for HPLC analysis. Oil samples (10-20 mg)

were solubilized in 6 mL of methyl-tert-butyl ether (MTBE) + methanol (1+1), filtered

through a 0.2 urn nylon filter, and injected on the HPLC immediately. Standard solutions

of carotenoids were made in hexane, while tocopherols were in methanol and their

concentrations were quantified spectrophotometrically using published extinction

coefficients (Britton et al., 1995; AOCS, 1998). Serial dilutions were made from the

stock solutions using MTBE + methanol (1+1).

HPLC conditions. Analysis of carotenoid and tocopherol were performed on a

polymeric YMC C 3o, 4.6 mm i.d. x 250 mm, 5 urn reversed-phase column (Waters,

Milford, MA), protected by a pre-column filter and a C 18 guard column (Vydac, Hisperia,

CA). The HPLC system consisted of a Hewlett Packard model 1050 (Santa Clara, CA)

solve- • '■-'.‘iv.- v system coupled with a coulometric array electrochemical detector (ESA

Model L Of Coularray™, Chemlsford, MA). Two analytical cells consisted of eight

channels were set between 200 to 620 mV in 60 mV increments. The ESA Coularray™

version 1 u 1 software was used for data collection.

Gradient elution was as follow; 0 to 5 min, 100% solvent A; 5 to 45 min, 15%

solvent A, 85% solvent B; 45 to 50 min 100% solvent B. Solvent A consisted of

methanol:MTBE:ammonium acetatewater (88:5:5:2), solvent B consisted of

methanol:MTBE:ammonium acetate (20:78:2). Authentic standards were used for the

identification of carotenoids and tocopherols. Carotenoid cis isomers were identified by

comparison to previously reported C 30 methods (Emenhiser et al., 1996a and 1996b,

Ferruzzi et al., 1998). Tocotrienols were identified by co-chromatography using

tocotrienol containing oils and comparison to reported data (Drotleff and Tesoriere,

1999).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.4. RESULTS AND DISCUSSION

Systematic investigation of the individual role of carotenoids and tocopherols on

the oxidative and thermal stability of RPO is not easy because of their similar properties

(e.g. solubility, highly oxidative) and the presence of other minor components (e.g.

sterols, minor phenolic compounds), which may act as synergists or antagonists during

the course of the study. This research was, therefore, carried out in a model system

consisting of RBDPO and palm carotenoids in the amount close to the commercially

available RPO. Our RPO model system contains 470 ^ /g carotenoids (37.2% and

62.8% a-carotene and 3-carotene, respectively) and 935 pg/g tocopherols (20.6 %. 10.9

%, 37.6 % and 31.0 % a-tocotrienol, 5-tocopherol, y-tocopherol and a-tocopherol,

respectively). A typical separation of major RPO carotenoids, their isomers, degradation

products and tocopherols is represented in Figure 4.1.

Oxidative stability. The oxidation of a-carotene and 3-carotene during RPO

storage followed zero-order reaction kinetics (Table 4.1, Figure 4.2.A), indicating that the

oxidation rate was constant throughout the study and independent of concentration.

Based on the reaction kinetics, storage time for the carotenoids to deplete 50% (ti/2) was

calculated as 12, 4 and 1.3 months at 25, 37 and 60 °C, respectively. Zero-order

reaction kinetics for 3 -carotene degradation was reported earlier for 3 -carotene

dissolved in toluene and exposed to continuous stream of oxygen (El-Tinay and

Chichester, 1970) and 3-carotene, 3 -cryptoxanthin and lycopene exposed to oxygen and

ozone in an aqueous system (Henry et al., 2000). More recently, Hackett (2001) studied

lycopene degradation in different tomato oleoresins and found zero-order kinetics in ail 55

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cases. Conversely, first-order kinetics was reported for carotenoid degradation in

crystalline p-carotene (Chen et al., 1994) and low moisture samples such as spray-dried

encapsulated carrot (Wagner and Warthesen, 1995), and spray-dried microalga (Orset

et al., 2000). These findings suggest that carotenoid degradation depends on factors

such as carotenoid source, type of matrix, and presence of other antioxidants.

The effect of other antioxidants on the reaction kinetics of carotenoid degradation

in RPO was shown in the following experiments. When tocopherols were removed,

palm carotenes degraded at greater rates following first-order reaction kinetics (Figure

4.2.B). At 60 °C, the half-life of palm carotenes in the absence of tocopherols occurred

after only 18 h of storage, while total loss occurred after 100h. Similar to our results,

Henry et al. (1998a) found first-order reaction kinetics for the oxidative degradation of

various carotenoids in tocopherol-stripped safflower oil.

The activation energies for a-carotene, p-carotene and total carotene oxidation in

RPO were found to be 8.10, 9.12 and 11.23 kcal/mole, respectively. The standard

deviations of the activation energies were between 4.6 and 6.9%. Our findings were

similar to published reports for various carotenoids (El-Tinay and Chichester, 1970,

Henry et al., 1998 and Hackett, 2001). These values suggest that oxidative degradation

of carotenoids in RPO is not temperature dependent.

At all temperatures studied, our results showed that a-carotene oxidation

proceeded at lower rates than p-carotene (Table 4.1.). This confirmed results of another

study (Anguelova and Warthesen, 2000) which compared the antioxidant effectiveness

of various carotenoids during oxidation of methyl linoleate. Using 2,2 -azinobis (3-

ethylbenzothiazoline- 6 -sulphonic acid diammonium sulfate (ABTS) as radical initiators.

Miller et al. (1996) also reported similar results. Experiments conducted in our

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. laboratory using a similar methodology showed no difference in antioxidant activity

between all -trans a-carotene and p-carotene (Bohm et al., 2001). However, Farombi

and Britton (1999) reported that a-carotene was a superior antioxidant than p-carotene

in a peroxy radical-mediated oxidation of phosphatidyl choline. The contradicting results

could partly be explained by the structural similarity between a-carotene and p-carotene.

Any difference in the reaction conditions such as the type of radical involved, the type of

solvent used and the presence of lipid would have a big effect on the reactions involving

a-carotene and p-carotene.

To better understand factors important for RPO stability, tocopherol degradation

was also examined during storage. Our data indicated that total tocopherols degraded

following zero-order kinetics (Figure 4.3.A) and they degraded 1.3 to 3.2 times faster in

RPO than in the control (Table 4.2.). Degradation rate for individual tocopherols were as

follows: '/-tocopherol > a-tocopherol > 5-tocopherol = a-tocotrienol. Haila et al. (1996)

reported that lutein was consumed faster in the absence of 7 -tocopherol. However, 7 -

tocopherol consumption was not affected when lutein was added. This difference may

be due to the lower lutein concentration (< 60 pg/g) used in the study. Tocopherols are

known to be more effective lipid antioxidants than carotenoids, and together they act

synergistically in preventing lipid oxidation (Haila et al., 1996; Henry et al., 1998b).

Perrin et al. (1996) reported that a-tocopherol plays a major role in protecting p-carotene

from oxidation. Moreover, they suggested that tocopherols retard the formation of

carotenoid degradation products (Perrin et al., 1996; Haila et al., 1996). Our RPO

contains tocopherols as well as tocotrienols. The effectiveness of palm tocotrienols as

antioxidants has been demonstrated (Gapor et al., 1989) and the activity increased with

increasing concentration.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The increase of oxidative stress in RPO during storage was apparent (Figure

4.4 ). Peroxide values increased and peaked after 4, 3 and 1 week storage at 25, 37

and 60 °C, respectively. No increase was observed in the control. These data

suggested that carotenoids induced hydroperoxide formation and that the rate of

hydroperoxide degradation increased with increasing temperature. Similar findings have

been reported by other investigators. Anguelova and Warthesen (2000) reported a

tendency of increased hydroperoxide formation with the increase of p-carotene

concentration in their methyl linoleate oxidation system. Furthermore, they suggested

that higher p-carotene concentrations did not offer additional antioxidant activity and p-

carotene could potentially become a prooxidant. Henry et al. (1998b) observed the

prooxidant effects of p-carotene and lycopene at concentrations greater than 500 |.ig/g in

tocopherol stripped-safflower oil. Steenson and Min (2000) reported that p-carotene

thermal degradation products, but not p-carotene, act as prooxidants by increasing the

autoxidation rate of soybean oil. The type of p-carotene thermal degradation products,

however, was not indicated.

Oxidation of p-carotene is a complicated series of reaction involving self-initiated

oxidation (El-Tinay and Chichester, 1970; Burton and Ingold, 1984) as well as reaction of

p-carotene with free radicals (Liebler, 1993). In the presence of lipid, p-carotene

oxidation is further complicated by the involvement of lipid peroxyl radicals (Tsuchihashi

et al. 1995). These radicals add to a double bond in p-carotene molecule to give a

resonance-stabilized radical, leading to several competing reactions. At higher p-

carotene concentration, autoxidation proceeded and eventually produced polymeric

products. In addition, lipid-p-carotene radical may also attack lipid molecules to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. generate lipid peroxyl radicals and induce a chain oxidation reaction (Tsuchihashi et al.

1995).

Thermal stability. Figure 4.5.A shows the degradation of carotenoids during

frying at 180 °C. At Day1, > 90% of the carotenoids degraded at the end of the come-up

time (15 min). The first few batches of french fries were bright orange and the color of

french fries from the later batches was darker. The same trend was observed for the

second and third days of frying. (3-Carotene has been reported as unstable at elevated

temperatures. Onyewu et al. (1986) showed 92% p-carotene degradation upon heating

in glycerol at 210 °C for 15 min, while Papadopoulou and Ames (1994) reported

complete destruction of p-carotene after heating in liquid paraffin at 210 °C for 15 min.

The presence of food constituents has also been reported to affect the degradation rate

of carotenoids. Marty and Berset (1990) indicated that prolonged heating of crystalline

p-carotene at 180 °C only caused a limited breakdown of the molecule. However, in the

presence of water and starch higher amount of p-carotene was lost at the same

condition. In addition, our data showed that more than 50% of carotenoids remained in

RPO after 5 min of heating (Figure 4.5.A). This suggests that RPO can still be used in

food preparation applying mild heating, such as in stir-frying or sauce preparation.

Tocopherol degradation during frying followed the same pattern as their oxidative

degradation in RPO. Tocopherol degraded faster in the presence of carotenoid,

especially after french fries were added to the hot oil (Figure 4.6.A). At the end of the

come-up time, 71.7 and 65.3% of tocopherols degraded in both RPO and control.

However, upon addition of french fries, tocopherols were completely degraded after the

5m frying in RPO. At the end of the 10m frying, the control oil still contained 15.7% of

tocopherol (Figure 4.6.B). After the second day of frying, no difference in tocopherol

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. degradation was detected between RPO and control. Most likely at this point, the level

of thermal degradation products of carotenoids, tocopherols and lipids already reached

the point where addition of fresh oil at the beginning of the day did not offer extra

protection. The degradation pattern of individual RPO tocopherol during frying was very

different from its oxidative degradation. After 15 min heating, 66.9, 60.3 and 60.9% u-

tocopherol, 5-tocopherol and y-tocopherol, respectively remained in the oil. The level

was drastically reduced to 31.4% when french fries were added to the hot oil. On the

contrary, a-tocotrienol was more stable than the tocopherols. After 15 min heating

76.7% remained in the oil and the addition of french fries only reduced the level to

66.9%. Our data also suggested that at frying conditions, tocopherols in RPO were

more stable than carotenoids.

The amount of heating applied to our RPO was not sufficient to significantly

increase the formation of total polar compounds. Many European countries set a limit of

25% total polar compounds in frying oil. Heating of corn oil for 6 day ( 8 h/d) at 190 °C

was required to reach that limit and oils with higher levels of unsaturated fatty acids

produced more polar compounds compared to the more saturated oils (Takeoka et al..

1997).

Degradation products of palm carotenes. During RPO oxidation, 9-c/s and

13-c/s p-carotene were identified. At 25 °C, the amount of the 9-cis isomer was relatively

constant at 58 ng/g throughout the study, while 13-c/s level increased from 13 to 40 pg/g

(Figure 4.7.A). At higher temperatures, both isomers present at similar levels (50 pg/g)

and the level dropped during storage. The decrease in a\\-trans p-carotene was not

accompanied by a concurrent increase in either the 9-c/s or 13-c/s isomers, suggesting

differences in the observed degradation and formation kinetics of the p-carotene

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. isomers. The 13-c/s p-carotene has been reported as the predominant isomer formed

upon heating, while the 9-c/s isomer was present in lower levels (Chandler and

Schwartz, 1988; Chen et al., 1994; Henry et al., 1998). Although the 9-c/s, 13-c/s and

13 -c/s isomers of a-carotene were detected in our samples, only the 9-c/s isomer was

quantified due to the low concentrations of the other two isomers. The formation and

degradation of 9-c/s a-carotene was similar to that of 9-c/s p-carotene (Figure 4.7.B).

The major isomers found during frying was similar to the isomers formed during

oxidation (Figure 4.5.B). Interestingly, their formation and degradation patterns were

different. The 9-c/s a-carotene and 9-c/s p-carotene were formed at the early stage of

heating and reached their maximum levels 1 min after heating. The 13-c/s p-carotene

was formed slower and did not reach its maximum level until 1 0 min after heating.

Epoxy p-carotene has been reported as one of the major oxidation products of p-

carotene (Marti and Berset, 1990; Handelman et al., 1991). A higher production of p-

carotene epoxide was noted when a higher concentration of p-carotene was present

during oxidation of methyl linoleate (Anguelova and Warthesen, 2000). We only

detected a small amount of 5,6 p-carotene epoxide in our RPO stored at 25 °C and only

trace amount was detected in the oil stored at 37 and 60 °C. However, up to 100 pg/g of

5,6 p-carotene epoxide was detected in other batches of RPO stored at -20 °C (Figure

4.1.). This suggests that the compound was formed and degraded at the same time,

and degradation was much faster at higher temperature.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.5. CONCLUSIONS

Our results suggested that a-carotene and p-carotene in RPO were stable during

storage. However, these compounds were very heat labile and RPO was not suggested

for deep-frying. Mild heating can still be applied to RPO while maintaining a beneficial

amount of carotenoids and tocopherols in the oil. The significant role of tocopherols in

maintaining carotenoid stability in RPO can be used by food processors adding

carotenoids to their products to provide additional antioxidant to minimize their

degradation.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Temperature Rate constant 9 R2 (°C) (cone week'1)

a-Carotene 25 3.518 ± 0.086 0.993 37 5.027 ±0.152 0.990 60 14.418 ± 1.222 0.952

(3-Carotene 25 6.176 ±0.373 0.961 37 7.561 ± 0.278 0.985 60 29.238 ± 2.509 0.951

Total Carotene 25 7.259 ± 0.685 0.911 37 16.093 ± 0.605 0.985 60 54.051 ± 2.924 0.980

a Means ± standard error of means.

Table 4.1. Kinetic parameters for a-carotene, (3-carotene and total carotene degradation in red palm oil during storage

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Temperature Rate constant 9 R2 (°C) (cone week'1)

Red palm oil 25 7.51 ± 2.46 0.700 37 7.50 ± 1.49 0.835 60 17.22 ±3.03 0.915

Control 25 11.68 ± 1.67 0.645 37 23.87 ± 1.98 0.839 60 22.79 ± 2.75 0.783

a Means ± standard error of means.

Table 4.2. Kinetic parameters for total tocopherol degradation in palm oil during storage

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. all-frans u-carotene 5.6-epoxy p-carotene i* j i! i1 all-frans p-carotene

13 -c/s a-carotene u 9-c/s a-carotene y-tocopherol

13-c/s a-carotene ij 9-c/s p-carotene

i a-tocopherol

10 20 30 Retention time (minute)

Figure 4.1. Separation of major RPO carotenoids and tocopherols on a C30 RP- HPLC with coulometric electrochemical array detection.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 700 — 600 i 500 400 s 300 ♦ 25 C

200 ■ 37 C 100 A60C

0 3 6 9 12 Storage Time (week)

900 Carotene 750 ♦ Beta-Carotene 600 • e ■Alpha-Carotene so S 450 ■ § 300 o c ♦ ♦ o o 150 ■

0 20 40 60 80 100 120 Storage Time (h)

Figure 4.2. A) Degradation of carotenoids in RPO with indigenous tocopherols during storage at 25, 37 and 60 °C. B) Degradation of carotenoids in RPO without indigenous tocopherols during storage at 60 °C.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1000

900 • e o 800 • 3 sc 700 ■ ♦ 25 C o« c ■ 37 C o 600 ■ o A60C 500 0 3 6 9 12

Storage Time (week)

B) 500 ♦ alpha-tocopherol ■ detta-tocopherol j j 400 A gamma-tocopherol ♦ alpha-tocotnenol 3. § 300 3 2 f£ 200 so c oo 100 0 • 0 3 6 9 12 Storage Time (week)

Figure 4.3. A) Degradation of tocopherols in RPO during storage at 25, 37 and 60 °C. B) Degradation of individual tocopherol in RPO during storage at 37 °C.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 20 25C 37C 16 60C I E 12 s

> 8 1 * 5 o 4

0 0 3 6 9 12 Storage Time (week)

B) 12

> 25C 4 37C s 60C a. 0 0 3 6 9 12 Storage Time (week)

Figure 4.4. Peroxide values (meq/kg) of A) RPO and B) control oil during storage at 25, 37 and 60 °C.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced Figure 4.5. A) Degradation of carotenoids in RPO during come-up time and frying frying and time come-up during RPO in carotenoids of Degradation A) 4.5. Figure Concentration (nfl/fl) Concentration (ng/fl) 700 600 • 600 50 40 500 300 • 300 20 200 0 1 100 - during come-up time and frying of french fries. french of frying and time come-up during of french fries. B) Formation and degradation of carotene isomers isomers carotene of degradation and Formation B) fries. french of Come-Up Come-Up Time Time ID 9cs (x-carotene 9-cis ■ [l-carotene 13-cis □ 9cs [i-carotene 9-cis □ □ Day 3 BDay 2 DayBDay 3 □ 1 Day □ Frying Frying m 69 1000

800

g 600

1 4 0 . l -

® v Day 1 r / / Day 3 n

Coma-Up Prying Time

Coma-Up Frying Tima

Figure 4.6. Degradation of tocopherols in A) RPO and B) control oil during come- up time and frying of french fries.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 400

'Si 300

Total 3 200 All-trans 13-cis g 100

0 3 6 9 12 Storage Time (week)

250

a 200 ■ a. c 150 ■ 3o S Total c 100- All-trans • o 9-cis O 50 ■ O t

0 3 6 9 12 Storage Time (week)

Figure 4.7. Formation and degradation of A) (3-carotene and B) a-carotene isomers in RPO during storage at 37 °C.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.6. REFERENCES

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Ong, A. S. H. and Y. M. Choo.Carotenoids and tocols from palm oil. Natural

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of p-carotene. J.Agric.Food Chem. 30: 1147-1151, 1982.

Orset, S., G. C. Leach, R. Morais, and A. J. Young. Spray-drying of the microalga

Dunaliella salina: Effects on p-carotene content and isomer composition.

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Papadopoulou, K. and J. M. Ames. Kinetics of all-trans p-carotene degradation on

heating with and without phenylalanine. J.Am.Oil Chem.Soc. 71: 893-896, 1994.

Perrin, D., H. El Oualja, and R. Martin. Antioxidant properties of all-trans-p-carotene/a-

tocopherol mixtures in the induced oxidation of ethyl linoleate. J.Chim.Phys. 93:

1462-1471, 1996.

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of tocopherols, carotenoids and chlorophylls in vegetable oils by direct injection

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Nutr.Bull. 15: 2-5, 1994.

Solomon, N. W. Plant sources of vitamin A and human nutriture: red palm oil does the

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van het Hof, K. H., C. Gartner, A. Wiersma, L. B. M. Tijburg, and J. A. Weststrate.

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Walking, A. E. and H. Wessels. Chromatographic separation of polar and non

components of frying fats. J.Am.Oil Chem.Soc. 64: 1329-1330, 1981.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5

p-Carotene Epoxides:In Vitro Digestive Stability, and Their Uptake and

Conversion to Vitamin A by Caco-2 Human Intestinal Cells

Ni Luh Puspitasari-Nienaber1, Mark L. Failla2 and Steven J. Schwartz1*

1 Department of Food Science and Technology

The Ohio State University

2015 Fyffe Road, Columbus, OH 43210-1007

2 Department of Human Nutrition

The Ohio State University

1787 Neil Avenue, Columbus, OH 43210-1295

Keywords: 0-carotene, bioavailability, Caco-2, p-carotene 15,15’-dioxygenase, epoxide.

epoxy p-carotene, in vitro digestion, vitamin A 77

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1. ABSTRACT

The objective of this study was to investigate the digestive stability, and small

intestinal cellular uptake and metabolism of p-carotene and several of its epoxides from

foods using a simulated gastric and small intestinal digestion procedure coupled with the

Caco-2 human intestinal cell line. Epoxy p-carotenes were synthesized and added to

either applesauce, fresh palm oil or used palm oil. 5,6-Epoxy p-carotene decreased

22.9 ± 9.7% immediately after addition to applesauce and rearranged to 5,8-epoxy p-

carotene. In contrast, 5,6-epoxy p-carotene was stable in both oils. Micellarization

efficiency of p-carotene, 5,6-epoxy p-carotene and 5,8-epoxy p-carotene when added to

the applesauce meal individually were 11.6 ± 0.6%, 16.6 ±1.8 % and 16.3 ± 2.3%,

respectively. The type of food matrix significantly affects micellarization of carotenoids

from the test meals. All carotenoids tested were better micellarized during digestion of

palm oil than during digestion of applesauce. Micellarization of p-carotene was

significantly lower (p < 0.05) when used palm oil was digested than fresh palm oil.

Micellarization of p-carotene epoxides was similar during digestion of used and fresh

palm oil. Micellarization of p-carotene was not altered significantly (p > 0.05) when

equivalent amounts of epoxy p-carotene were present in applesauce. In contrast,

micellarization of 5,6-epoxy p-carotene and 5,8-epoxy p-carotene were significantly (p <

0.0001) suppressed by 74 and 62%, respectively when equimolar of p-carotene was

present. Cellular accumulation of p-carotene epoxides was evaluated using 78

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. differentiated cultures of the parent line (HTB37) and the TC7 clonal line of Caco-2

human intestinal cells. Cellular accumulation of [3-carotene epoxides (1.9 ± 0.1 and 2.3

± 0.2 pmol/mg protein for 5,6- and 5,8-epoxy p-carotene, respectively) was significantly

lower (p < 0.001) than that of p-carotene (3.5 ± 0.2 pmol/mg protein). The intracellular

conversion of 5,6-epoxy p-carotene and 5,8-epoxy p-carotene to retinol and retinyl

palmitate was only 10% that of p-carotene. These results show that p-carotene

epoxides are accessible for uptake into intestinal cells, although the degree of

micellarization depends on food matrix and interactions with other carotenoids during in

vitro digestion. In addition, Caco-2 cells accumulated and converted p-carotene

epoxides to vitamin A less efficiently than p-carotene.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2. INTRODUCTION

Epoxycarotenoids, such as (3-carotene and lutein epoxides, violaxanthin,

neoxanthin and fucoxanthin, are widely distributed in nature. In plants,

epoxycarotenoids serve as precursors of the plant growth regulator abscisic acid (Rock

and Zeevaart, 1991). Their presence in fruits and green vegetables (Khachik et al..

1991; Barua and Olson, 1998; Barua and Olson 2001), red palm oil (Ong and Tee. 1992)

and brown algae (Sugawara et al., 2002) has been reported. In addition,

epoxycarotenoids are well-known oxidation products of carotenoids (Kennedy and

Liebler, 1992, Handelman et al., 1991) that form during food processing and storage

(Marti and Berset, 1988; Tonucci et al., 1995; Puspitasari-Nienaber et al., 2000). The

possibility that epoxycarotenoids may be generated in vivo is supported by their

appearance when solutions of p-carotene are exposed to cigarette smoke (Baker et al..

1999) and during incubation of p-carotene with rat intestinal mucosa homogenates

(Tang et al., 1991). Moreover, 5,6-dihydroxy-5,6-dihydrolycopene is present in human

sera (Khachik et al., 1995) and human milk (Khachik et al., 1997) of human subjects.

The amount of this compound in tomato products consumed by these subjects was too

low to account for the concentration present in sera, suggesting oxidation of lycopene to

5,6-lycopene epoxide and subsequent reduction to 5,6-dihydroxy-5,6-dihydrolycopene in

vivo.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The biological activities of epoxy carotenoids remain unclear. Several reports

suggest that epoxy carotenoids have chemopreventive properties. Okuzumi et al.

(1990) demonstrated the inhibitory effects of fucoxanthin in human malignant tumor

cells. Also, neoxanthin inhibited DMBA-induced carcinogenesis in the hamster buccal

pouch model (Chang et al., 1995). More recently, Kotake-Nara et al. (2001) reported

that neoxanthin and fucoxanthin were cytotoxic for human prostate cancer cells in vitro,

and Duitsman et al. (1999) showed that p-carotene epoxides induced the differentiation

of NB4 leukemia cell line. However, in vitro binding of benzo[a]pyrene metabolites to

DNA was increased in the presence of p-carotene epoxides, suggesting that the epoxy

carotenoids can act as co-carcinogens (Salgo et al., 1999).

Available information on human absorption of epoxycarotenoids is conflicting.

Efficient absorption of 5,6-epoxy and 5,8-epoxy p-carotene by human subjects was

reported (Barua, 1999), although epoxyxanthophylls such as 5,6-epoxy lutein and

5,6,5’,6-diepoxy zeaxanthin were not absorbed by humans (Barua and Olson, 2001).

Similarly, Khachik et al. (1995) suggested that lycopene epoxides present in sera were

generated post-absorption.

We have used an in vitro digestion method that simulates gastric and small

intestinal processes to screen the digestive stability and bioaccessibility of carotenoids

and chlorophylls from foods (Garrett et al., 1999; Garrett et al., 2000; Ferruzzi et al.,

2001). The amount of test compounds transferred from the food matrix to the aqueous

fraction was used to estimate pigment bioaccessibility from foods. The availability of the

micellarized lipophiles was evaluated by determining accumulation by the Caco-2 human

intestinal cells. Differentiated monolayers of Caco-2 cells exhibit similar morphological

and biochemical characteristics of absorptive enterocytes (Pinto et al., 1981). In

addition, the TC7 clone of the Caco-2 cell line possesses p-carotene 15,15 -dioxygenase 81

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity and it has been used to study the provitamin A activity of p-carotene (During et

al., 1998 and 2001). Combination of the in vitro digestion procedure and the TC7 cell

model provides a tool for the systematic investigation of factors affecting bioaccessibility

and provitamin A activity of various carotenoids from foods.

The objective of this study was to examine the stability and micellarization of p-

carotene epoxides during in vitro digestion, and the accumulation and conversion of

micellarized epoxides to vitamin A by human intestinal Caco-2 cells.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.3. EXPERIMENTAL PROCEDURES

Chemicals and Standards. Certified HPLC and ACS grades organic solvents

(Fisher Scientific, Fair Lawn, NJ) were used for extraction, synthesis and

chromatography. p-Carotene was purchased from Sigma Chemical Co. (St. Louis, MO)

and 3-chloroperoxy benzoic acid was from Aldrich (Milwaukee, Wl). Refined bleached

deodorized palm oil (RBDPO) was purchased from Arista Industries Inc. (Darien, CT).

All other chemicals were of analytical grade. Used palm oil was prepared as described

in a separate study (Puspitasari-Nienaber et al., 2000).

Synthesis and Purification of p-Carotene Epoxides. p-Carotene epoxides

(Figure 5.1.) were synthesized as described by Barua and Olson (1998). Briefly, p-

carotene (2 mmol) was dissolved in 100 mL methyl tert-butyl ether (MTBE) before the

addition of 2 mmol 3-chloroperoxy benzoic acid. Synthesis was performed in the dark at

room temperature for 1 h. The reaction was terminated by the addition of water and

neutralization of the mixture with 0.1 N NaOH. p-Carotene epoxides were separated

using a preparatory (19 x 300 mm) ^ondapak C i8 HPLC column (Waters, Milford, MA).

Methanol MTBE (85:15) was used as the mobile phase at a flow rate of 5.0 mL/min. The

extract obtained from synthesis was injected with a 1 mL sample loop. Individual

fractions were dried under nitrogen and stored at -80 °C. In addition to their retention

times on the HPLC column, the identity of epoxides was confirmed using their UV-vis

spectra and chemical derivatization for epoxide group (Britton, 1995). 83

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Preparation of Test Meals. p-Carotene and/or its epoxides were dissolved in a

small amount of acetone to ensure complete solubilization when added to palm oil.

Solvent residue was removed under a stream of nitrogen gas. The carotenoid enriched

oils were either tested directly or added (10%, w/w) to applesauce (Mott's Inc., Stamford.

CT). Applesauce was chosen as a test food because it contains no detectable levels of

carotenoids. Test samples were subjected immediately to digestion in vitro. The

concentration of p-carotene derivatives used in this study ranged between 20-30 mmol/g

test meal.

In Vitro Digestion. Digestive stability and micellarization of p-carotene epoxides

were evaluated using an in vitro digestion method designed to mimic the gastric and

small intestinal phases of the in vivo process as described by Garrett et al. (1999 and

2000). For the gastric phase, a portion of test meals were acidified to pH 2 with 0.1 N

HCI before the addition of porcine pepsin (3 mg/mL). The mixture was incubated at

37°C for 1 h in a shaking water bath. The gastric digestate was then neutralized with 1

mol/L NaHCC >3 to initiate the small intestinal phase. Porcine bile extract (final

concentration = 2.4 mg/mL), porcine pancreatin (0.4 mg/mL) and lipase (0.2 mg/mL)

were added and the final pH was adjusted to 7.0 with 1.0 mol/L NaOH. The samples

were incubated at 37 °C for 2 h in a shaking water bath. This mixture was designated

as digesta. The aqueous micellar fraction was isolated from the digesta by

centrifugation at 167 000 x g at 4 °C for 35 min (Beckman model L7-65 Ultracentrifuge,

Fullerton, CA) and filtered (0.22 pm pores, cellulose acetate) to remove nonmicellarized

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aggregates. Samples of the test meal, digesta and micellarized fraction were stored

under nitrogen at -80 °C until analysis.

Cellular Uptake and Retention of Micellarized p-Carotene Epoxides.The

parent Caco-2 cells (HTB37) was acquired from the American Type Culture Collection

(Rockville, MD), while the TC7 clone was kindly provided by Dr. Monique Rousset,

INSERM, Cedex, France. Stock cultures were maintained according to Han et al.

(1994). Complete medium contained high-glucose DMEM (Sigma, St. Louis, MO), 10%

heat inactivated fetal bovine serum (F6S), nonessential amino acids (10 mL/L medium),

L-glutamine (2 mmol/L), amphotericin B (0.5 mg/L), gentamicin (50 mg/L), HEPES (15

mmol/L), and sodium bicarbonate (44 mmol/L). Cells were grown in a humidified

atmosphere of air/C02 (95:5 v/v) at 37 °C in 6-well plastic dishes. Highly differentiated

monolayers were used 11-14 days postconfluency (passages 27-35 for HTB37 and

passages 70-80 for TC7). Medium was replaced with fresh complete medium every 48

h and 24 h before initializing experiments.

To begin the experiment, spent medium was removed and monolayers were

washed twice with 1 mL of Hank’s balanced salt solution (HBSS). Filtered aqueous

fraction from the in vitro digestion procedure was diluted 1:4 with basal DMEM

(designated as Medium 1x) and 2 mL was added to the monolayer. Previous studies

have shown that the diluted aqueous fraction of digestate has no adverse effects on

cellular integrity and metabolism (Garrett et al., 1999 and 2000). Cultures were

incubated at 37 °C and cells were harvested at indicated times. After removing the test

media, monolayers were washed three times with HBSS containing 5.0 mmol/L sodium

taurocholate. Cells were collected in 1 mL ice-cold phosphate-buffered saline (PBS) and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. centrifuged at 300 x g to collect the cell pellet. Pellets were blanketed with N2 and

stored at -80 °C until analysis.

In studying the effect of carotenoid concentrations on cellular uptake, a control

medium containing micelles but no carotenoids was prepared by subjecting test meals

without test compounds to in vitro digestion procedure and diluting the micellar fraction

1:4 with DMEM. Appropriate volumes of this control medium were added to aliquots of

medium containing micellarized carotenoids to decrease carotenoid concentration

without reducing the micellar content of medium.

To examine cellular retention of carotenoids, monolayers initially were incubated

for 4 h in DMEM containing the test micellarized compounds obtained from in vitro

digestion. Following accumulation, monolayers were washed with phosphate buffered

saline and fresh DMEM without micelles or test compound. Monolayers were incubated

for an additional 18 h and epoxy p-carotene and p-carotene were quantified.

Conversion of p-Carotene Epoxides to Retinol and Retinyl Esters.The TC7

clone of the Caco-2 cells was grown in 75 cm2 flasks. Once monolayers reached

confluency, cultures were maintained in serum-free DMEM supplemented with

glutamine, non-essential amino acids and antibiotics as above for 14 days. We, like

During et al. (1998), found that the serum-free condition was required for p-carotene

15,15'-monooxygenase activity in the Caco-2 cells. Elimination of serum from medium

for monolayers that reached 100 % confluency did not affect general cell morphology or

decrease the number and diameter of domes.

Carotenoids were delivered to the cells as Tween micelles prepared according to

During et al., (1998). Carotenoids (in hexane), a-tocopherol (in methanol) and Tween 40

(20%) in acetone were mixed in a glass vial. Tocopherol was added as an antioxidant at 86

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10x the carotenoid concentration. The volume of Tween 40 was 5 pL per mL medium.

Solvents were evaporated under a stream of N2 gas and the residue was solubilized in

10% of the final volume of DMEM. The suspension was sonicated for 30 s (3x), added

to the rest (90% volume) of DMEM, and filter sterilized (0.22 pm diameter pores) before

addition to cells.

Monolayers were washed twice with 20 mL sterile phosphate buffered saline

before addition of 12.5 mL DMEM containing the test compounds in Tween 40 micelles.

Cultures were incubated at 37 °C for 48 h. The spent medium was collected, centrifuged

at 300 x g to remove any cell debris and the supernate blanketed with N2 and stored at -

80 °C. Monolayers were washed with 20 mL ice-cold phosphate buffered saline. Cells

were collected, centrifuged and the cell pellet stored at -80 °C until analysis. The

amount of retinol, epoxy (3-carotene and p-carotene were quantified in cells and spent

medium.

Transport of Accumulated p-Carotene Epoxide. The transport of accumulated

p-carotene epoxides across the basolateral membrane was studied using differentiated

TC7 clone of the Caco-2 cells (13 days postconfluence) grown on cell-culture inserts

(0.4 pm pores, Beckton Dickinson, Franklin Lakes, NJ). Carotenoids were delivered to

the cells as Tween micelles similar to the vitamin A conversion study. Cells were loaded

by addition of 1.5 mL DMEM containing 500 pmol/L phenol red and the test compound to

the apical compartment and 2.5 mL phenol red-free DMEM in the basolateral

compartment. After the cultures were incubated at 37 °C for 4 h, medium was removed

form the apical and basolateral compartments. Monolayers were washed twice with

phenol red-free DMEM and inserts were transferred to new wells.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apical medium for overnight incubation contained Tween micelles with oleic and

taurocholic acids but no carotenoids. Oleic and taurocholic acids have been shown to

stimulate chylomicron synthesis (Luchoomun and Hussain, 1999). The apical medium

was prepared as described by Nayak et al. (2001) with slight modifications. Briefly, 160

nL Tween 40 (20% in acetone) was pipetted into a 50 mL glass beaker. Acetone was

evaporated by a continuous stream of N2 and 10 mL of DMEM containing 500 |.imol/L

phenol red was added. The mixture was sonicated for 30 sec three times. In a separate

glass beaker, 0.5 mmol/L taurocholic acid was added to 30 mL DMEM containing 500

pmol/L phenol red and the mixture was stirred for 15 min. After addition of 1.5 mmol/L

oleic acid (sodium salt), the mixture was added for an additional 20 min. This mixture

was then added to the DMEM containing Tween 40 micelles, stirred for 2 h, and sterile

filtered (0.22 pm pores).

Medium with oleic and taurocholic acids containing Tween micelles (1.5 mL) and

phenol red-free DMEM (2.5 mL) were added to the apical and basolateral

compartments, respectively. Cultures were incubated for 18 h at 37 °C. Apical and

basolateral media were collected after 18h incubation, centrifuged at 300 x g for 5 min.

supernate was collected, blanketed with N2, and stored at -80 °C until analysis.

Monolayers were washed (2x) with cold PBS, collected, pelleted, blanketed with N2, and

stored at -80 °C until analysis. Monolayer integrity was monitored by calculating the flux

of phenol red from the apical to the basolateral compartments samples from the

experiments. Monolayers exhibiting a flux of phenol red less than 0.0002 %/h/cm2

phenol red flux were analyzed.

Extraction off Test Meal, Digesta and Micellar Fraction. Samples were

extracted similar to that described by Ferruzzi et al. (2001). Briefly, 1 mL of test meal, 88

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. digesta or micellar fraction was extracted with 3 mL of petroleum ether + acetone (2+1)

containing 0.01% BHT and vortexed for 1 min. After centrifugation (2000 x g) for 1 min,

petroleum ether layer was collected. Extraction was performed 3 times and the pooled

petroleum ether layers were dried under a stream of nitrogen, redissolved in HPLC

solvent, and analyzed by HPLC immediately.

Extraction of Caco-2 Cell. Extraction of p-carotene and its epoxides from

Caco-2 cells was done following the method of Peng and Peng (1992). Cell pellet was

digested with 200 ^L protease solution (100 mg protease/10 mL PBS) for 30 min at 37

°C. SDS-ethanol solution (0.5 mL) was then added, and the sample was vortexed for 1

min. Carotenoids were extracted into 1 mL of petroleum ether/acetone (2:1) containing

0.01% BHT. The mixture was then vortexed for 1 min and centrifuged at 2000 x g for 1

min. The extraction was performed three times and the pooled petroleum ether layers

were dried under nitrogen, redissolved in HPLC solvent, and analyzed immediately.

HPLC Method for Carotenoid Analysis. This methodology was applied for the

analysis of p-carotene and its epoxides during in vitro digestion, cellular uptake and

transport studies. The use of an electrochemical detector allows for the detection of low

levels of p-carotene and its epoxides in the cells. p-Carotene and its epoxides were

separated on a YMC C30 column, 3.9 mm x 150 mm (Waters, Milford, MA) coupled with

a coulometric electrochemical array detector according to Puspitasari-Nienaber et al.

(2002). The eight channels of the ESA Model 5600 Coularray electrochemical detector

(Chelmsford, MA) were set at 200 to 620 mV in 60 mV increments. A linear gradient

elution consisting of solvent A (methanol:MTBE:ammonium acetate:water 88:5:5:2) and

B (methanol:MTBE: ammonium acetate 20:78:2) was applied as follows: 0-5 min, 100% 89

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. solvent A; 5-45 min, 15% solvent A, 85% solvent B; 45-50 min 100% solvent B.

Ammonium acetate (1.0 mol/L, pH 4.6) was prepared with Ci8 Sep-Pak® (Waters,

Milford, MA) treated water. Figure 5.2. illustrates a typical separation of a\\-trans (V

carotene, its isomers (13-c/s and 9-c/s), and epoxides (5,6- and 5,8-epoxy, 5,6,5 ,6 - and

5,8,5',8’-diepoxy p-carotene). In addition, p-apocarotenal was also separated and

detected (data not shown). Current voltage curves (CVC) of these compounds were

used to assist in carotenoid identification throughout the study.

HPLC Method for Retinol, Retinyl Palmitate and Carotenoid Analysis. This

methodology was applied for the analysis of retinol, retinyl palmitate, p-carotene and its

epoxides during the conversion study. Separation was performed on a Vydac 201TP54

Ci8 column, 5 nm, 4.6 mm x 250 mm (Hesperia, CA) using a Waters 2695 Separation

Module (Milford, MA) equipped with a photodiode array detector (Waters 990). Solvent

A consisted of methanol.water:ammonium acetate (88:15:2), while solvent B consisted

of methanol:MTBE:ammonium acetate (85:13:2). Ammonium acetate was prepared as

described in the previous paragraph. The following linear gradient elution was

performed: 0-5 min, 100% solvent A; 5-15 min, 100% solvent B; 15-25 min, 100%

solvent B; 25-30 min, 100% solvent A. Detector was set between 300 and 550 nm to

monitor the presence of retinoids other than retinol and carotenoids. Quantitation of

retinol and retinyl palmitate were perfomed based on their absorption at 325 nm, while p-

carotene and its epoxides were at 450 nm. A typical separation of retinol, epoxy p-

carotene and p-carotene is shown in Figure 5.3.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Data analysis. Data are presented as the mean ( SEM (n=3-6). Analysis of variance

(ANOVA) was used to determine significant (p < 0.05) differences for groups of samples

using a statistical package StatView 5.0 (SAS Institute, Cary, NC).

5.4. RESULTS AND DISCUSSION

Stability of p-Carotene Epoxides in Test Meals. A stock solution containing 15-

carotene, 5,6-epoxy p-carotene and 5,8-epoxy p-carotene (1.0 : 0.8 : 0.7) was added to

fresh palm oil or used palm oil. In addition, a portion of the carotenoid enriched fresh

palm oil was added to applesauce (10% w/w). The three test meals (applesauce, fresh

palm oil and used palm oil) were sampled 10 minutes after mixing for analysis of

carotenoid (Figure 5.4.). p-Carotene was stable in all test meals (Figure 5.4.A.), and

5,6-epoxy and 5,8-epoxy p-carotene were stable in the oil samples (Figure 5.4.B. and

C). In contrast, the amount of 5,6-epoxy p-carotene decreased 22.9 ± 9.7% (p < 0.05)

immediately after addition to applesauce (Figure 5.4.B.). This decrease in 5,6-epoxy p-

carotene was accompanied by a significant increase (p < 0.05) in 5,8-epoxy p-carotene

(Figure 5.4.C) in applesauce. Rearrangement of p-carotene 5,6-epoxide to its 5,8-

epoxide upon low acid treatment has been reported (Britton, 1995). The pH of

applesauce, fresh palm oil and used palm oil test meals were 3.8, 6.7 and 6.6,

respectively.

Stability of p-Carotene Epoxides duringIn Vitro Digestion. Test meals

(applesauce, fresh palm oil or used palm oil) containing p-carotene, 5,6-epoxy p-

carotene and 5,8-epoxy p-carotene (1.0 : 0.8 : 0.7) were subjected to simulated gastric

and small intestinal phases of digestion 10 min after preparations. Qualitative changes 91

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of carotenoids in fresh and used palm oils were similar, but differed from that of

applesauce (Figure 5.5.A). Recoveries of p-carotene in digested fresh and used palm oil

were similar, but the amount of p-carotene remained in fresh palm oil after digestion was

significantly lower (p < 0.05) than the test meal. The recovery of 5,6-epoxy p-carotene in

both oils after digestion was significantly (p < 0.05) below 100%, indicating its

degradation during the digestion. In contrast, the recovery of 5,8-epoxy p-carotene in

fresh and used palm oil was greater than 100%, although the difference was significant

(p < 0.05) only for used palm oil. Since 5,6-epoxy and 5,8 epoxy p-carotene were stable

in palm oil test meals (Figure 5.4 ), this observation indicates that rearrangement of 5.6-

epoxy to 5,8-epoxy p-carotene occurred during in vitro digestion. The results for

applesauce were not consistent with the oils. The amount of 5,6-epoxy p-carotene after

digestion increased significantly (p < 0.05) but 5,8-epoxy p-carotene remained

unchanged (Figure 5.5.A). The reason for this inconsistency is unclear. Diepoxy p-

carotene was not detected in the test meals, but was present in all digesta samples

(Figure 5.5.B). Diepoxy p-carotene was generated from oxidation of epoxy p-carotene.

The amount of 5,6,5 6 - and 5,8,5’,8-diepoxy p-carotene present in the digesta ranged

from 19.9 ± 3.9 to 32.4 ± 3.7 % of the corresponding 5,6-epoxy and 5,8-epoxy p-

carotene in the test meals.

Human gastric fluid and the acidic pH of the stomach have been suggested to

enhance oxidations of lipid and dietary constituents such as p-carotene (Kanner and

Lapidat, 2001). With the exception of fresh palm oil, our data showed that p-carotene

was stable during digestion. The same trend was reported in earlier studies (Garrett et

al., 1999 and Ferruzzi et al., 2001). Our data also suggested that epoxy p-carotene was

oxidized more readily than p-carotene during digestion. It is interesting to note that

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Barua (1999) demonstrated the present of 5,6-epoxy, but not 5,8 epoxy (3-carotene, in

plasma samples of subjects consuming 5,6-epoxy p-carotene.

Micellarization of p-Carotene Epoxides. Micellarization was defined as the

relative efficiency of transfer of p-carotene derivatives from the food matrix to the

aqueous fraction. Concentrations of the carotenoid in the aqueous fraction were divided

by that in the digesta and multiplied by 1 0 0 % to calculate the micellarization efficiency.

Figure 5.6. A and B show that micellarization of p-carotene, 5,6-epoxy p-carotene and

5,8-epoxy p-carotene when added to the applesauce meal individually were 11.6 +

0.6%, 16.6 ± 1.8 % and 16.3 ± 2.3%, respectively. When 5,6-epoxy p-carotene was

added to applesauce, 5,8-epoxy p-carotene was generated (Figure 5.6.B).

Micellarization efficiency of the generated 5,8-epoxy p-carotene was similar to that of the

added 5,8-epoxy p-carotene. Micellarization efficiencies of both 5,6-epoxy and 5,8-

epoxy p-carotene were significantly higher (p < 0 .0 1 ) than that of p-carotene.

Factors affecting the transfer of various carotenoids from foods and biological

emulsions to micelles have been extensively studied (Borel et al., 1996; Tyssandier et

al., 2001), and include carotenoid polarity, interaction with other carotenoids, pH and bile

concentration. Since pH and concentration of bile were identical throughout the

digestion process for all test samples, their possible effects on micellarization efficiency

could be eliminated. Borel et al. (1996) indicated that carotenoid polarity governed their

distributions between the core and surface of biological emulsions. Non- polar

carotenoids such as p-carotene and lycopene were found almost exclusively in the core

of biological emulsion, while polar carotenoids such as p-cryptoxanthin and lutein were

distributed between the core and surface. In line with this finding, Garrett et al. (1999

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and 2 0 0 0 ) reported the micellarization of lutein > p-carotene > lycopene from digested

test meals. The polarity of p-carotene epoxides is greater than p-carotene, as apparent

from their retention on the reversed-phase HPLC column (Figure 5.2.). In our system,

the relative retention times of 5,6-epoxy p-carotene and 5,8-epoxy p-carotene compared

to p-carotene were 0.82 and 0.84, respectively. The greater polarity of epoxy p-carotene

is likely the basis for the more efficient micellarization than p-carotene.

The presence of p-carotene and one or both of its epoxy derivatives in the same

applesauce meals affected the efficiency of caroteoid micellarization. The efficiency cf

micellarization of p-carotene increased by 37% (from 9.79 ± 0.5 to 13.4 ± 0.8 %) and

51% (from 9.79 ± 0.5 to 14.8 ±1.5 %) in digested samples containing 5,8-epoxy p-

carotene and both 5,6-epoxy and 5,8-epoxy p-carotene, respectively. In contrast, the

micellarization efficiency of 5,6-epoxy and 5,8-epoxy p-carotene were significantly

suppressed (p < 0.0001) by 74% (from 16.6 ± 1.8 to 3.4 ± 0.7) and 62% (from 16.3 ± 2.3

to 6.3 ± 0.7 %), respectively (Figure 5.6.B) in digested samples containing p-carotene.

Also, micellarization efficiency of 5,8-epoxy p-carotene generated from 5,6-epoxy p-

carotene during digestion was suppressed by 36% (from 18.4 ± 2.8 to 11.7 ± 1.7 %) in

the presence of p-carotene.

Interactions between carotenoids in diets high in carotenoid-rich foods has been

thoroughly investigated with conflicting results. Tyssandier et al. (2001) reported that

transfer from emulsion lipid droplet to micelle of the more polar xanthophyll was not

significantly affected by the presence of carotenes and other xanthophylls in vitro In a

subsequent study, these authors observed competition between lutein, p-carotene and

lycopene for incorporation into chylomicrons but found no adverse effect on plasma

levels of these carotenoids after 3 weeks (Tyssandier et al., 2002). White et al. (1996)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and Paetau et al. (1997) found that p-carotene reduced canthaxathin absorption in

human subjects when the two carotenoids were consumed simultaneously.

Next, we studied the effect of different food matrices on the transfer efficiency of

p-carotene epoxides to the aqueous fraction (Figure 5.7 ). The three test meals

(applesauce, fresh palm oil and used palm oil) contained 5,6-epoxy, 5,8-epoxy and -

carotene together, and were subjected to in vitro digestion. Micellarization of p-carotene

and its epoxides from fresh and used palm oil were significantly greater (p < 0 .0 0 0 1 )

than from applesauce. This agrees with previous findings which showed that p-carotene

from RPO is more efficiently absorbed and converted to vitamin A than from other plant

sources (Solomon, 1998, de Pee et al., 1995 and 1998). Soluble fiber has been

reported to reduce p-carotene bioavailability and conversion to vitamin A in Mongolian

gerbils (Deming et al., 2000). Pasquier et al. (1996) demonstrated that apple pectin

reduced emulsification of dietary lipid, mainly due to its high viscosity. Soluble fiber may

also physically bind and entrapp bile salts, thereby reducing micellarization of fat-soluble

compounds (Lairon, 1996). We also observed a slight, but significantly lower (p < 0.05)

micellarization of p-carotene from used palm oil than fresh palm oil (Figure 5.7). Used

frying oil consisted of triglycerides and its degradation products such as free fatty acids,

monomeric, dimeric and polymeric compounds (Chang et al., 1978). Used frying oil has

been reported to lower fat absorption, due to the inhibition of pancreatic lipase in

hydrolizing polymerized triglycerides (Henderson et al., 1993). A diet containing 15%

oxidized frying oil significantly lower a-tocopherol, cholesterol and triglycerides

concentration in plasma and tissues or rats than a diet with similar level of fresh soybean

oil (Liu and Huang, 1995). It is possible that formation of polymeric compounds in used

frying oil impaired fat absorption and caused lower micellarization.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.7. also shows that micellarization efficiencies of 5,8-epoxy (3-carotene

were consistently higher than that of 5,6-epoxy p-carotene regardless of the food matrix.

This difference cannot be explained by the polarity difference between the p-carotene

epoxides, since no substantial difference was observed between the polarity of 5,6- and

5.8-epoxy p-carotene. We demonstrated earlier that 5,6-epoxy p-carotene rearranged to

5.8-epoxy p-carotene when exposed to acidic environment. Rearrangement of 5,6-

epoxy p-carotene to 5,8-epoxy p-carotene decreased the amount of 5,6-epoxy p-

carotene and at the same time increased the amount of 5,8-epoxy p-carotene available

for micellarization. It appeared that rearrangement continued to occur during in vitro

digestion and was faster in oil samples when insoluble food matrix was not present.

Effect of pH on the Stability of p-Carotene Epoxides DuringIn Vitro

Digestion. Next, we examined the effect of gastric pH on the rearrangement of 5.6-

epoxy to 5,8-epoxy p-carotene (Figure 5.8). Applesauce test meal (with 10% oil)

contained 5,6-epoxy p-carotene and p-carotene and has a pH value of 3.8. After pH

adjustment to 2, 4 or 6 , this test meal was subjected to the gastric phase of the in vitro

digestion procedure. The amount of p-carotene was not affected by pH during the

gastric phase. At pH 2, about one-half of the 5,6-epoxy p-carotene originally present in

the food was converted to 5,8-epoxy p-carotene. When the pH of the gastric phase was

adjusted to 4 or 6 , rearrangement was minimal and distribution of the test compounds

was very similar to the starting material. Furthermore, at pH 2 the total amount of

carotenoids present in the gastric digesta was significantly lower (p < 0.05) than when

digestion was performed at higher pH. This decrease in the total amount of carotenoids

was primarily due to loss of the diepoxides.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Effect of Bile Salt on the Micellarization Efficiency of p-Carotene Epoxides.

Data shown in Figure 5.9 demonstrate the requirement for bile salt in the micellarization

of carotenoids from the test meals. The amount of bile salt present during the small

intestinal phase of the standard procedure was 2.4 mg/mL. In the absence of bile

extract, very low amounts of p-carotene and 5,6-epoxy p-carotene were present in the

micellar fraction. The 5,8-epoxy p-carotene was less affected by the absence of bile

salts. Micellarization of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene when the bile

extract was 2.4 mg/mL in the digesta was 15.4 ± 2.4, 6 . 8 ± 1.3, and 18.6 ± 2.1 %,

respectively. Increasing bile extract to 3.6 mg/mL increased the efficiency of

micellarization approximately 3 fold. Garrett et al. (1999) noted an insignificant increase

(p > 0 .0 1 ) in efficiency of micellarization of lutein, a-carotene and p-carotene during

digestion of meal when the amount of bile salt was increased from 2.4 to 3.6 mg/mL.

The difference between present and previous results could be due to the amount of fat

present in the respective test meals. The fat content of the applesauce meal was 10%.

while test meals used by Garret et al. (1999) contained 3% (w/w) fat. Dietary fat is an

important factor for the efficient carotenoid digestion and absorption. Increased fat

content increases the size of the bile salt micelles, therefore increasing the amount of

carotenoids that can be solubilized in the aqueous fraction (Furr and Clark, 1997).

Stability of p-Carotene Epoxides in Cell Culture Environment.The stability

of micellarized p-carotene and its epoxides derived from in vitro digestion was examined

in cell-free, tissue culture conditions. Each carotenoid was solubilized in corn oil and

subjected to in vitro digestion procedure. Aqueous micellar fractions of the digestate

were diluted 1:4 with DMEM. Micellarized p-carotene was relatively stable under tissue

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. culture conditions (Figure 5.10) with 89.1 ± 5.2 and 86.7 ± 8.2 % recovered after 6 h and

24 h incubation, respectively. Only 9-cis p-carotene, but no epoxy p-carotene, was

detected after 24 h incubation. The relative amount of 9-cis p-carotene to p-carotene in

the medium at the beginning of incubation was 2.2 ± 0.3 % and increased to 3.98 ± 0.4%

and 12.2 ± 1.5% after 6 h and 24 h, respectively. These observations suggest the

isomerization, but not epoxidation, of p-carotene in cell culture environment.

Both 5,6-epoxy and 5,8-epoxy p-carotene was present in the micelles generated

by digestion of corn oil containing 5,6-epoxy p-carotene. Only 50.3 ± 3.2 and 53.4 ± 2.3

% of micellarized 5,6- and 5,8-epoxy p-carotene remained in the media after 3 h

incubation (Figure 5.10). The amount of 5,6-epoxy p-carotene in the media continued to

decline with only 15.7 ± 1.4% of originally micellarized 5,6-epoxy p-carotene recovered

after 24 h incubation. The decline in 5,6-epoxy p-carotene at 3 h was accompanied by a

significant (p < 0.05) increase in 5,8-epoxy p-carotene to 72.2 ± 1.1% after 24 h

incubation. Diepoxy p-carotene was detected during incubation, but the amount

generated did not account for the loss of monoepoxy p-carotene (data not shown).

Monoepoxy p-carotene may degrade to other compounds not detected by our HPLC

methodology.

Cellular Accumulation and Retention of p-Carotene Epoxides.Intestinal cell

accumulation of micellarized p-carotene and epoxy p-carotene was studied using the

Caco-2 human cell line. Cellular accumulation of p-carotene and its epoxide from

medium containing micelles was monitored for 0- 6 h. Exposure of cells to micellarized

P-carotene and its epoxides during this period was not toxic since cell viability remained

high (>95%) and large numbers of domes were maintained in the monolayer. 98

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Accumulation of p-carotene and its epoxy derivatives by differentiated cultures of

the Caco-2 HTB37 cell line and the TC7 clone are shown in Figure 5.11. and 5.12.,

respectively. Medium added to the Caco-2 HTB37 and TC7 monolayers contained

approximately 60 nM and 20 nM of each test compound, respectively. The

concentrations of 5,6-epoxy and 5,8-epoxy p-carotene in the HTB37 parent and TC7

clonal lines of Caco-2 increased linearly in monolayers exposed to micellarized

compounds for as long as 4 h incubation (Figure 5.11 and 5.12 Panel A) and with

increasing concentrations of micellarized compounds (Figure 5.11 and 5.12 Panel B).

The cellular concentration of 5,8-epoxy p-carotene increased more rapidly than the 5.5-

epoxy p-carotene in both cell lines (Figure 5.11 and 5.12 Panel A), suggesting that

rearrangement of 5,6-epoxy p-carotene to its 5,8-epoxy derivative may occur in the

micelles and/or once transferred into the cell. After 6 h, the percentage of micellarized

5,6- and 5,8-epoxy p-carotene from the medium that had accumulated in the HTB37 (5.3

± 0.8% and 7.2 ±1.1%, respectively) cell line was significantly lower (p < 0.005) than in

the TC7 clone (14.3 ± 1.1% and 15.3 ± 0.3%, respectively). It is apparent from Figure

5.12.A that accumulation of epoxy p-carotenes (14.3 ± 1.1 % and 11.5 ± 0.9% for 5,6-

epoxy and 5,8-epoxy p-carotene, respectively) after 6 h was significantly lower (p <

0.001) than p-carotene (22.9 ± 1.0%) in the TC7 clone. Although cellular accumulation

of p-carotene was not studied in the HTB37 cell line, its relative accumulation was

expected to be similar to its epoxy derivatives and lower than in the TC7 clone. Ferruzzi

et al. ( 2 0 0 1 ) reported a higher accumulation of micellarized p-carotene and lutein from

digested spinach by the TC7 clone than the HTB37 line. Since the TC7 clone, but the

HTB37 cell line, have 15,15 -dioxygenase activity, one might expect lesser accumulation

if the carotenoids were being converted to retinol and its derivatives.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Factors such as hydrophobicity, ionization and molecular size have been

reported to affect the physicochemical properties of a compound, and therefore its

accessibility and absorption across the intestinal epithelium (Chan and Stewart, 1996).

Hydrophobicity affects the solubilization of carotenoids in micelles, which was a

requirement for apical uptake by intestinal cells. It is doubtful that the difference in

hydrophobicity between (3-carotene and its epoxides resulted in significant difference in

uptake by the Caco-2 cells. There was no significant difference in Caco-2 accumulation

of p-carotene and p-cryptoxanthin, although the later has one additional hydroxyl group

in its molecule (Sugawara et al. 2001). The preferential uptake of p-carotene over its

epoxy derivatives may be explained by the relative stability of p-carotene in cell culture

conditions (Figure 5.10). More p-carotene than epoxy p-carotene was available in cell

culture medium for cell uptake.

The diepoxy derivatives of p-carotene were not detected in Caco-2 cells,

although they were detected in the micellar fraction after digestion. Assuming that their

relative accumulation was similar to the monoepoxy derivatives, i.e. 1 1 to 14%, our

methodology lacked sufficient sensitivity to detect the presence of diepoxy p-carotene in

the cell extract with confidence.

The cellular retention of epoxy p-carotene and (3-carotene were assessed to

determine the possible metabolism of these compounds by Caco-2 cells (Figure 5.1 1 .

and 5.12. Panel C). Retention of pre-loaded 5,6-epoxy p-carotene in HTB37 cell line

(69.7 ± 6 .6 %) was similar to that in TC7 clone (61.1 ± 3.4%), whereas retention of 5,8-

epoxy p-carotene in HTB37 cell line (60.1 ± 2.9%) was significantly lower (p < 0.001)

than in the TC7 clone (91.5 ± 2.9%). As for p-carotene, the majority (89.4 ± 1.5%)

remained in the cell after 18 h incubation in p-carotene free medium. The possible fate

100

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of p-carotene and its epoxy derivatives once they enter the cell may include retention in

its originally accumulated form, structural rearrangement, metabolism to one or more

products (e.g. cleavage, epoxidation, etc.). In mammals, provitamin A carotenoids are

metabolized to vitamin A by the p-carotene 15,15-dioxygenase. When grown in seru.n

free medium, the TC7 clone of Caco-2 cells have been reported to express the p-

carotene 15,15 -dioxygenase (During et al., 1998 and 2001). Our standard cell culture

conditions were not conducive for the expression of this enzyme (see later discussion on

vitamin A conversion). Consequently, the decrease in the level of p-carotene

accumulated in the cells was unlikely due to vitamin A conversion. The low cellular

retention of 5,6-epoxy p-carotene may be partly explained by its rearrangement to 5,8-

epoxy p-carotene. The possibility of other unknown metabolic processes or oxidative

degradation of carotenoids in Caco-2 can not be eliminated. Sugarawa et al. (2002)

reported enzymatic reduction of fucoxanthin to fucoxanthinol during uptake by Caco-2

cells and during absorption in mice.

Cleavage of p-Carotene Epoxides to Retinol and Retinyl Esters.The ability

of epoxy p-carotene to serve as substrates for 15,15 -dioxygenase was investigated

using the fully differentiated TC7 clone of the Caco-2 cells. Because the conversion

efficiency in this cell line was low (During et al. 1998) and our results (Figure 5.11)

showed low accumulation of micellarized p-carotene epoxides by Caco-2 cells, high

levels of p-carotene epoxides had to be loaded to the cells to ensure detection of their

metabolites in the cell. While presentation of p-carotene epoxides using micellar fraction

obtained from in vitro digestion provides a physiologically relevant process, preparation

101

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the amount of pure epoxy (3-carotene required was not feasible. Cell loading was,

therefore, undertaken using Tween 40 micelles as described earlier (During et al. 1998).

Figure 5.3. showed a typical separation of (3-carotene and retinol extracted from

TC7 cell after 48 h incubation before and after saponification. The HPLC methodology

was able to detect the presence of retinyl palmitate in the cell extract without

saponification. However, cis isomers of (3-carotene and cis isomers of retinol, were not

baseline separated. The reported values of p-carotene and retinol (Table 5.1.) were the

total amount of p-carotene isomers (monitored at 450 nm) and the total amount of retinol

isomers (monitored at 325 nm). Cis p-carotene contributed 36.3 ± 1.7 % of the total p-

carotene and cis retinol contributed 14.9 ±0.1 % of the total retinol.

p-Carotene and 5,6-epoxy p-carotene in Tween micelles were stable in cell

culture environment with more than 80% recovered after 48 h incubation (Table 5.1). No

5,8-epoxy p-carotene was detected in the medium containing 5,6-epoxy p-carotene. We

demonstrated earlier that 5,6-epoxy p-carotene in natural micelles (generated via in vitro

digestion procedure) was not stable and partly rearranged to 5,8-epoxy p-carotene

during digestion (Figure 5.12). Lack of exposure to acidic digestive environment may

explain the excellent stability of 5,6-epoxy p-carotene in Tween micelles. In contrast,

only 29.6 ± 1.4 % 5,8-epoxy p-carotene was recovered after 48 h incubation (Table 5.1).

Since 5,8-epoxy p-carotene was not converted to vitamin A efficiently, its low recovery

cannot be attributed to vitamin A conversion. The different composition of natural and

Tween micelles may partly explain the different stability of carotenoids in cell culture

environments.

Assuming that central cleavage of one molecule of p-carotene generates two

molecules of retinol, conversion efficiency was defined as one-half the amount of retinol

102

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the cell after 48 h incubation divided by the sum of 50% retinol and p-carotene (Table

5.1). Conversion efficiency of p-carotene was significantly higher (p < 0.0001) than that

of 5,6- and 5,8-epoxy p-carotene; no significant difference was found for conversion of

the two epoxides. In contrast, retinol was not detected when 5,6,5'6-diepoxy p-carotene

was used as the substrate, confirming the enzyme s high substrate specificity for

carotenoids with p-ionone ring (Wirtz et al., 2001). Lack of provitamin A activity of

5,6,5'6 -diepoxy p-carotene was reported previously in rats (Heinonen et al., 1995).

Since the monoepoxides have a single p-ionone ring, it is reasonable to expect that their

conversion efficiencies would be about one-half that of p-carotene. However, the

conversion efficiencies of both monoepoxides were only about 1 0 % that of p-carotene.

Thus, factors in addition to the presence of p-ionone ring also are important for

conversion. Wirtz et al. (2001) concluded that any deviation from the rod -like' structure

of p-carotene has the likelihood of affecting substrate recognition by the binding pocket

of 15,15 -dioxygenase. For example, the efficiency of retinol production from a-carotene

with its single p-ionone ring and one distorted end group was 8 % that of p-carotene.

The ratio of retinol to p-carotene in the cell after 48 h incubation was 1 .1 (Table

5.1). This value is much higher than the reported value of 0.094 from During et al.

(1998). The discrepancy can be attributed to the difference in cell differentiation in the

two studies. Our experiments were conducted with cells at 12-14 days postconfluency,

while During et al. (1998) conducted their experiment with cells at 7 days

postconfluency. In the absence of serum, dioxygenase activity of the TC7 clone of the

Caco-2 cells increased linearly up to 19 days postconfluency (During et al., 1998).

Dioxygenase activity at 7 days postconfluency was about one-half that at 14 days

postconfluency.

103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The enzyme p-carotene 15,15'-dioxygenase has been reported to exert both

central (Glover, 1960; Nagao et al., 1996) and asymmetric (Wang et al., 1991; Kiefer et

al., 2001) cleavages of p-carotene. Retinol and retinyl palmitate were the only products

observed. We, like During et al. (1998 and 2001), did not detect apocarotenals, the

asymmetric products of p-carotene in cells. The presence of sufficient amounts of

antioxidants results in central cleavage of carotenoids (Yeum et al., 2000). The amount

of a-tocophero! used in our study was 1 0 times that of p-carotene.

Transport of Accumulated p-Carotene Epoxides. Caco-2 cell monolayers

grown on inserts were used to examine the possible transport of p-carotene and its

epoxides across the transepithelial membrane. Medium (1.5 mL) containing 3 pM p-

carotene or epoxy p-carotene was added to the apical compartment of the inserts and

cells were incubated for 4 h and referred to as loading. Following loading, cells were

washed and incubated in fresh DMEM containing Tween micelles, taurocholic and oleic

acid in the absence of test compounds for 18 h. Supplementation of the apical media

with oleic and taurocholic acid has been shown to induce chylomicron secretion by

Caco-2 cells (Luchoomun and Hussain, 1999) and successfully applied to examine the

secretion of retinyl esters (Nayak et al., 2001) and p-carotene isomers (During et al.,

2002).

More than 94% of p-carotene and its epoxides remain in the cell after 18 h

incubation. The amount of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene present in

the basolateral media was 2.8 ± 0.2, 2.6 ± 0.3 and 1.7 ± 0.2 % of the total carotenoid,

respectively (Figure 5.13). Our were lower than published results (During et al., 2002)

where 1 1 % of the p-carotene was absorbed and 80 % was incorporated in the

104

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chylomicron. Although the composition of apical media in the two studies were similar,

several differences were noted. During et al. (2002) as well as other researchers

(Luchomun and Hussain, 1999; Nayak et al, 2001) used older cells (21 days) than our

study (13 days). Also, the previous studies used inserts with larger membrane pore size

(3 nm) than our study (0.4 nm). These differerences may contribute to the differing

results. The lack of carotenoid secretion into the basolateral medium may be due to lack

of chylomicron formation. Since chylomicron formation was not monitored in the present

study, this aspect merits further investigation.

5.5. CONCLUSIONS

Epoxy p-carotene was micellarized from foods during in vitro digestion

suggesting their bioaccessibility. The degree of micellarization was affected by the type

of food matrix, the presence of other carotenoids in the food, and the pH and bile extract

content during the gastric and small intestinal phases of digestion, respectively. When

present together with p-carotene, micellarization efficiency of p-carotene epoxide was

suppressed. Accumulation of these micellarized compounds by Caco-2 cells indicates

their potential for absorption. The relative accumulation of p-carotene epoxides was

lower than p-carotene. Furthermore, p-carotene epoxides were less efficiently cleaved

to vitamin A in the Caco-2 cells. Together, these results demonstrate the usefulness of

the coupled in vitro digestion/Caco-2 cell model to address problems related to the

bioavailability and bioconversion of provitamin A carotenoids.

105

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All-frans [3-carotene

5,6-epoxy [3-carotene 5,8-epoxy [3-carotene

5,6,5’,6-diepoxy [$-carotene 5,6,5’,6'-diepoxy [3-carotene

Figure 5.1. Structures of all-trans [3-carotene and its epoxy derivatives

106

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13-c/s (3-carotene

5,6-epoxy (3-carotene

All-frans |3-carotene 5.8-epoxy (3-carotene

5.8,5',8'-diepoxy [3-carotene 9 -c/s |3-carotene 5,6,5',6'-diepoxy [3-carotene

440 mv 380 mV 320 mV 260 mV

5.0 10.015.0 20.0 25.0 30.0 35 0

Retention time (minutes)

Figure 5.2. Separation of (3-carotene isomers and (3-carotene epoxides on a C30 RP-HPLC with coulometric electrochemical array detection

107

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Retinyl Palmitate-

Retinol Isomers B-Carotene Isomers

Unknown

0 10 20 30

Retention time (minutes)

Figure 5.3. Separation of retinol, retinyl palmitate and (3-carotene of Caco-2 cell extracts on a C18 RP-HPLC. Cell extracts were A) not saponified (325 nm), B) saponified (325 nm) and C) saponified (450 nm).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.4. Concentration of A) [3-carotene, B) 5,6-epoxy p-carotene and C) 5,8-epoxy p-carotene in applesauce, fresh palm oil and used palm oil test meals 10 min after preparation. The broken horizontal line represents the amount of test compounds added to the test meal. Preparation of test meal, digestion and analyses were described in Materials and Methods. Data represent mean ± SEM (n=3-6). Test meals for which carotenoid contents differed significantly (p < 0.05) from the added amount are indicated by an asterisk (*)•

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 A) p-carotene

40 34.5 30

B) 5,6-epoxy p-carotene o 40 E E, c 30 9o 27.5 20 C o c 10 oo 0

C) 5,8-epoxy p-carotene 40 r

30 23.5 20 WA: 10 H r::.::";

0 Applesauce Fresh Used Palm Oil Palm Oil

110

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 200 B BC □ 5,6-epoxy BC □ 5,8-epoxy BC J

Applesauce Fresh Palm Oil Used Palm Oil

B) 20 ■ 5,6,5',6'-diepoxy BC § 15 □ 5,8.5'.8'-diepoxy BC £ E a a | 10 S o § 5 o

miitiApplesauce Fresh Palm Oil Used Palm Oil

Figure 5.5. A) Recovery of p-carotene and its epoxy derivatives in the digesta following in vitro digestion of applesauce, fresh palm oil and used palm oil. Data represent the percentage of carotenoids recovered from the digested to the starting test meal which is arbitrarily set to 100%. B) Generation of diepoxy p-carotene in the digesta following in vitro digestion of applesauce, fresh palm oil and used palm oil.

Digestion was started 10 min after stock solutions were added to the raw materials. Digestion and analyses were described in Materials and Methods. Data represent mean ± SEM (n=3-6). Different letters above the error bars indicate statistically significant difference (p < 0.05) between test meals. The asterisks (*) within the bars indicate that the recovery of these carotenoids differed significantly (p < 0.05) from the test meal (100%).

111

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 - 0e •e1 15- l » . *

BC w/ 5,6-epoxy BC w/ 5.8-epoxy BC w/ both epoxides

(1:1) (1:1) (1:1:1)

□ 5,6-Epoxy BC 5,8-Epoxy BC

5.6-Epoxy BC 5.6-Epoxy BC w/ 5,8-Epoxy BC 5.8-Epoxy BC w/ BC BC

Carotenoid Added

Figure 5.6. Micellarization of p-carotene and its epoxy derivatives from applesauce test meals. A) Micellarization of p-carotene in the absence or presence of different p-carotene epoxides. B) Micellarization of various p-carotene epoxides in the absence or presence of equivalent amounts of p-carotene. Applesauce test meals contain 10% (w/w) oil and indicated p-carotene derivatives (25-35 mmol/g). Data represent mean ± SEM (n=6). Different letters above the bars indicate statistically significance different (p < 0.05) between samples.

112

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BC 5,6-epoxy BC 5,8-epoxy BC

Figure 5.7. Micellarization of (3-carotene and its epoxides in applesauce (AS), fresh palm oil (FPO) and used palm oil (UPO) test meals following in vitro digestion. Apple sauce test meal contained 10% com oil with the test compounds. All test meals contained 21-37 mmol/g of (3-carotene, 5,6-epoxy and 5,8-epoxy p-carotene. Data represent mean ± SEM (n=3-6). Different letter above the bars indicate statistically significance different (p <0.05) between test meals.

113

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 □ BC ■ 5,6-epoxy BC □ 5,8-epoxy BC ■Total

Food pH 2 pH 4 pH 6 pH during gastric phase

Figure 5.8. Effect of pH on the conversion of 5,6-epoxy p-carotene to 5,8-epoxy p- carotene during the gastric phase of in vitro digestion procedure. Applesauce test meal contained 10% corn, 5,6-epoxy p-carotene and p- carotene. Gastric pH was adjusted with 1.0 mol/L HCI or 1.0 mol/L NaOH before addition of pepsin. Samples were incubated at 37 °C for 1 h, and neutralized before analysis. Data represent mean ± SEM (n=3-6). The asterisk (*) indicate that concentration of these carotenoids differed significantly (p < 0.05) from the food.

114

% Micellarization 0 H 60 40 70 50 0 - 30 20 10 applesauce meal to the aqueous micellar fraction. Preparation of raw raw of Preparation fraction. micellar aqueous the to meal applesauce concentration when bile acid concentration was 0 mg/mL. 0 was concentration acid bile significantly when differed concentration carotenoids these of Methods. and concentration Materials in described were analyses and digestion material, Data represent mean ± SEM (n=3-6). The asterisk (*) indicate that the the that indicate (*) asterisk The (n=3-6). SEM ± mean represent Data 0

BC 5,6-epoxy BC 5,8-epoxy BC 5,8-epoxy 5,6-epoxyBC 115 ■ 2.4 mg/mL 2.4 ■ mg/mL 0 ■ □ 3.6 mg/mL 3.6 □

(p < 0.05) from the the from 0.05) < 150

125 5.6-epoxy BC

5.8-epoxy BC a 100

0 0 6 12 18 24 Time (h)

Figure 5.10. Stability of micellarized p-carotene and its epoxides in cell culture environment. The concentration of each test compound in the medium at 0 h was 60 nmol/L. Data represent the percentage of carotenoids relative to the raw material (arbitrarily set to 100%). Data represent mean ± SEM (n=3-6). Different letters above the bar indicate statistically significance different (p < 0.05) within time points. The asterisk (*) indicate that the remaining (%) concentration of these carotenoids differed significantly (p < 0.05) from the previous time points.

116

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.11. Cellular accumulation and retention of micellarized epoxy p-carotenes in differentiated cultures of Caco-2 HTB37 cells. Each test compound was solubilized in corn oil and digested in vitro. Aqueous fractions were diluted (1:4) with DMEM before addition to cell monolayers. A) Cellular accumulation after various periods of incubation (0-6 h). Initial concentrations of 5,6-epoxy and 5,8-epoxy p-carotene was 60 nmol/L. P) Cellular accumulation after 4h incubation in medium with different concentrations of epoxy p-carotenes. C) Retention of epoxy p-carotene by cells pre-loaded with test compound during 4h of incubation and subsequently incubated in medium free of test compounds for 18 h. Data represent mean ± SEM (n=3).

117

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 5 5,6-epoxy BC 4 5,8-epoxy BC O ^ S a e P 3 p• £& g ® 5 | 2 ■ ° |i 1

0 0 1 2 3 4 5 6 Incubation Time (h)

B) 5 c 5.6-epoxy BC o _ 4 5,8-epoxy BC 25 -3 c o u® an 3 = o 3 J 2 m O ii o 1 O 0 0 50 100 150 Medium Concentration (pmoVwell)

C) * c o 3 3.

5.6-epoxy BC 5,8-epoxy BC

118

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.12. Cellular accumulation and retention of micellarized p-carotene and epoxy p- carotenes in the TC7 clone of the Caco-2 cells. Each test compound was solubilized in corn oil and digested in vitro. Aqueous fractions were filtered and diluted (1:4) with DMEM before addition to cell monolayers. A) Cellular accumulation after various periods of incubation (0-6 h). Initial concentrations of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene was 20 nmol/L. B) Cellular accumulation after 4h incubation in medium with different concentrations of p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene. C) Retention by cells pre-loaded with p-carotene, 5,6-epoxy and 5,8-epoxy p-carotene during 4h of incubation and subsequently incubated in medium free of test compounds for 18 h. Data represent mean ± SEM (n=3). Different letters above the bars indicate statistically significance different (p < 0.05) between samples.

119

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 BC A) ___0 _ ■2 .£ S.6-epoxy BC Sc 5 o 5,8-epoxy BC *u a« 3

— S S i 2 a O

1 i 1 o 0 0 1 2 3 4 5 6 Incubation Time (h)

8 B) oc _ BC 5,6-epoxy BC 6 S 2 5.8-epoxy BC §o a S £ a 4 3 6 a O 3 6 2 =o a u 0 0 5 10 15 20 25 30 Medium Concentration (pmoL/well)

120

% Total 100 -| 120% 0 - 60% 0 - 40% - 80% 20 0% - % - % to the apical compartment for 4 h. Cell monolayer was washed and and washed was monolayer Cell h. 4 for compartment apical the to addition of DMEM containing individual test compound as Tween micelles micelles Tween as compound by test loaded were individual Cells containing DMEM inserts. of on grown addition Caco-2 of clone TC7 the of medium were collected and analyzed as described in Material and and Material in described as analyzed and collected were medium Methods. Data represent mean ± SEM (n=3). SEM ± mean represent Data basolateral and Methods. medium apical Cells, DMEM. fresh in h 20 for incubated BC 5,6-epoxy BC 5,8-epoxy BC 5,8-epoxy BC 5,6-epoxy 121 medium Apical ■ Basolateral□ Medium Cell□ n.d. n.d. Efficiency Conversion 0.05 ± 0.004 ± 0.05 n.d. Retinol In cells In (nmol/flask) 0.1cd Carotene 2.8 ±0.32.8 a ±0.15 3.1 ±0.017 0.36 1.3 ± ± 1.3 (nmol/flask) 2.9 ±0.22.9 ab 0.01± 0.2 0.001 ± 0.03 48 h (%) h 48 82.4 ±3.1 82.4 a 4 ± 1 29.6 C c 0.1 ± 1.5 0.01 ± 0.2 92.3 ± 2.4 a 8 a 2.4 ± 92.3 Recovery after after Recovery n.d n.d. n.d. n.d. 12 7 ±7 0512 30.2 ± 0 4 0 ± 30.2 44.0 ± 0.4 ± 44.0 25 3 ±0 3 25 2 d 5 3 ± 53.9 (nmol/flask) In medium In Carotene at 48 h 48 h at Carotene n.d. 36.8 ± ± 1.3 36.8 47.8 ± ± 1.9 47.8 43.3 ± 3 5 3 ± 43.3 47.6 ± 8 3 ± 47.6 (nmol/flask) Carotene at 0 h h 0 at Carotene in the cells 48 h after incubation. Values are mean ± SEM (for n = 3-5). Different letters indicate statistically days postconfluency. Conversion efficiency was calculated as the ratio of(1/2 retinol) to (1/2 retinol + carotene) flasks in complete DMEM. Once monolayers were confluent, cells were grown in serum-free medium for 12-14 significance difference (p < 0.0001) n.d. = not detected. medium added to the cell cell the to added Type of carotene carotene of Type BC 5,6-epoxy BC 5,6-epoxy BC 5,8-epoxy 5,6,5’,6-diepoxy BC 5,6,5’,6-diepoxy Control Tabel 5.1. Conversion efficiency of p-carotene epoxides in the TC7 clone of Caco-2 cells. Cells were grown in T75 cm2

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Cancer Let. 55: 75-81.

Ong, A. S. H. & Tee, E. S. (1992) Natural sources of carotenoids from plants and oils.

Methods Enzymol. 213: 142-167.

Paetau, I., Chen, H., Goh, N. M. -Y. & White, W. S. (1997) Interactions in the

postprandial appearance of p-carotene and cantaxanthin plasma triacylglycerol-

rich lipoprotein in humans. Am. J. Clin. Nutr. 66: 1133-1143.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pasquier, B., Armand, M , Castelain, C., Guillon, F., Borel, P., Lafont, H. & Lairon, D.

(1996) Emulsification and lipolysis of triacylglycerols are altered by viscous

soluble dietery fibers in acidic gastric medium in vitro. Biochem. J. 314: 269-

275.

Peng, Y.-S. & Peng, Y. M. (1992) Simultaneous liquid chromatographic determination of

carotenoids, retinoids, and tocopherols in human buccal mucosal cells. Cancer

Epidemiol. Biomarkers Prev. 1: 375-382

Pinto, M., Robine-Leon, S., Appay, M. D , Kedlinger, M., Triadou, N., Dussaulx, E ,

Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., & Zweibaum, J. (1981)

Enterocyte-like differentiation and polarization in the human colon carcinoma cell

line Caco-2 in culture. Biol. Cell. 47: 323-330.

Puspitasari-Nienaber, N. L., Ferruzzi, M. G., & Schwartz, S. J. (2002) Simultaneous

detection of tocopherols, carotenoids and chlorophylls in vegetable oils by direct

injection on RP-HPLC with coulometric electrochemical detection. J. Am.Oil

Chem. Soc. 79:633-640.

Puspitasari-Nienaber, N. L., Min, D. B., & Schwartz, S. J. (2000) High concentration of

a- and O-carotenes increase oxidation and tocopherol degradation in red palm

oil. Inform 11: S121.

Quick, T. C. & Ong, D. E. (1990) Vitamin A metabolism in the human intestinal Caco-2

cell line. Biochem. 29:11116-11123.

Rock, C. D. & Zeevaart, J. A. D. (1991) The aba mutant of Arabidopsis thaliana is

impaired in epoxy-carotenoid biosynthesis. Proc. Natl. Acad. Sci. 88: 7496-

7499.

128

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Salgo, M. G., Cueto, R., Winston, G. W., & Pryor, W. A. (1999) p-Carotene and its

oxidation products have different effects on microsome mediated binding of

benzo[a]pyrene to DNA. Free Rad. Biol. Med. 26: 162-173.

Solomon, N. W. (1998) Plant sources of vitamin A and human nutriture: red palm oil

does the job. Nutr. Rev. 56: 309-311.

Sugawara, T , Baskaran, V., Tsuzuki, W. & Nagao, A. (2002) Brown algae fucoxanthin

is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells

and mice. J. Nutr. 132:946-951.

Sugawara, T., Kushiro, M., Zhang, H., Nara, E., Ono, H. & Nagao, A. (2001)

Lysophosphatidylcholine enhances carotenoid uptake from mixed micelles by

Caco-2 human intestinal cells. J. Nutr. 131: 2921-2927.

Tang, G. W., Wang, X.-D., Russell, R. M., & Krinsky, N. I. (1991) Characterization of

beta-apo-13-carotenone and beta-apo-14'-carotenal as enzymatic products of the

excentric cleavage of beta-carotene. Biochem. 30: 9829-9834.

Tonucci, L. H., Holden, J. M., Beecher, G. R., Khachik, F., Davis, C. S., & Mulokozi, G.

(1995) Carotenoid content of thermally processed tomato-based food products.

J. Agric. Food Chem. 43: 579-586.

Tyssandier, V., Cardinault, N., Caris-Veyrat, C., Amiot, M.J., Grolier, P., Bouteloup. C..

Azais-Braesco, V. & Borel, P. (2002). Vegetable-born lutein, lycopene, and beta-

carotene compete for incorporation into chylomicrons, with no adverse effect on

the medium-term (3-wk) plasma status of carotenoids in human. Am. J. Clin.

Nutr. 75: 526-534.

Tyssandier, V., Lyan, B. & Borel, P. (2001) Main factors governing the transfer of

carotenoids from emulsion lipid droplet to micelles. Biochim. Biophys. Acta.

1533: 285-292.

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conversion of beta-carotene into beta-apocarotenals and retinoids by human,

monkey, ferret, and rat tissues. Arch. Biochem. Biophys. 285: 8-16.

White, W. S., Stacewicz-Sapuntzakis, M. & Erdman Jr., J. W. (1994) Pharmakokinetics

of (3-carotene and cantaxanthin after ingestion of individual and combined doses

by human subjects. J. Am. Coll. Nutr. 13: 665-671.

Wirtz, G. M., Bornemann, C., Giger, A., Muller, R. K., Schneider, H., Schlotterbeck, G .

Schiefer, G. & Woggon, W.-D. (2001) The substrate specificity of p, p-carotene

15,15 -monooxygenase. Helv. Chim. Acta. 84:2301-2315.

Yeum, K.-J., Ferreira, A. L. A., Smith, D., Krinsky, N. I., & Russell, R. M. (2000) The

effect of a-tocopherol on the oxidative cleavage of b-carotene. Free Rad. Biol.

Med. 29: 105-114.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6

Mutagenicity of Epoxy (3-Carotene inSalmonella typhimurium

Ni Luh Puspitasari-Nienaber, Polly D. Courtney and Steven J. Schwartz'

Department of Food Science and Technology

The Ohio State University

2015 Fyffe Road, Columbus, OH 43210-1007

Keywords: Ames assay, bacterial reverse mutation assay, p-carotene, epoxide, epoxy

p-carotene, mutagencity 131

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.1. ABSTRACT

The purpose of this study was to investigate the potential mutagenicity of epoxy

p-carotene using the miniscreen bacterial reverse mutation assay. Salmonella

typhimurium histidine-dependent strains TA98, TA100, TA97 and TA102 were used, p-

Carotene epoxides were synthesized, purified and tested in acetone at 0.5 to 20

nmol/well. The 5,6-epoxy p-carotene was mutagenic in the TA102 strain with and

without activation with S-9 mix and 5,8-epoxy p-carotene was mutagenic in the same

strain but with activation only. Mutagenicity of the epoxides was not detected in the

other strains tested.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.2. INTRODUCTION

Epoxy compounds are highly reactive compounds because of the electron

deficient sites in their molecules. These electrophilic centers are highly reactive and can

covalently bind to cellular macromolecules with nucleophilic sites such as proteins and

nucleic acids. Binding of epoxides to DNA has been reported to result in cytotoxicity

(Dypbukt et al., 1992), mutagenicity (Ehrenberg and Hussain, 1981) and carcinogenesis

(Pitot and Dragan, 1996). Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (Jeffrey et al..

1976) and 2,3-epoxy aflatoxin B1 (Essigman et al., 1977) have been reported to react

with the guanine base pair of DNA. These epoxy compounds are metabolites of

benzo[a]pyrene and aflatoxin Bi after oxidation via the cytochrome P-450 pathways.

However, not all epoxy compounds are formed in vivo.

Epoxycarotenoid is a class of compounds widely distributed in nature, such as

fruits and green vegetables (Khachik et al., 1991; Barua and Olson, 1998; Barua and

Olson 2001), red palm oil (Ong and Tee, 1992) and brown algae (Sugawara et al..

2002). Epoxycarotenoids in plants serve as precursors of abscisic acid, a plant growth

regulator (Rock and Zeevaart, 1991). In addition, epoxycarotenoids formed during food

processing and storage as the result of oxidation (Marti and Berset, 1988; Handelman et

al., 1991; Lieblerand Kennedy, 1992; Tonucci etal., 1995). Baker et al. (1999) showed

the formation of p-carotene epoxide when p-carotene reacted with cigarette smoke,

while Tang et al. (1991) during the incubation of (3-carotene with rat intestinal mucosa

homogenates. These observations suggest that epoxycarotenoids may also be formed

133

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in vivo. The presence of a 5,6-dihydroxy-5,6-dihydrolycopene in the serum of human

subjects (Khachik et al., 1995) and human milk (Khachik et al., 1997) was described.

The authors suggested in vivo oxidation of lycopene into 5,6-lycopene epoxide, which

then undergoes metabolic reduction to 5,6-dihydroxy-5,6-dihydrolycopene.

We previously reported the oxidative and thermal stability of red palm oil

containing 500 pg/g of a - and p-carotene, and found that epoxy p-carotene accumulated

during storage (Puspitasari-Nienaber et al., 2000). Epoxy p-carotene accumulated up to

100 pg/g, depending on the storage time and temperature. Furthermore, p-carotene

epoxides were,accumulated but poorly converted to vitamin A by Caco-2 human

intestinal cells (Puspitasari-Nienaber et al., 2002). Palm fruit carotene has been shown

to demonstrate no mutagenic activity in a series of in vitro mutagenicity assays (Masuda

et al., 1995). However, whether the presence of epoxycarotenoids in red palm oil would

affect the safety of the oil is unknown. In this study the potential mutagenicity of epoxy

p-carotene was investigated using the bacterial mutation assay.

6.3. EXPERIMENTAL PROCEDURES

Chemicals and Standards. p-Carotene was purchased from Sigma Chemical

Co. (St. Louis, MO) and 3-chloroperoxy benzoic acid was from Aldrich (Milwaukee, Wl).

Organic solvents were of certified HPLC and ACS grade (Fisher Scientific, Fair Lawn.

NJ). The positive controls benzo[a]pyrene (B[a]P) and 2-aminoanthracene (AA) were

purchased from Moltox (Boone, NC).

134

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Synthesis and Purification of (3-Carotene Epoxides. (3-Carotene epoxides

were synthesized as previously described (Barua and Olson, 1998). Briefly, p-carotene

was dissolved in 100 mL methyl tert-butyl ether (MTBE) before the addition of an

equimolar amount of 3-chloroperoxy benzoic acid. The mixture was stirred in the dark at

room temperature for 1 h. At this point, 5,6-epoxy p-carotene was the primary product of

oxidation and the mixture existed in 1:1 ratio with the corresponding p-carotene. The

reaction mixture was then divided into two 50 mL portions. To the first portion, 2-3 drops

of glacial acetic acid was added and the mixture was stirred for an additional 30 min. An

acidic condition was required for the rearrangement of 5,6-epoxy p-carotene to its 5,8-

epoxy derivative. Reactions in both portions were terminated by the addition of water

and neutralization with 0.1 N NaOH. Fractions containing individual p-carotene epoxide

were separated using a preparatory (19 x 300 mm) pBondapak C is HPLC column

(Waters, Milford, MA). Methanol:MTBE (85:15) was used as the mobile phase at a flow

rate of 5.0 mL/min. The extract obtained from synthesis was injected with a 1 mL

sample loop. Individual fractions were then dried under nitrogen and stored at -80 °C.

In addition to their retention times on the HPLC column, the identity of epoxides was

confirmed using their UV-vis spectra and chemical assay for epoxide group (Britton.

1995).

Bacterial mutation assay. Samples were tested for mutagenicity by the

modified microscreen assay (Diehl et al., 2000), based on the Salmonella histidine

reverse mutagenicity assay (Maron and Ames, 1983). Six-well cell-culture dishes were

used instead of standard plates. Salmonella typhimurium histidine-dependent strains

TA98, TA100, TA97 and TA102 were used as tester strains (Moltox, Boone, NC).

135

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bacterial cultures were grown in 10 mL of nutrient broth (Oxoid No.2, Unipath,

Ogdensburg, NY) containing ampicilin and incubated in a 35 ± 2 °C shaker for 12 h or

until the optical density reached 1.0. Pre-poured six-well cell-culture dishes (5 mL

Vogel-Bonner minimal glucose agar), top agar (2% agar), and S-9 fraction of arocior

1254-induced rat liver microsomes were purchased from Moltox (Boone, NC). The S-9

mix was prepared in a glucose-6-phosphate and NADP solution (4%, v/v) immediately

before use and kept on ice throughout the experiment. Each carotene derivative and

positive control was dissolved in acetone. Both positive and negative (acetone) controls

were run concurrently with each assay.

Plate incorporation test with preincubation was conducted as follows. Into a 15

mL sterile vial 100 pL S-9 mix, 25 pL bacterial culture and 25 pL test compound were

added. This order of addition was followed when solvent other than water or DMSO was

used, to avoid exposure of bacteria with undiluted solvent (Maron et al., 1981). Mixtures

were then preincubated for 30 min in a 37 °C incubator. After the addition of 500 pL

molten top agar, mixtures were poured onto the six-well dishes, and incubated inverted

in a 37 °C incubator for 48 h.

6.4. RESULTS AND DISCUSSION

Oxidation products of (3-carotene, i.e. 5,6-epoxy and 5,8-epoxy (3-carotene, were

screened for their potential mutagenicity using the bacterial mutagenicity assay. The

modified microscreen method of Diehl et al. (2000) was applied instead of the standard

plate assay (Maron and Ames, 1983). In the microscreen assay six-well cell-culture

dishes were used instead of the standard plate, thus reducing the amount of required

136

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. test compounds. Very good agreement for positive and negative mutagenic responses

between the two methods was reported in Salmonella typhimurium TA 98, TA100, TA

102 and E. coli (Diehl et al., 2000).

(3-Carotene and its epoxy derivatives were dissolved in acetone. The commonly

used solvent for bacterial mutagenicity assay, i.e. dimethyl sulfoxide (DMSO) was not

used because at high concentration (3-carotene epoxide preparations crystallized out of

solution in the present study. Acetone was one of the 12 solvents considered suitable

for Ames assay (Maron et al., 1981). (3-Carotene and its epoxy derivatives were tested

at concentrations between 0.5 to 20 nmol/well (or 0.3 to 10.8 ng/well). Positive controls

were tested at concentrations previously reported to give positive responses (Diehl et al..

2000). B[a]P was tested at 15 ^ig/well and AA was tested at 12.5 fig/well.

The 5,6-epoxy p-carotene was highly mutagenic in S. typhimurium TA102 without

and with activation (Table 6.1 and 6.2). Without activation and at lower concentrations

(0.3 to 2.7 ng/well), the numbers of revertant per well were significantly higher (p < 0 05)

than the positive controls. The numbers of revertant per well at the greater

concentration was less as compared with that at lower concentrations, suggesting that

5,6-epoxy p-carotene at greater concentration was toxic to the cell. In some cases, the

reduction in the background lawn of bacterial growth was observed at greater

concentrations. The results for 5,8-epoxy p-carotene were positive within the same

strain but only with activation. At the level tested, the number of revertant per well was

not different from the positive controls.

It appeared that S. typhimurium TA102 was the most sensitive strain in detecting

the mutagenicity of epoxy p-carotene. Results of the present study suggest that 5,6-

epoxy p-carotene is a direct acting mutagen at all concentrations tested. Interestingly,

137

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the 5,8-epoxy p-carotene required activation to demonstrate its mutagenicity. Most of

chemical carcinogens requires activation to their ultimate carcinogenic forms, via

hydroxylation or epoxidation reactions by cytochrome P45 enzymes (Pitot and Dragan,

1996). Two step epoxidation was required for benzo[a]pyrene to form its ultimate

carcinogen form. Further investigation using other mutagenicity assays is required to

confirm and explain results of the present study. In addition, mutagenicity assessment

of epoxy p-carotene in red palm oil is suggested to better understand the safety of red

palm oil as a source of provitamin A carotenoids and antioxidants.

6.5. CONCLUSION

In conclusion, epoxy p-carotene was found mutagenic in S. typhimurium TA102

but not in TA98, TA100 and TA97. Further experiments to confirm the mutagenicity of

epoxycarotenoids are warranted.

138

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Concentration Revertant/well Compound (pg/well) TA98 TA100 TA97 TA102

5,6-Epoxy p-carotene 0.3 1 ± 1 1 ± 1 - 29 ±9* 1.3 1 ± 1 3 ± 1 - 31 ±5* 2.7 - 1 ± 1 - 39 ± 9**

5.4 - - - 16 ± 2 10.7 - - - 9 ± 2 5,8-Epoxy p-carotene 0.3 - - -- 1.3 --- - 2.7 - - -- 5.4 -- -- 10.7 - - -- p-Carotene 0.3 -- -- 1.3 -- -- 2.7 -- -- 5.4 - - -- 10.7 “ Positive Control: '

Benzo[a)pyrene 15.0 3 ± 1 3 ± 1 3 ± 1 14 r 1 2-Aminoanthracene 12.5 5 ± 2 5 ± 1 6 ± 2 26 ±2 Negative control 1 ± 1 1 ± 1 - 2 ± 1

Table 6.1. Mutagenicity of epoxy p-carotene to S. typhimurium TA98, TA100, TA97 and TA102 without activation. Values are mean ± SEM (n=3-7).

- Negative * Significantly higher from benzo[a]pyrene (p < 0.05) * Significantly higher from 2-aminoanthracene (p < 0.05)

139

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Concentration Revertant/well Compound (pg/well) TA98 TA100 TA97 TA102

5,6-Epoxy p-carotene 0.3 - - - 1 ± 1 1.3 - 2 ± 2 - 29 + 9 2.7 1 ± 1 1 ± 1 - 14 ± 3

5.4 1 ± 1 - - - 10.7 - - - - 5,8-Epoxy p-carotene 0.3 2 ± 1 -- 27 ± 9 1.3 3 ± 1 - - 44 ± 5 2.7 1 ± 1 -- 34 i 5 5.4 - -- 20 r 5 10.7 - - - -

p-Carotene 0.3 - - - 1 + 1 1.3 1 ± 1 - - 1 + 1 2.7 1 ± 1 - - 1 ± 1 5.4 1 ± 1 - - 1 ± 1 10.7 - -- - Positive Control:

Benzo[a]pyrene 15.0 16 ± 4 72 ± 4 30 ± 6 37 r 8

2-Aminoanthracene 12.5 249 ± 53 102 + 3 362 ± 32 55 r 6

Negative control 1 ± 1 -- 1 r 1

Table 6.2. Mutagenicity of epoxy p-carotene to S. typhimurium TA98, TA100, TA97 and TA102 with activation. Values are mean ± SEM (n=3-7).

- Negative * Significantly higher from benzo[a]pyrene (p < 0.05) * Significantly higher from 2-aminoanthracene (p < 0.05)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.6. REFERENCES

Baker, D. L., Kroll, E. S., Jacobsen, N., & Liebler, D. C. (1999) Reactions of (3-carotene

with cigarette smoke oxidants. Identification of carotenoid oxidation products and

evaluation of the prooxidant/antioxidant effect. Chem. Res. Toxicol. 6: 535-543.

Barua, A. B. & Olson, J. A. (1998) Reversed-phase gradient high-performance liquid

chromatographic procedure for simultaneous analysis of very polar to nonpolar

retinoids, carotenoids and tocopherols in animal and plant samples. J.

Chromatogr. B 707: 69-79.

Barua, A. B. & Olson, J. A. (2001) Epoxyxanthophylls, unlike mono-epoxy carotenes, are

not absorbed by humans. J. Nutr. 131: 3212-3215.

Britton, G. (1995) UV/Visible Spectroscopy. In: Carotenoids Vol. 1B Spectroscopy

(Britton, G., Liaaen-Jensen, S., & Pfander, H., eds ), pp. 13-59. Birkhauser,

Basel.

Diehl, M. S., Willaby, S. L. & Snyder, R. D. (2000) Comparison of the results of a

modified miniscreen and the standard bacterial reverse mutation assays.

Environ. Mol. Mutagen. 35: 72-77.

Dypbukt, J. M., Costa, L. G , Manzo, L., Orrenius, S. & Nicotera, P. (1992) Cytotoxic

and genotoxic effects of styerene-7,8-oxide in neuroadrenergic Pc 12 cells.

Carcinogenesis 13: 417-424.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ehrenberg, L. and Hussain, S. (1981) Genetic toxicity of some important epoxides.

Mutation Res. 86:1-113.

Essigman, J. M., Croy, R. G., Nadzan, A. M., Busby Jr., W. F„ Reinhold, V. N., Buchi,

G., & Wogan, G. N. (1977) Structural identification of the major DNA adduct

formed by aflatoxin Bi in vitro. Proc. Natl. Acad. Sci. 74: 1870-1879.

Handelman, G. J., van Kuijk, F. J. G. M., Chatterjee, A., & Krinsky, N. I. (1991)

Characterization of products formed during the autoxidation of p-carotene. Free

Rad. Biol. Med. 10: 427-437.

Jeffrey, A. M., Jennete, K. W., Blobstein, S. H„ Weinstein, I. B., Beland, F. A., Harvey,

R. G., Kasai, H., Miura, I. & Nakashini, K. (1976) Benzo[a]pyrene-nucleic acid

derivative found in vivo: Structure of a benzo[a]pyrene-tetrahydrodiol epoxide-

guanosine adduct. J. Am. Chem. Soc. 98:5714-5719.

Khachik, F., Beecher, G. R., Mudlagiri, B. G., Lusby, W. R., & Goli, M. B. (1991)

Separation, identification and quantification of carotenoids in fruits, vegetables

and human plasma by high performance liquid chromatography. Pure Appl.

Chem. 63: 71-80.

Khachik, F., Beecher, G. R , & Smith Jr., J. C. (1995) Lutein, lycopene, and their

oxidative metabolites in chemoprevention of cancer. J. Cell. Biochem. 22: 236-

246.

Khachik, F., Spangler, C. J., Smith Jr., J. C., Canfield, L. M., Steck, A. & Pfander, H.

(1997) Identification, quantification, and relative concentrations of carotenoids

and their metabolites in human milk and serum. Anal. Chem. 69:1873-1881.

Liebler, D. C. & Kennedy, T. A. (1992) Epoxide products of p-carotene antioxidant

reactions. Methods Enzymol. 213: 472-479.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Maron, D. M. & Ames, B. N. (1983) Revised methods for the Salmonella mutagenicity

test. Mutation Res. 113:173-215.

Marty, C. & Berset, C. (1988) Degradation products of trans-p-carotene produced during

extrusion cooking. J. Food Sci. 53: 1880-1886.

Masuda, M., Haresaku, M., Takei, T., & Nakamura, T. (1995) Mutagenicity studies with

palm fruit carotene. J. Toxicol. Sci. 20: 619-627.

Ong, A. S. H. & Tee, E. S. (1992) Natural sources of carotenoids from plants and oils.

Methods Enzymol. 213: 142-167.

Pitot III, H. C. and Dragan, Y. P. (1996) Chemical Carcinogenesis. In: Casarett &

Doulls' Toxicology, The Basic Science of Poison (Klaassen, C. D., ed ). pp. 210-

221. McGraw Hill, New York.

Puspitasari-Nienaber, N. L , Min, D. B., & Schwartz, S. J. (2000) High concentration of

a- and (3-carotenes increase oxidation and tocopherol degradation in red palm

oil. Inform 11: S121.

Puspitasari-Nienaber, N. L., Ferruzzi, M. G., & Schwartz, S. J. (2002) Simultaneous

detection of tocopherols, carotenoids and chlorophylls in vegetable oils by direct

injection on RP-HPLC with coulometric electrochemical detection. J. Am.Oil

Chem. Soc. Accepted for publication.

Rock, C. D. & Zeevaart, J. A. D. (1991) The aba mutant of Arabidopsis thaliana is

impaired in epoxy-carotenoid biosynthesis. Proc. Natl. Acad. Sci. 88: 7496-

7499.

Sugawara, T., Baskaran, V., Tsuzuki, W. & Nagao, A. (2002) Brown algae fucoxanthin

is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells

and mice. J. Nutr. 132: 946-951.

143

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tang, G. W., Wang, X.-D., Russell, R. M., & Krinsky, N. I. (1991) Characterization of

beta-apo-13-carotenone and beta-apo-14'-carotenal as enzymatic products of the

excentric cleavage of beta-carotene. Biochem. 30: 9829-9834.

Tonucci, L. H., Holden, J. M., Beecher, G. R., Khachik, F., Davis, C. S., & Mulokozi, G.

(1995) Carotenoid content of thermally processed tomato-based food products.

J. Agric. Food Chem. 43: 579-586.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISSERTATION SUMMARY

The research described in the previous documents was designed to evaluate the

storage and thermal stability of carotenoids in red palm oil (RPO), by understanding the

role of indigenous carotenoids and tocopherols. The digestibility, absorption, conversion

to vitamin A and mutagenicity of (3-carotene epoxides, a class of compounds formed

during RPO storage, were also examined.

The first study was conducted to develop an analytical methodology suitable for

the separation and identification of carotenoids and tocopherols in vegetable oils, such

as red palm oil. A C30 RP-HPLC with electrochemical detection was employed for the

simultaneous analysis of tocopherols, carotenoids and chlorophylls. Aliquots of

vegetable oils were dissolved in appropriate solvents and injected directly without

saponification, thus preventing sample loss or component degradation. Effective

separation of tocopherols, carotenoids and chlorophylls was achieved. Detection wai

performed using a coulometric electrochemical array detector set between 200 and 620

mV. For a 25 pL injection, the respective detection limits for carotenoids, tocopherols

and chlorophylls were 1 fmol, 0.15 pmol and 0.5 pmol, representing 1000, 25 and 5 fold

enhancement over the UV-vis methodology.

The second study was performed to investigate the effects of carotenoids (500

ng/g) and tocopherols (900 pg/g) on the oxidative and thermal stability of red palm oil

(RPO). RPO was stored at different temperatures in the dark for 12 weeks. Ten

145

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. batches of frozen french fries were fried per day for three consecutive days at 180 °C.

Refined bleached and deodorized palm oil (RBDPO) was the control oil. Oxidative

degradation of carotenoids in RPO followed zero-order reaction kinetics. Storage time

for the carotenoids to deplete 50% (ti/2) was calculated as 12, 4 and 1.3 months at 25,

37 and 60 °C, respectively. Up to 100 ^ / g epoxy p-carotene, the oxidation product of p-

carotene, accumulated during storage at lower temperatures. Tocopherols degraded 1.3

to 3.2 times faster in RPO than control. When used for frying, > 90% carotenoids was

degraded at the end of the come-up time (15 min). These observations suggest that

RPO is stable during storage but not suitable for frying. In addition, tocopherol played

an important role in maintaining the oxidative and thermal stability of RPO.

In the third study, the focus was on the digestion, absorption and vitamin A

conversion of epoxy p-carotene, using a simulated gastric and small intestinal digestion

coupled with Caco-2 cells. The presence of epoxy p-carotene in applesauce did not

affect the micellarization of p-carotene. In contrast, micellarization of p-carotene

epoxides was significantly reduced in the presence of p-carotene. The type of food

matrix had a profound effect on micellarization of carotenoids. Greater micellarization of

all carotenoids tested from palm oil than applesauce was found. Furthermore,

micellarization efficiencies of p-carotene from used palm oil was lower than from fresh

palm oil. Accumulation of p-carotene epoxides was evaluated using fully differentiated

HTB37 parent line and TC7 clone of the Caco-2 human intestinal cells. Cellular

accumulation of p-carotene epoxides was lower than that of p-carotene. The TC7 clone

was also used to evaluate the conversion of the different p-carotene epoxides to vitamin

A. The conversion efficiency of 5,6-epoxy p-carotene and 5,8-epoxy p-carotene was

only 10% that of p-carotene. These results suggest that p-carotene epoxides were

146

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. readily accessible for absorption but the degree of micellarization depended on the

presence of other carotenoids and the food matrix. In addition, p-carotene epoxides

were poorly absorbed and poorly converted to vitamin A.

In the last study, the potential mutagenicity of epoxy p-carotene was investigated

using the modified miniscreen bacterial reverse mutation (Ames) assay. Salmonella

typhimurium histidine-dependent strains TA98, TA100, TA97 and TA102 were used, p-

Carotene epoxides were tested in acetone at 0.5 to 20 nmol/well. The 5,6-epoxy p-

carotene was mutagenic in the TA102 strain with and without activation with S-9 mix and

5,8-epoxy p-carotene was mutagenic in the same strain but with activation only.

Mutagenicity of the epoxides was not detected in the other strains tested.

The overall conclusion of this study was that RPO is a good source of provitamin

A carotenoids and tocopherols. RPO is stable during storage at room temperature and if

stored in the absence of light its shelf-life reached one year. RPO, however, is not

suitable for frying. Oxidation products of carotenoids, mainly p-carotene epoxides,

accumulated during storage. These compounds are readily accessible for uptake by the

enterocytes, but accumulation was limited and depended on the presence of other

carotenoids as well as the type of food matrix. Epoxy p-carotene was converted less

efficiently to vitamin A than p-carotene. Also, epoxy p-carotene showed mutagenic

activity in one Salmonella strain.

147

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