Β-Apocarotenoids: Occurrence in Cassava Biofortified with Β-Carotene and Mechanisms of Uptake in Caco-2 Intestinal Cells

β-Apocarotenoids: Occurrence in Cassava Biofortified with β-Carotene and Mechanisms of Uptake in Caco-2 Intestinal Cells

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Boluwatiwi O. Durojaye

Graduate Program in Human Nutrition

The Ohio State University

2015

Master's Examination Committee:

Earl H. Harrison, Ph.D., Advisor

Steven J. Schwartz, Ph.D.

Joshua A. Bomser, Ph.D.

Copyrighted by

Boluwatiwi O. Durojaye

2015

Abstract

Biofortification is defined as the enrichment of staple crops with essential

micronutrients. At present, it is one of the strategies used to alleviate vitamin A

deficiency (VAD) by breeding staple crops with β-carotene. Staple crops that

have been successfully biofortified with β-carotene under the HarvestPlus

program are cassava, maize (corn) and sweet potato. Recently, β-

apocarotenoids have been identified and quantified in cantaloupe melons and

orange-fleshed honeydew. These cleavage products of β-carotene are formed by

chemical and enzymatic oxidations. However, there are no detailed analyses of

these compounds in biofortified foods and little is known about their bioavailability

and intestinal absorption. Hence, this research focused on the analysis of β-

apocarotenoids in biofortified cassava and kinetics of cell uptake and metabolism

of β-apocarotenoids.

In the first study, we analyzed the β-carotene and β-apocarotenoids

content of conventionally bred cassava biofortified with β-carotene. Using a

previously described high performance liquid chromatography-tandem mass

spectrometry (HPLC-MS/MS) method, we identified and quantified β-apo-13-

carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and

β-apo-8′-carotenal in hexane/acetone extracts of raw, boiled, and fried roots of

ii the two biofortified cassava varieties investigated. The levels of β-apocarotenoids in roots of non-biofortified cassava varieties were lower than those of biofortified varieties and some of these values approached the limit of detection (LOD) or limit of quantification (LOQ).

The purpose of the second study was to determine kinetics of uptake and metabolism of β-apocarotenoids using Caco-2 intestinal cells as a model. We hypothesized that these compounds are directly absorbed from the diet similarly to β-carotene. Pure β-apocarotenoids were delivered to fully differentiated monolayers of Caco-2 cells using tween-40 micelles. Carotenoids were extracted from media and cells with acetone:hexanes (1:4 v/v) and analyzed by HPLC. We observed that there was rapid uptake of β-apo-8′-carotenal into cells. We detected two unidentified metabolites (X and Y) of β-apo-8′-carotenal. The formation of compound X increased with time corresponding with a decrease in

β-apo-8′-carotenal. Also, cellular uptake of β-apo-13-carotenone was rapid and this compound was extensively degraded over time.

Understanding the mechanisms of absorption and metabolism of β- apocarotenoids relative to their quantities in foods is critical in exploring the functions of these metabolites, some of which have been shown to be potent antagonists of vitamin A.

Keywords: Biofortification; HPLC-MS/MS; cell uptake; carotenoids metabolism

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Acknowledgements

First and foremost, I would like to express my sincere gratitude to my advisor and mentor, Dr. Earl H. Harrison. Thank you for the funding, support and constructive criticism throughout this research project.

Also, I would like to thank Dr. Steven J. Schwartz for serving on my thesis advisory committee and for his expertise and collaboration on this project. I also would like to thank Dr. Joshua A. Bomser for serving on my committee. I am also grateful to Dr. Mark L. Failla for his expertise and Dr. Chureeporn

Chitchumroonchokchai (Julie C) for her important laboratory training and contributions to the success of this project.

To present and past members of the Harrison laboratory group – Carlo,

Sara, Yan and Vanessa, thank you for the training and time spent to troubleshoot numerous unsuccessful experiments. It has been a pleasure working with you all.

I am most especially grateful to my parents Kolawole and Olufeyisara

Durojaiye for whom none of this would be possible. Thank you for your prayers, support and words of encouragement that got me through my most difficult days.

Vita

Education

2013-Present...……..……………M.S. Human Nutrition, The Ohio State University

2012-2014.……….………………...………M.Sc. Biochemistry, University of Lagos

2007-2010…………………..……B.Sc. (Honors) Biochemistry, University of Lagos

Experience and Awards

2014-Present…………...Graduate Teaching Associate, The Ohio State University

2013-Present……………..EHE Graduate scholarships, The Ohio State University

2013…………………...University Graduate Fellowship, The Ohio State University

Fields of Study

Major Fields: Human Nutrition; Biochemistry

Table of Contents

Abstract ...... ii

Acknowledgements ...... iv

Vita ...... v

Table of Contents ...... vi

List of Tables ...... x

List of Figures ...... xi

List of Abbreviations ...... xiv

Introduction ...... 1

Specific Aims ...... 4

Aim 1 ...... 4

Aim 2 ...... 5

Chapter 1. Review of Literature ...... 6

1.1 Biofortification as a strategy to alleviate VAD ...... 7

1.2 Methods of biofortification for provitamin A cassava ...... 9

1.3 Retention, bioaccessibility and bioavailability of β-carotene in provitamin

A cassava ...... 11 vi

1.4 Metabolism of β-carotene ...... 14

1.4.1 Digestion ...... 14

1.4.2 Intestinal absorption ...... 15

1.4.3 Cellular Uptake ...... 18

1.5 Cleavage products of β-carotene: How are they formed? ...... 19

1.5.1 Chemical reactions of carotenoids yielding β-apocarotenoids ...... 19

1.5.2 Enzymatic cleavage of carotenoids...... 20

1.6 Occurrence of β-apocarotenoids in foods and in vivo ...... 23

1.7 Biological activity of β-apocarotenoids ...... 24

Chapter 2. Analysis of β-carotene and β-apocarotenoids in cassava biofortified with β-carotene ...... 28

2.1 Abstract ...... 29

2.2 Introduction ...... 30

2.3 Materials and Methods ...... 35

2.3.1 Chemicals and Supplies ...... 35

2.3.2 Processing of cassava varieties ...... 35

2.3.3 Extraction and HPLC-MS/MS analysis of β-carotene and β-

apocarotenoids ...... 37

2.4 Results ...... 40

vii

2.4.1 Profile of β-apocarotenoids in cassava samples ...... 40

2.4.2 Determination of total content of β-carotene and β-apocarotenoids in

cassava ...... 44

2.4.3 Effect of boiling and frying on β-apocarotenoids content ...... 48

2.5 Discussion ...... 54

2.6 Conclusion ...... 56

2.7 Acknowledgements ...... 56

Chapter 3. Kinetics of uptake and metabolism of β-apo-8′-carotenal and β- apo-13-carotenone by Caco-2 human intestinal cells ...... 57

3.1 Abstract ...... 58

3.2 Introduction ...... 59

3.3 Materials and Methods ...... 64

3.3.1 Chemicals and Supplies ...... 64

3.3.2 Cell culture conditions ...... 64

3.3.3 Preparation of Tween-40 micelles ...... 64

3.3.4 Stability of β-apocarotenoids during incubation in cell culture

conditions ...... 65

3.3.5 Uptake of β-apocarotenoids by Caco-2 cells ...... 66

3.3.6 Extraction of β-apocarotenoids ...... 67

3.3.7 HPLC Analysis ...... 68 viii

3.4 Results ...... 69

3.4.1 Stability of β-apo-8′-carotenal and β-apo-13-carotenone ...... 69

3.4.2 Kinetics of uptake of β-carotene: effects of incubation time and

concentration ...... 75

3.4.3 Effects of concentration of β-apo-8′-carotenal and incubation time on

the uptake and metabolism of β-apo-8′-carotenal ...... 80

3.4.4 Kinetics of uptake of β-apo-13-carotenone: effects of concentration

and incubation time ...... 89

3.5 Discussion ...... 95

3.6 Conclusion ...... 99

3.7 Acknowledgements ...... 99

Chapter 4. Epilogue ...... 100

References ...... 106

Appendix A. Additional figures for Chapter 2 ...... 113

Appendix B. Additional figures for Chapter 3 ...... 128

List of Tables

Table 2.1. Varieties of cassava investigated in this project ...... 36

Table 2.2. Source parameters for analysis of cassava samples ...... 39

Table 2.3. Relative total β-apocarotenoids in raw, boiled, and fried cassava ..... 47

Table 3.1. Stability of β-apo-8′-carotenal in cell culture environment ...... 72

Table 3.2. Stability of β-apo-13-carotenone in cell culture environment ...... 74

Table 3.3. Recovery of β-carotene during cell culture experiment ...... 79

Table 3.4. Recovery of β-apo-8′-carotenal during cell culture experiment ...... 88

Table 3.5. Recovery of β-apo-13-carotenone during cell culture experiment ..... 94

List of Figures

Figure 1.1. Overview of intestinal absorption of β-carotene ...... 17

Figure 1.2. Enzymatic cleavage of carotenoids ...... 22

Figure 2.1. Products of eccentric cleavage pathway of β-carotene ...... 33

Figure 2.2. Chemical structures of some β-apocarotenoids ...... 34

Figure 2.3. Representative chromatogram of profile of β-apocarotenoids

standards mixture ...... 41

Figure 2.4. Profile of β-apocarotenoids in raw roots of Clone 03-15 variety ...... 42

Figure 2.5. Profile of β-apocarotenoids in raw roots of Saracura variety ...... 43

Figure 2.6. Total β-carotene content of raw, boiled and fried roots of all cassava

varieties ...... 45

Figure 2.7. Total β-apocarotenoids content of raw, boiled and fried roots of all

cassava varieties ...... 46

Figure 2.8. Determination of β-apocarotenoids content of raw, boiled, and fried

roots of Clone 03-15 cassava variety ...... 49

Figure 2.9. Determination of β-apocarotenoids content of raw, boiled, and fried

roots of IAC 265-97 cassava variety ...... 50

Figure 2.10. Determination of β-apocarotenoids content of raw, boiled, and fried roots of IAC 576-70 cassava variety ...... 52

Figure 2.11. Determination of β-apocarotenoids content of raw, boiled, and fried roots of Saracura cassava variety ...... 53

Figure 3.1. Products of eccentric cleavage pathway of β-carotene ...... 62

Figure 3.2. Overview of intestinal absorption of β-carotene ...... 63

Figure 3.3. Stability of β-apo-8′-carotenal in cell culture conditions ...... 71

Figure 3.4. Stability of β-apo-13-carotenone in cell culture conditions ...... 73

Figure 3.5. Representative chromatogram of time dependent uptake of β-

carotene in Caco-2 cells ...... 76

Figure 3.6. Time course of uptake of β-carotene in Caco-2 cells ...... 77

Figure 3.7. Dose dependent uptake of β-carotene in Caco-2 cells ...... 78

Figure 3.8. Representative chromatogram of time dependent uptake of β-apo-8′-

carotenal in Caco-2 cells ...... 82

Figure 3.9. Kinetics of uptake and conversion of β-apo-8′-carotenal in Caco-2 cells ...... 83

Figure 3.10. Time course of uptake of β-apo-8′-carotenal in Caco-2 cells ...... 84

Figure 3.11. Time course of conversion of β-apo-8′-carotenal into an unknown metabolite (compound X) ...... 85

Figure 3.12. Dose dependent uptake of β-apo-8′-carotenal in Caco-2 cells ...... 86

Figure 3.13. Dose dependent conversion of β-apo-8′-carotenal into an unknown metabolite (compound X) ...... 87

Figure 3.14. Representative chromatogram of uptake of β-apo-13-carotenone in

Caco-2 cells ...... 91

xii

Figure 3.15. Time course of uptake of β-apo-13-carotenone in Caco-2 cells ..... 92

Figure 3.16. Dose dependent uptake of β-apo-13-carotenone in Caco-2 cells ... 93

Figure A.1. Profile of β-apocarotenoids in boiled roots of Clone 03-15 variety . 114

Figure A.2. Profile of β-apocarotenoids in fried roots of Clone 03-15 variety .... 115

Figure A.3. Profile of β-apocarotenoids in raw roots of IAC 265-97 variety ...... 116

Figure A.4. Profile of β-apocarotenoids in boiled roots of IAC 265-97 variety .. 117

Figure A.5. Profile of β-apocarotenoids in fried roots of IAC 265-97 variety ..... 118

Figure A.6. Profile of β-apocarotenoids in raw roots of IAC 576-70 variety ...... 119

Figure A.7. Profile of β-apocarotenoids in boiled roots of IAC 576-70 variety .. 120

Figure A.8. Profile of β-apocarotenoids in fried roots of IAC 576-70 variety ..... 121

Figure A.9. Profile of β-apocarotenoids in boiled roots of Saracura variety ...... 122

Figure A.10. Profile of β-apocarotenoids in fried roots of Saracura variety ...... 123

Figure A.11. Standard curves of pure β-apocarotenoids ...... 124

Figure A.12. Standard curves of pure β-apocarotenoids ...... 125

Figure A.13. Standard curves of pure β-apocarotenoids ...... 126

Figure A.14. Standard curves of pure β-carotene ...... 127

Figure B.1. Standard curves of pure β-apocarotenoids ...... 129

Figure B.2. Standard curves of pure β-carotene ...... 130

xiii

List of Abbreviations

APCI……………………………………….atmospheric pressure chemical ionization

atRA……………………………………………………….……….all-trans retinoic acid

BCO1………………………………………………β, β-carotene 15, 15′-oxygenase 1

BCO2……………………………………………….....β-carotene 9′, 10′-oxygenase 2

CD36………………………………………………………….....cluster determinant 36

CIAT……………………………………….international center for tropical agriculture

DHS……………………………………….…………..demographic and health survey

DMEM……………………………….....………Dulbecco’s minimum essential media

DW…………………………………..…………………………………………dry weight

DXS…………………………………………1-deoxy-xylulose-5-phosphate synthase

EM……………………………..……………………………efficiency of micellarization

EMBRAPA………………..………………Brazilian agricultural research corporation

FBS…………………………………………………………………...fetal bovine serum

FF……………………….………………………………………………..fermented flour

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FW…………………….……………………………………………….…….fresh weight

HPLC……………………………………….high performance liquid chromatography

HPLC-MS/MS...... high performance liquid chromatography- tandem mass spectrometry

IITA…………………………………….....international institute of tropical agriculture

LBD……………………………………………………………....ligand binding domain

LC/MS……………………………….……liquid chromatography/mass spectrometry

LOD……………………………………………………………….…….limit of detection

LOQ………………………………………………………………..limit of quantification

LRAT……………………………...…………………….lecithin:retinol acyltransferase

MRM………………………………………………………multiple reaction monitoring m/z………………………………………………………………....mass to charge ratio

NEAA……………………………………………………...... non-essential amino acids

NFF…………………………………………………………...…….non-fermented flour

NHANES………………………..…national health and nutrition examination survey

NPC1L1………………………………….Niemann-Pick disease type C1 gene-like 1

PBS…………………………………..…………..…………….phosphate buffer saline

PPAR……………………………………..peroxisome proliferator-activated receptor xv

RALDH………………………………………………………...... retinal dehydrogenase

RAR………………………………………………………….…….retinoic acid receptor

RDH…………………………………………………………...... retinol dehydrogenase

RXR……………………………………………………….………….retinoid X receptor

RBP…………………………………………………….…………retinol binding protein

S/N…………………………………………………………..………signal to noise ratio

SNP………………………………………………..….single nucleotide polymorphism

SR-B1………………………………………...……scavenger receptor class B type 1

STRA6…………………………………..…stimulated by retinoic acid gene 6 protein

TTR…………………………………..…….….thyroxine binding protein transthyretin

VAD………………………………..……………………………….vitamin A deficiency

WT………………………………...……………………………………….…….wild type

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Introduction

Vitamin A Deficiency (VAD) is a global health problem with an estimated

100 million people affected across the world [1, 2]. According to various reports,

VAD is the leading cause of preventable blindness in children. Vitamin A has extensively been shown to play an important role in vision both as a chromophore of the visual pigment rhodopsin and as a hormone necessary for the maintenance of the corneal epithelium [3]. Also, VAD contributes to the high prevalence of maternal and child (< 5 years of age) mortality in developing countries [4, 5]. This is because it decreases the body’s resistance and ability to fight diseases such as diarrhea, measles, and respiratory infections [4, 6]. In addition, VAD primarily results from insufficient intake of dietary preformed vitamin A or reduced bioavailability of provitamin A carotenoids such as β- carotene [5].

Vitamin A is a general term for structurally related compounds that have the functional activity of retinol [3]. De novo synthesis of these compounds with vitamin A activity occurs almost exclusively in plants and some microorganisms such as algae and fungi [7, 8]. Therefore, these essential micronutrients must be provided in the human diet. Dietary vitamin A can be obtained in two major forms: preformed vitamin A (retinyl esters and retinol) and provitamin A

carotenoids (β-carotene, α-carotene, and β-cryptoxanthin) [3, 7]. Preformed vitamin A is present in animal derived products such as liver, eggs and dairy products. In contrast, plant food sources (mostly fruits and vegetables) account for significant intake of dietary provitamin A carotenoids [7, 8]. Carrots, squash, and dark-leafy vegetables provide high amounts of β-carotene; oranges, papaya, and peppers are excellent sources of β-cryptoxanthin; and sweet potatoes and pumpkin are important sources of α-carotene [8, 9].

In the United States, data of the 2001-2002 national health and nutrition examination survey (NHANES) estimates that 70 – 75% of vitamin A is consumed as preformed vitamin A, primarily provided from animal products, meat and cold cereals [3]. However, in developing countries, demographic and health surveys (DHS) reveal that the diet of children (6 months and older) consists mainly of grains and staple crops, with some fruits and vegetables but animal products constitute a minor percentage of overall food intake [10-13]. In these countries, it has been reported that about 70% or more of vitamin A intake is in the form of provitamin A carotenoids [14]. However, widely consumed staple crops such as cassava, maize, potato and rice contain very low amounts of provitamin A carotenoids and do not supply individuals in developing countries with the daily requirements for vitamin A [1, 9].

There are three main strategies have been used in the prevention and treatment of VAD. These are direct supplementation of an individual with vitamin

A capsules, diet diversification and food fortification. However, figures from DHS

records show that the percentage of vulnerable children and women who receive supplementation in the six months prior to their survey is below 60% in Nigeria,

Bangladesh, Uganda, and India [10-13]. Also, these strategies are not particularly successful in rural areas because of inadequate economic resources and infrastructure [15]. Recently, a new approach has focused on increasing the concentrations of β-carotene and other micronutrients in staple crops of developing countries. This approach is called biofortification [16]. Staple crops that have been successfully biofortified with β-carotene under the HarvestPlus program are cassava, maize (corn) and sweet potato [17].

β-Carotene can undergo oxidative cleavage at bonds other than the central 15, 15′ bond to yield retinoid-like compounds called β-apocarotenoids.

These cleavage products are naturally occurring in the diet and may be directly absorbed in the intestine, or formed as a result of enzymatic or non-enzymatic oxidation of the parent carotenoids [18]. The processes involved in the absorption of carotenoids involves the following steps: (1) disruption of food matrix to release carotenoids, (2) incorporation of carotenoids into mixed micelles, (3) uptake of carotenoids by intestinal cells, (4) packaging of absorbed carotenoids into chylomicrons, and (5) secretion of chylomicron-packaged carotenoids into the lymph [8, 19]. However, little is known about the bioavailability and intestinal absorption of these β-apocarotenoids. Further comprehension of mechanisms of intestinal absorption and metabolism of these

β-apocarotenoids is important in studying the functions of these metabolites, some of which have been shown to be potent antagonists of vitamin A [20].

Thus, my research was focused on determining the extent of formation of these β-apocarotenoids in conventionally bred biofortified cassava and understanding the mechanisms involved in the intestinal absorption and metabolism of β-apocarotenoids. Studies involving chemical, molecular and cellular approaches were conducted to address current gaps in literature with the following specific aims.

Specific Aims

Aim 1

To evaluate the β-carotene and β-apocarotenoid content of two lines of conventionally bred cassava varieties commonly consumed in Brazil.

a. To determine the β-carotene content of two conventionally-bred biofortified

cassava varieties (Clone 03-15 and IAC 265-97) compared to two non-

biofortified varieties (IAC 576-70 and Saracura).

b. To quantify amount of β-apocarotenoids (relative to β-carotene) present in

the four cassava varieties.

c. To investigate the effects of boiling and frying on the amounts of total β-

carotene and β-apocarotenoids in cassava varieties.

Aim 2

To elucidate the mechanisms of intestinal absorption of β-apocarotenoids using

Caco-2 cells in culture.

a. To determine the effects of concentration and incubation time on the

kinetics of cellular uptake of β-apo-8′-carotenal and β-apo-13-carotenone

in Caco-2 cells and compare those to β-carotene.

b. To investigate the metabolic conversion and identify metabolic products of

β-apo-8′-carotenal and β-apo-13-carotenone in Caco-2 human intestinal

cells following uptake.

Chapter 1. Review of Literature

1.1 Biofortification as a strategy to alleviate VAD

There are three strategies that have been successful in the prevention and

treatment of VAD in developing countries and these are fortification, dietary

diversification, and direct supplementation of individuals with vitamin A [1].

Fortification involves the direct addition of preformed vitamin A to foods

commonly consumed by high risk or deficient populations in an attempt to

increase its nutritional value and prevent a nutritional deficiency [21]. Dietary

diversification aims to change existing dietary practices of food production,

purchase, preparation, and consumption so as to increase the quantity and

variety of vitamin A rich foods in an individual’s diet [21, 22]. Direct

supplementation of individuals with vitamin A has been a widespread practice in

several countries to treat and prevent VAD by providing mega doses of vitamin A

in pills, capsules or syrups [21].

However, these strategies have not been as successful in rural areas

especially those areas with limited access to fortified foods or supplements due

to inadequate economic resources and infrastructure [1, 15]. In addition, β-

carotene and provitamin A carotenoids are major sources of vitamin A for most

individuals in these rural areas because of their plant based diets [1, 14].

Nonetheless, the low quantity of provitamin A carotenoids in commonly consumed staple crops such as cassava, maize, potato and rice does not supply these individuals with the daily requirements for vitamin A [1, 9]. Hence, there is the need for strategies that address the concerns mentioned above without 7

causing major changes to dietary practices in these communities and

biofortification is an example of such strategy. Biofortification is a rural area

focused approach that involves the enrichment of staple crops in a country or

region with essential vitamins and minerals such as β-carotene (as a precursor of vitamin A), zinc, and iron [15].

There are several advantages biofortification offers over the other different

approaches as discussed in Bouis et al [15]. First, a biofortification program

emphasizes working with the farmers in rural and VAD populations of developing

countries to develop crop varieties that are disease and drought resistant, rich in

β-carotene, and also high yielding. This accessibility of the program is especially important because a significant number of VAD individuals live in rural and poor

communities. This will eventually benefit the families and communities of these

farmers because fortified foods and vitamin A supplements are more accessible

in urban areas. Second, biofortification is sustainable. It is proposed that after

initial investments to ensure these crop varieties are developed, the farmers can

continue to grow them and their consumption by VAD populations will ensure

there is a demand. Furthermore, biofortification is very cost effective after the

huge investments to develop several crop varieties are made.

However, there are also limitations and challenges to the success of

biofortification in vitamin A deficient regions [15]. The primary challenge is

convincing farmers to adopt and grow these micronutrient-dense staple crops in

large quantities. This will involve assuring farmers that biofortified crops do not

compromise the yields and profit margins observed with present commercial

varieties [23]. In addition, biofortification can lead to visible changes (e.g. color change associated with increased levels of β-carotene) of staple crops and in other cases; the introduced traits are not obvious (e.g. higher concentrations of iron or zinc). Therefore, consumers need to be properly educated and be willing to accept any sensory changes associated with some of these biofortified crops in order to make a behavior change [23].

Furthermore, the benefits of biofortification at the public health level

depend on the quantity of biofortified foods consumed, baseline vitamin A stores

of vitamin A deficient individuals, and vitamin A requirements which are impacted

by daily losses and physiological processes such as growth, pregnancy and

lactation. Thus, the benefits of this intervention vary throughout the lifecycle and

this needs to be considered when determining target breeding levels [15, 23].

1.2 Methods of biofortification for provitamin A cassava

Presently, there are two common approaches to increasing the provitamin

A content of high yield cassava varieties and these are conventional breeding

and transgenic/genetic engineering [1, 24]. Biofortification accomplished through

conventional breeding involves crossing parental varieties with high provitamin A

content over multiple generations to produce plants with superior and preferred

traits [24]. There are continuous efforts being made by various collaborating

9 agricultural agencies in South America and Africa, which are focused on breeding new cassava varieties with high concentrations of β-carotene [25].

Research at IITA, Nigeria has shown crossbred cassava roots with all-trans-β- carotene concentrations up to 27.9 µg/g dry weight (DW) and 40.9 µg/g DW total

β-carotene [26]. Similar studies at EMBRAPA, Brazil and CIAT, Colombia have revealed cassava varieties with 30 µg/g DW total β-carotene [27] and 13.50 µg/g

DW β-carotene respectively [28].

The transgenic/genetic engineering approach allows for the direct introduction of new genetic information into the genome of the plant that will enable de-novo synthesis of provitamin A carotenoids such as β-carotene [24,

29]. The best known example of this approach is the development of Golden

Rice, although this same approach has now been used in the development of

BioCassava Plus [30]. The BioCassava Plus program seeks to increase the β- carotene content in cassava by directing flux into the carotenoid biosynthesis pathway through two different methods.

In the first method, de novo synthesis of provitamin A carotenoids occurs through the expression of bacterial phytoene synthase gene crtB which is regulated by the root specific promoter for the potato patatin gene. The second method utilizes the co-expression of two transgenes, the crtB gene and

Arabidopsis 1-deoxy-xylulose-5-phosphate synthase (DXS) gene placed separately under the potato patatin gene promoter [30]. The available data from published literature show that transgenic cassava lines engineered to co-express

crtB and DXS yielded up to 26.65 µg/g DW all-trans-β-carotene and 32.10 µg/g

DW total carotenoids [1]. A search of existing literature databases yielded no research articles on β-carotene concentrations in transgenic cassava lines engineered to express the crtB gene alone. However, a recent review by Sayre et al [30] based on unpublished data, reports that the highest carotenoid producing cassava lines had 25 µg/g DW total carotenoid.

1.3 Retention, bioaccessibility and bioavailability of β-carotene

in provitamin A cassava

In addition to the different biofortification approaches being able to

increase the concentrations of β-carotene in cassava roots, there are also

several factors to consider before biofortification can reach its goal of providing

physiologically relevant amounts of the provitamin in an individual’s diet [31, 32].

Therefore, it is important to examine the retention, bioaccessibility and

bioavailability of β-carotene in biofortified cassava cultivars after various methods

of processing and local preparations. Bioaccessibility of carotenoids is defined as

the amount of carotenoids transported from the digested food matrix to mixed

micelles [33]. In addition, bioavailability is defined as the quantity of carotenoids

absorbed and incorporated into chylomicrons and secreted into lymph/blood [34].

There is profound interest in the bioaccessibility and bioavailability of carotenoids

from different food matrices.

Thakkar et al [26] studied the effects of three different local Nigerian

preparations on three crossbred high β-carotene cassava varieties. The methods

are boiling, roasting after fermentation (gari), and boiling after fermentation (fufu).

It was observed that 92% of β-carotene content was retained after fermentation but further roasting to prepare gari resulted in significantly decreased content of all-trans-β-carotene with a corresponding increase in its 13-cis isomer. The loss of β-carotene also increased with increasing temperature and cooking time.

However, there were no significant losses in the other two preparation methods.

Also, bioaccessibility of all-trans and cis isomers in fufu was significantly lower as compared to gari and boiled cassava [26].

Many populations of the world also utilize cassava in various other different food dishes and recipes [31]. Therefore, Failla et al [1] investigated the effects of the three popular preparation methods mentioned above as well other methods such as fermented flour (FF) and non-fermented flour (NFF) on β- carotene content of transgenic cassava roots compared to wild type (WT) based on DW. Similarly to what was reported by Thakkar et al [26], gari preparation resulted in the greatest loss of β-carotene but FF and NFF decreased β-carotene content by 30% and 20% respectively.

It was also observed that there was significantly decreased retention of β- carotene in WT as compared to the transgenic lines. The quantity of total β- carotene in micelle fractions of boiled cassava, fufu, and gari preparations of transgenic cultivars digested were significantly higher than in WT. In addition, the

27 – 30% efficiency of micellarization (EM) of β-carotene in processed WT

cassava was significantly lower than the 30 – 45% EM obtained for transgenic

roots. However, contrary to a previous report that used traditionally bred

biofortified cassava for their preparations [26], the % EM of fufu prepared from

transgenic cassava was significantly higher than boiled cassava and gari.

Furthermore, fully differentiated Caco-2 human intestinal cells were used to study availability of β-carotene in the mixed micelles from digested boiled cassava, fufu and gari for uptake across the intestinal brush border membrane

[1]. The quantity of β-carotene accumulated in cells from these digested food

prepared from transgenic roots ranged from about 18 – 90 ng/mg protein

compared to that obtained from WT which varied from about 0.3 – 1.2 ng/mg

protein. More recently, Berni et al [31] investigated the effects of genotype and

local Brazilian cooking methods such as boiling, frying after boiling, and frying on

both the retention and bioaccessibility of β-carotene in sweet cassava varieties

based on fresh weight (FW).

It was observed that the genotype and cooking method had a significant

interaction on both retention and efficiency of micellarization (% EM) of β- carotene isomers. Boiling of cassava resulted in significantly decreased total β- carotene per 1 g FW in all offspring cultivars. The % EM of all-trans and cis-β-

carotene from boiled cassava varied from 9 – 40% and 8 – 25% respectively

whereas % EM of all-trans-β-carotene from fried cassava roots was significantly increased for most of the cultivars and ranged from 19 – 40% for the cis-β-

carotene. The estimated bioaccessible total β-carotene per 100 g FW of boiled cassava varied from 30 – 258 µg while that of fried cassava varied from 73 – 352

µg.

Also, the amount of all-trans and total β-carotene in micellar fractions of boiled and fried cassava was highly correlated with the β-carotene content of processed cassava. The Caco-2 human intestinal cells as described by Failla et al [1] used to examine bioavailability showed that the amount of accumulated all- trans-β-carotene from fried cassava (86 – 158 ng/mg cell protein) was significantly higher than that accumulated from boiled cassava (48 – 95 ng/mg cell protein) for the cultivars investigated.

1.4 Metabolism of β-carotene

1.4.1 Digestion

Fruits and vegetables are an excellent source of β-carotene and other carotenoids such as α-carotene, β-cryptoxanthin and lutein. Carrots, kale, pumpkin, cantaloupe and orange-fleshed honeydew have very high amounts of

β-carotene and other provitamin A carotenoids [2, 7]. β-Carotene is an excellent

precursor of vitamin A and exhibits this provitamin A activity because it has two

unsubstituted β-ionone rings, the correct number, and position of methyl groups

in its long hydrocarbon chain [7]. β-Carotene is a lipophilic micronutrient and as such, its digestion is presumed to follow the digestion pattern of lipids in the 14 gastrointestinal tract [35]. After ingestion of β-carotene rich foods, β-carotene and other carotenoids are released from the food matrix [18].

Available data suggest that only the free form of this micronutrient is absorbed. Thus, any possible esterified forms of carotenoids containing hydroxyl groups such as β-cryptoxanthin are hydrolyzed by pancreatic enzymes

(cholesterol ester hydrolase, pancreatic triglyceride lipase and pancreatic lipase- related protein 2) that cleave retinyl esters during digestion of preformed vitamin

A [35]. Subsequently, β-carotene is incorporated into mixed micelles along with other lipids and are transported to the brush border membrane of enterocytes for uptake. There are several factors that can affect the incorporation of β-carotene into mixed micelles as detailed in the review by Reboul et al [35].

1.4.2 Intestinal absorption

The uptake of β-carotene at the apical membrane (Figure 1.1) occurs through facilitated transport involving scavenger receptor class B type 1 (SR-B1), cluster determinant 36 (CD36) and Niemann-Pick disease type C1 gene-like 1

(NPC1L1) although some β-carotene can be absorbed through passive diffusion at supraphysiological doses [7, 35, 36]. After uptake by the enterocyte, β- carotene is either converted to retinol by β, β-carotene 15,15′ oxygenase 1

(BCO1) and retinal reductase or absorbed intact. The retinol or intact β-carotene are then incorporated into chylomicrons and secreted into the lymph [7]. As with digestion of β-carotene, there are also factors that could affect the intestinal absorption of β-carotene and other carotenoids as discussed in Story et al [37]. 15

These factors include food matrix, food processing methods, consumption of dietary lipids, other carotenoids, concentration of β-carotene, and single nucleotide polymorphisms (SNPs).

LUMEN ENTEROCYTE LYMPH/BLOOD

Chylomicrons

Retinyl Esters apoB β-carotene

BCO1

β-CAROTENE RETINAL SRB1 β-CAROTENE β-CAROTENE

Figure 1.1. Overview of intestinal absorption of β-carotene

The absorption of β-carotene by intestinal cells is a facilitated process mediated by scavenger receptor class B type 1 (SR-B1). Following uptake, β-carotene is converted to retinal and subsequently to retinol by β,β-carotene 15,15′- oxygenase (BCO1) and retinal reductase. Retinol is then esterified and incorporated into chylomicrons. Also, β-carotene can be absorbed intact and incorporated into chylomicrons. These chylomicrons containing retinyl esters and intact β-carotene are then secreted into the lymph [7].

1.4.3 Cellular Uptake

The newly absorbed chylomicrons then undergo further degradation and

the remnants are absorbed by the liver where retinyl esters are stored until its

metabolites are required by other tissues. The retinyl esters are hydrolyzed into

retinol which could be utilized by hepatocytes or re-esterified by lecithin:retinol

acyltransferase (LRAT) and stored until further required [18]. In order for vitamin

A to be delivered to peripheral tissues, stored retinyl esters are hydrolyzed to

retinol, packaged with retinol binding protein (RBP) to form a retinol-RBP complex (holo RBP) and is secreted from the liver. In the blood, holo RBP interacts with thyroxine binding protein transthyretin (TTR) and is then delivered to target tissues [18]. The holo RBP-TTR complex is transported into cells via

receptor mediated uptake. The stimulated by retinoic acid gene 6 protein

(STRA6) was identified as the receptor for this function [38]. Retinol is then

converted to the metabolically active all-trans-retinoic acid (atRA) by retinol

dehydrogenases (RDHs) and retinal dehydrogenases (RALDHs) [18]. Some β-

carotene also accumulates in the liver and can be converted to retinal via

cleavage by BCO1. While much is known about the intestinal absorption and

cellular uptake of intact β-carotene, there are no studies on the absorption and

uptake of its eccentric cleavage products (β-apocarotenoids).

1.5 Cleavage products of β-carotene: How are they formed?

Carotenoids are highly unsaturated compounds which are prone to

degradation by oxidation and losses of carotenoids during food processing and

storage are well documented [25, 39]. However, there have been limited studies

to elucidate the pathways and fundamental mechanisms of carotenoid oxidation

[39]. There are two types of oxidative cleavage of unsaturated bonds in

carotenoids that can lead to the formation of β-apocarotenoids. These are

chemical reactions in carotenoid containing foods and enzymatic cleavage of full

length carotenoids [18, 40].

1.5.1 Chemical reactions of carotenoids yielding β-apocarotenoids

β-Apocarotenoids can be produced in foods by heat, light and oxidants

[41]. Rodriguez and collaborators [39] used two chemical reaction methods to

oxidize β-carotene to yield β-apocarotenals and they include oxidative cleavage

with excess potassium permanganate (KMnO4) and autoxidation (in presence

and absence of light) in model systems and processed foods. The β-

apocarotenoids observed from reaction with excess KMnO4 in the presence of

water after 12 hours were β-apo-15-carotenal, β-apo-14′-carotenal, β-apo-12′- carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal.

Autoxidation of β-carotene in low moisture (in presence and absence of light) and aqueous (in presence of light) model systems also yielded similar products although no β-apo-8′-carotenal was detected in the low moisture

system. β-Apo-15-carotenal and β-apo-12′-carotenal were also detected in processed mango juice but they were not identified in the other samples investigated. Recently, Gurak et al [41] used similar methods to investigate oxidation products from oxidation of β-carotene and detected similar products as reported by Rodriguez and colleagues [39] as well as cis-β-apo-8′-carotenal and apo-8′-β-carotenone.

In addition, there has been research [42] to investigate the oxidation products of β-carotene (dissolved in toluene) formed at 110°C for 30, 60, and 90 minutes. It was observed that optimal separation of products occurred at 30 minutes and the β-apocarotenals detected are 13-β-apo-carotenone, β-apo-15- carotenal, β-apo-12′-carotenal, 5, 6-epoxy-12′-β-apo-12′-carotenal, and 5, 6- epoxy-8′-β-apo-8′-carotenal. Studies to elucidate the detailed mechanisms of pathways leading to these oxidation products are still required because it will help in assessing the relevance of these compounds in physiological processes

[39, 41].

1.5.2 Enzymatic cleavage of carotenoids

Two important enzymes, BCO1 and β-carotene 9′, 10′ oxygenase (BCO2),

catalyze the oxidative cleavage of carotenoids to produce biologically active

metabolites (Figure 1.2) such as atRA [40]. BCO1, localized in the cytosol,

cleaves β-carotene to give two molecules of retinal, through the much discussed

central pathway, and this could be further converted to atRA by RALDH or

reduced to retinol by retinol dehydrogenase (RDH). On the other hand, BCO2, 20

localized in the mitochondria, cleaves β-carotene to yield β-apo-10′-carotenal through the eccentric pathway [34, 40, 43]. This β-apocarotenoid can also be converted to retinal by BCO1 [7, 44]. Several provitamin A carotenoids such as

α-carotene and β-cryptoxanthin are also substrates for BCO1 while xanthophylls such as lutein and zeaxanthin are also substrates for BCO2 [18]. Peroxidases and lipoxygenases have also been suggested as enzymes that could also catalyze the formation of β-apocarotenoids other than β-apo-10′-carotenal [43].

Figure 1.2. Enzymatic cleavage of carotenoids

The main product of the eccentric cleavage of β-carotene is β-apo-10′-carotenal and this reaction is catalyzed by β-carotene 9′, 10′-oxygenase 2 (BCO2). In addition, β-carotene can undergo chemical and enzymatic oxidations in vivo and in foods to yield other long-chain and short chain β-apocarotenoids. The exact mechanism and enzymes responsible for these other products are largely unknown [7].

1.6 Occurrence of β-apocarotenoids in foods and in vivo

At present, there is no published evidence of the occurrence of β-

apocarotenoids in conventionally bred and transgenic biofortified cassava

varieties. However, several of these apocarotenoids have been identified in

cantaloupe melons and orange-fleshed honeydew and have been reported [2].

The quantities of β-apo-13-carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal,

β-apo-10′-carotenal, and β-apo-8′-carotenal were detected using LC/MS and were found at concentrations totaling approximately 1.5% of β-carotene in both melons [2, 18]. Also, unpublished data from our laboratory show that these β-

apocarotenoids have been quantified in pumpkin, sweet potato and yellow

squash and the levels were 2.48%, 0.35% and 2.96% of β-carotene respectively.

Therefore, these β-apocarotenoids are present in low but significant quantities in β-carotene rich foods [43] and studies evaluating their presence and quantities in various fruits, vegetables and biofortified foods are required. There are studies that have reported that β-apocarotenoids are formed as products of eccentric cleavage of β-carotene in vivo. Shmarakov et al [45] investigated the absorption of β-carotene and its cleavage to various metabolites in serum and liver of BCO1 deficient (BCO1-/-) mice fed a β-carotene containing diet compared

to WT mice. Using LC/MS, concentrations of β-apo-14′-carotenal, β-apo-12′-

carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal were measured in serum

and liver of BCO1 deficient and WT mice. It was observed that β-apo-12′-

carotenal and β-apo-10′-carotenal were detected but only β-apo-12′-carotenal

concentrations in liver of BCO1-/- mice was significantly higher than those of WT.

Furthermore in a pilot study [46] to examine the plausibility of cleavage of

β-carotene to β-apocarotenoids in humans, one healthy human volunteer

consumed an oral dose of 14C labeled all-trans-β-carotene. The plasma, plasma fractions, urine and feces of the volunteer were screened for 14C labeled

metabolites. β-Apo-8′-carotenal was detected in plasma after 3 days of dosing. A

second 14C analyte was also found at this time point but could not be identified

because of the lack of standard compound. More recently, Eroglu et al [20]

detected biologically significant amounts of β-apo-13-carotenone in plasma of six free-living individuals using sensitive LC/MS techniques. The technique was assessed for specificity and sensitivity by using multiple reaction monitoring.

In summary, the studies discussed above demonstrate that β-

apocarotenoids can be formed from eccentric cleavage of β-carotene by BCO2

and other unknown mechanisms. It also suggests the possibility of the absorption of β-apocarotenoids directly from the diet [43].

1.7 Biological activity of β-apocarotenoids

The detection and quantification of few β-apocarotenoids in human plasma might corroborate the hypothesis that these biomolecules have some physiological functions and modulate some of the global functions of vitamin A 24

through gene regulation of transcription factors similarly to other β-carotene

cleavage products such as atRA and 9-cis-RA [40, 47]. It has been established

that atRA is a ligand for a family of transcription factors called retinoic acid

receptors (RARs) which activate genes having retinoic acid response elements

(RAREs) as part of their promoter sequence [20, 47].

RAR must form a heterodimer with another nuclear receptor and this

dimer is co-activated in a stepwise process in order to elicit any transcriptional

responses as described in details in the mini review by Ziouzenkova and Plutzky

[48]. Also according to in vitro studies, 9-cis-RA is a ligand for retinoid X

receptors (RXRs) which serve as heterodimer partners for several nuclear

transcription factors including RARs [47, 48]. To this effect, a number of studies

have investigated the binding affinity and effects of β-apocarotenals, β-

apocarotenones and β-apocarotenoic acids on these nuclear receptors.

Marsh et al [49] tested the hypothesis that β-apo-14′-carotenoic acid, β-

apo-8′-carotenoic acid, β-apo-13-carotenone and short chain β-apocarotenoids

will activate RARs and promote transcription. Transactivation assay studies

revealed that the short and long chain β-apocarotenoids examined did not

significantly transactivate α and β isoforms of RAR as compared to atRA. Also, in

an earlier study, Tibaduiza et al [50] examined if β-apocarotenoic acids inhibition of tumor growth was mediated through RARs. It was observed that there was no competitive inhibition of tritium labeled atRA for the ligand binding domain (LBD) of RARs by the β-apocarotenoic acids.

However, more recent work from Eroglu et al [20] has confirmed that β-

apocarotenoids are antagonists of α, β and γ isoforms of RARs. In particular, β- apo-14′-carotenoic acid, β-apo-14′-carotenal and β-apo-13-carotenone significantly decreased activation of all three isoforms. Also, these three β- apocarotenoids effectively decreased atRA-induced upregulation of genes, RARβ and cytochrome P450-26A1. Ziouzenkova et al [47] also investigated the effects of β-apocarotenals on activation and transcriptional responses of peroxisome proliferator-activated receptors (PPARs) and RXR alpha (RXRα).

PPAR isoforms (α, β/δ and γ) play different roles that are crucial to the

regulation of lipid and glucose metabolic flux [48, 51]. Similarly to RARs, PPARs

must form a heterodimer usually with RXR in order to elicit any transcriptional

response [51]. There was a marked inhibition of adipogenesis and activation of

RXRα and PPAR isoforms in mouse fibroblast 3T3 cells. These cells were co-

treated with β-apo-14′-carotenal and strong agonists of these nuclear receptors.

This inhibition increased with increasing β-apo-14′-carotenal concentrations.

Also, β-apo-14′-carotenal inhibitory effects of PPAR transcriptional response of target genes were observed in cell culture experiments.

In addition, Eroglu et al [52] examined the effects of β-apocarotenoids on

RXRα activation and signaling. It was observed that β-apo-13-carotenone significantly inhibited the 9-cis-RA induced activation of RXRα and its transcriptional response. Molecular model studies and ligand binding studies also showed that β-apo-13-carotenone was able to directly bind to LBD of RXRα

which suggests that competitive inhibition might be the mechanism by which this

cleavage product antagonizes RXRα activation and gene response.

The biological functions of these β-apocarotenoids are still largely unknown but these studies show that they may play a role in modulating retinoid and nuclear receptors signaling. Thus, understanding mechanisms of absorption and bioavailability of these compounds relative to their quantities in foods [14] is

critical in exploring these functions and may have wider impacts for further

understanding the metabolic effects of β-carotene.

Chapter 2. Analysis of β-carotene and β- apocarotenoids in cassava biofortified with β-

carotene

2.1 Abstract

Biofortification is defined as the enrichment of staple crops with essential

micronutrients [15]. At present, it is one of the strategies used to alleviate VAD by

breeding staple crops with provitamin A carotenoids. Staple crops that have been

successfully biofortified with β-carotene under the HarvestPlus program are cassava, maize (corn) and sweet potato [17]. Recently, β-apocarotenals and β- apo-13-carotenone have been identified and quantified in cantaloupe melons and orange-fleshed honeydew [2]. In this study, we quantified the concentrations of β-

apocarotenoids in cassava biofortified with β-carotene. Using an optimized, previously described high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, we were able to resolve and identify β- apo-13-carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′- carotenal, and β-apo-8′-carotenal in hexane/acetone extracts of raw, boiled, and fried roots of the two biofortified cassava varieties investigated. All of the β- apocarotenoids of interest were quantified in all roots of cassava varieties biofortified with β-carotene.

Keywords: Biofortification; cassava varieties; HPLC-MS/MS

2.2 Introduction

Vitamin A deficiency (VAD) is a major public health concern in several

developing nations in Africa, Southeast Asia, and South America [1]. It is

estimated that VAD affects more than 100 million people, particularly in rural

areas, with many of those affected being pre-school children and pregnant

women [2]. VAD primarily results from insufficient intake of dietary preformed vitamin A or reduced bioavailability of provitamin A carotenoids e.g. β-carotene

[5]. In addition, β-carotene and provitamin A carotenoids are major sources of

vitamin A for most individuals in these rural areas because of their plant-based

diets but the low quantity of these carotenoids in staple crops such as cassava

does not supply these individuals with the daily requirements for vitamin A [1, 9].

Fortification, dietary diversification, and direct supplementation of

individuals with vitamin A have been successful in the prevention and treatment

of VAD in developing countries [1]. However, these approaches are not

successful in rural areas with limited access to fortified foods or supplements due

to inadequate economic resources and infrastructure [1, 15]. Hence, strategies

that address the concerns raised above without changes to dietary practices are

required and biofortification is an example. Biofortification is defined as the

enrichment of staple crops with essential vitamins and minerals such as β-

carotene (as a source of vitamin A), zinc, and iron through agricultural techniques

[15]. Presently, conventional breeding and genetic engineering are the

biofortification approaches used to increase β-carotene in high yield cassava varieties [24, 31].

β-apocarotenoids are cleavage products of β-carotene and are formed by chemical and enzymatic oxidations (Figure 2.1) [18]. Recently, β-apocarotenoids

have been identified in cantaloupe melons and orange-fleshed honeydew and

have been reported [2]. The quantities of β-apo-13-carotenone, β-apo-14′- carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal were detected using LC/MS and were found at concentrations approximately 1.5% of

β-carotene in both melons [2, 18]. Also, unpublished data from our laboratory show that these β-apocarotenoids have been quantified in pumpkin, sweet potato and yellow squash and the levels were 2.48%, 0.35% and 2.96% of β-carotene respectively. The structures of β-apocarotenoids that we have detected are presented in Figure 2.2.

However, nothing is known about the occurrence of β-apocarotenoids in raw roots of conventionally bred and transgenic biofortified cassava varieties and the effect of cooking preparations on their formation in consumed cassava-based dishes. The objective of the present study was to determine the β-carotene and

β-apocarotenoids content of raw roots of biofortified and non-biofortified cassava

31 varieties. Also, we compare the effect of two cooking methods (boiling and frying) on the levels of β-apocarotenoids in these cassava varieties.

Figure 2.1. Products of eccentric cleavage pathway of β-carotene

Figure 2.2. Chemical structures of some β-apocarotenoids

These are structures of several naturally occurring β-apocarotenals and β-apo-

13-carotenone. Using sensitive HPLC-MS/MS methods, we have identified and quantified these β-apocarotenoids in melons [2]. Also, unpublished data from our laboratory show that these β-apocarotenoids are present in other fruits and vegetables containing high β-carotene.

2.3 Materials and Methods

2.3.1 Chemicals and Supplies

Unless otherwise stated, all chemicals and supplies were purchased from

Sigma-Aldrich (St. Louis, MO, U.S.A) or Fisher Scientific (Pittsburgh, PA, U.S.A).

All standard compounds of β-apocarotenoids (except β-apo-8′-carotenal) were synthesized by Dr. Robert W. Curley Jr. at The Ohio State University (Columbus,

OH, U.S.A).

2.3.2 Processing of cassava varieties

Raw and processed (boiled and fried) roots of different cassava varieties

were a generous gift from Dr. Mark L. Failla of The Ohio State University,

Columbus, Ohio. The detailed procedure for the preparation of boiled and fried

cassava have been published recently [31]. Upon receipt, samples were wrapped in aluminum foil and stored at -80°C before extraction and analysis. The cassava varieties investigated in this project are listed in Table 2.1. For this preliminary

study, the determination of concentrations of β-carotene and β-apocarotenoids in all cassava varieties were from a single sample (i.e. no replicates).

Variety Type Processing method

Line 1

Saracura Non-biofortified Boiling, Frying

Clone 03-15 Biofortified Boiling, Frying

Line 2

IAC 576-70 Non-biofortified Boiling, Frying

IAC 265-97 Biofortified Boiling, Frying

Table 2.1. Varieties of cassava investigated in this project

Raw and processed (boiled and fried) roots of different cassava varieties were a generous gift from Dr. Mark L. Failla of The Ohio State University, Columbus,

Ohio. The detailed procedure for the preparation of boiled and fried cassava have been published recently [31].

2.3.3 Extraction and HPLC-MS/MS analysis of β-carotene and β-

apocarotenoids

Extraction of β-carotene and β-apocarotenoids

Raw, fried and boiled cassava samples were homogenized to a fine pulp using a mortar and pestle. An aliquot of 1 g of sample was weighed into 11 ml glass tube. 5 ml of methanol was added, tube was briefly vortexed and sample was probe sonicated. The sample was centrifuged at 2000 x g for 5 minutes at room temperature. The methanol extract was transferred into a 44 ml glass vial and 5 ml of hexane/acetone (1:1 v/v) was added to pellet. The tube was briefly vortexed, sample was probe sonicated and centrifuged at 2000 x g for 10 minutes. The hexane/acetone extraction was repeated until residue became white and the hexane/acetone supernatant was colorless (~ 2 additional times).

10 ml of saturated aqueous NaCl solution was added to the pooled extracts to

induce phase separation and the extract was shaken by gentle tilting. The

hexane extract was brought up to 25 ml with hexane. Aliquots were transferred

into glass vials, dried under nitrogen gas and analyzed immediately.

High Performance Liquid Chromatography

Cassava samples were analyzed by HPLC-MS/MS for levels of β-apo-13-

carotenone, retinal, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-

carotenal, β-apo-8′-carotenal, and β-carotene. Hexane extracts were solubilized

in 300 µL of methanol/methyl tert-butyl ether (1:1 v/v), nylon syringe filtered (0.22

µm pores) and injected into the HPLC system. Separation of carotenoids was

achieved using a reversed-phase C30 column (4.6 µm x 250 mm, 3 µm; YMC

America Inc., Allentown, PA). The following elution profile was used with 90/8/2 methanol/water/2% aqueous ammonium acetate (solvent A) and 78/20/2 methyl tert-butyl ether/methanol/2% aqueous ammonium acetate (solvent B): the initial

HPLC conditions were 100% solvent A and this was increased linearly through

100% solvent B over 20 minutes, held at 100% B for 2 minutes and re- equilibrated at initial conditions for 2.5 minutes. The flow rate used was 1.3 mL/minute at a constant column temperature of 45°C.

Tandem mass spectrometry

The HPLC was integrated with a QTRAP 5500 hybrid quadrupole/ion trap mass spectrometer (AB SCIEX, Foster City, CA) using an atmospheric pressure chemical ionization (APCI) in positive ion mode. Retinal, β-apo-13-carotenone, β-

apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, β-apo-8′-carotenal were ionized as their respective pseudo-molecular cations of 285.2, 259.2,

311.23, 351.26, 377.28, and 417.31 mass to charge ratio (m/z). Each parent ion was fragmented to daughter ions and monitored by multiple reaction monitoring

(MRM). Source parameters for this analysis are listed in Table 2.2. The resulting

chromatograms were integrated with Analyst 1.5.1 software (AB SCIEX, Foster

City, CA). The transitions that gave the highest signal to noise ratio (S/N) were

used for quantitation and these were 161.1, 175.1, 159.1, 161.1, 95.1, and 119.1

m/z for retinal, β-apo-13-carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal, β- apo-10′-carotenal, β-apo-8′-carotenal respectively.

ID Q1 Q3 Dwells D.P. Entrance Collision (msec) (volts) potential energy (volts) (electron volts) 13one 259.2 175.1 22 60 10 21 Retinal 285.2 161.1 22 60 10 15 14′AL 311.23 159.1 22 60 10 15 12′AL 351.26 161.1 22 80 10 27 10′AL 377.28 95.1 22 80 10 27 8′AL 417.31 119.1 22 80 10 37 βC 537.45 269.27 22 80 10 20 Source temperature for analysis was 400°C. Collision cell exit potential was 11 volts Ion spray voltage was 5500 volts Curtain gas was 30 psi

Table 2.2. Source parameters for analysis of cassava samples

Abbreviations are D.P., declustering potential; 13one, β-apo-13-carotenone;

14′AL, β-apo-14′-carotenal; 12′AL, β-apo-12′-carotenal; 10′AL, β-apo-10′- carotenal; 8′AL, β-apo-8′-carotenal; βC, β-carotene.

2.4 Results

2.4.1 Profile of β-apocarotenoids in cassava samples

In order to accurately identify β-apocarotenoids in samples, we prepared a

mixture of β-apocarotenoids standards and injected into the HPLC system. Using

a previously optimized LC-MS/MS method, we established a profile for the

separation of the different β-apocarotenoids. Figure 2.3 shows a representative

chromatogram of the profile of a mixture of pure β-apocarotenoids compounds.

β-Apo-14′-carotenal and β-apo-12′-carotenal had several peaks which is indicative of the presence of isomers of these compounds. Figure 2.4 and Figure

2.5 are representative chromatograms of the profile of β-apocarotenoids in raw roots of biofortified and non-biofortified cassava varieties respectively.

We successfully identified and quantified β-apo-13-carotenone, β-apo-14′- carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal in raw, boiled and fried roots of Clone 03-15 and IAC 265-97 cassava varieties

(Figure 2.4; Appendix A). In contrast, all of the β-apocarotenals of interest were below either the limit of detection (LOD) or the limit of quantification (LOQ) in roots of the Saracura variety (Figure 2.5; Appendix A). However, we were able to quantify the levels of β-apo-13-carotenone in the raw root of this cassava variety.

We also identified and quantified all β-apocarotenoids in the IAC 576-70 variety except for β-apo-8′-carotenal, β-apo-12′-carotenal in boiled root, and β-apo-14′- carotenal in boiled and fried roots of this variety (Appendix A).

Figure 2.3. Representative chromatogram of profile of β-apocarotenoids standards mixture

A mixture of pure β-apocarotenoids compounds was prepared and dried down under a stream of nitrogen. The dried residue was solubilized in methanol/methyl tert-butyl ether (1:1 v/v), injected and analyzed by HPLC-MS/MS.

Figure 2.4. Profile of β-apocarotenoids in raw roots of Clone 03-15 variety

Carotenoids were extracted from raw root of Clone 03-15 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

Figure 2.5. Profile of β-apocarotenoids in raw roots of Saracura variety

Carotenoids were extracted from raw root of Saracura cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

2.4.2 Determination of total content of β-carotene and β-

apocarotenoids in cassava

Figures 2.6 – 2.11 summarize the results of the analysis of β-carotene and

β-apocarotenoids of all non-biofortified and biofortified cassava varieties

investigated in this project. Figure 2.6 and Figure 2.7 describe the total β-

carotene and β-apocarotenoids content in raw, boiled and fried roots of

biofortified and non-biofortified cassava varieties. As anticipated, the total

concentrations of β-apocarotenoids in the cassava varieties biofortified with β- carotene (Clone 03-15 and IAC 265-97) were higher than non-biofortified varieties (Saracura and IAC 576-70) (Figure 2.6). Clone 03-15 cassava variety had the highest amount of total β-carotene and β-apocarotenoids.

More importantly, all of the β-apocarotenals of interest and β-apo-13- carotenone were present in the cassava varieties biofortified with β-carotene

(Clone 03-15 and IAC 265-97) (Figure 2.7). The total concentration of β- apocarotenoids (µg/kg FW) in raw roots of cassava varieties compared to their total concentration of β-carotene (mg/kg FW) for Saracura, Clone 03-15, IAC

576-70 and IAC 265-97 was 0.8%, 0.3%, 0.2%, and 0.5% respectively (Table

2.3). We also determined the relative total content of β-apocarotenoids in boiled and fried roots of each cassava variety and the data are also presented in Table

2.3.

Figure 2.6. Total β-carotene content of raw, boiled and fried roots of all cassava varieties

Carotenoids (including β-carotene) were extracted from a known weight (approx.

1 gram) of homogenized roots of all cassava varieties as described above. The extracts were analyzed by HPLC-MS/MS.

Figure 2.7. Total β-apocarotenoids content of raw, boiled and fried roots of

all cassava varieties

Carotenoids and β-apocarotenoids were extracted from a known weight (approx.

1 gram) of homogenized roots of all cassava varieties as described above. The extracts were analyzed by HPLC-MS/MS.

Sample name Total β- Total β-carotene % β-carotene apocarotenoids (mg/kg FW) that are β- (µg/kg FW) apocarotenoids Saracura Raw 14 2 0.8 Boiled 0 2 0.0 Fried 1 1 0.1 Clone 03-15 Raw 85 31 0.3 Boiled 113 47 0.2 Fried 88 16 0.6 IAC 576-70 Raw 18 10 0.2 Boiled 6 7 0.1 Fried 8 2 0.5 IAC 265-97 Raw 82 17 0.5 Boiled 49 15 0.3 Fried 27 4 0.6

Table 2.3. Relative total β-apocarotenoids in raw, boiled, and fried cassava

2.4.3 Effect of boiling and frying on β-apocarotenoids content

We compared the effect of boiling and frying on the concentration of total

β-apocarotenoids of all four cassava varieties (Table 2.3; Figure 2.7). Boiling and frying of cassava roots was associated with a decrease in the total β- apocarotenoids content in IAC 265-97, IAC 576-70, and Saracura. However, in

Clone 03-15 cassava variety, boiling and frying did not have any effect on the levels of total β-apocarotenoids. In fact, these two processing methods appeared to increase the amounts of total β-apocarotenoids in this variety. We then determined the impact of boiling and frying on the amounts of individual β- apocarotenoids in biofortified cassava varieties.

Figure 2.8 and Figure 2.9 present the quantity of individual β- apocarotenoids analyzed in raw, boiled and fried roots of cassava varieties biofortified with β-carotene (Clone 03-15 and IAC 265-97). Generally, boiling or frying did not have any effect on the levels of 4 of 5 β-apocarotenoids of interest in Clone 03-15 variety. However, boiling or frying was associated with a decrease in the concentration of β-apo-12′-carotenal in this variety. In contrast, boiling decreased the quantities of 4 of the 5 β-apocarotenoids of interest in IAC 265-97 variety. In addition, boiling appeared to increase the levels of β-apo-13- carotenone in this variety. Frying decreased the quantities of all β- apocarotenoids in IAC 265-97 variety.

Figure 2.8. Determination of β-apocarotenoids content of raw, boiled, and fried roots of Clone 03-15 cassava variety

Carotenoids and β-apocarotenoids were extracted from a known weight (approx.

1 gram) of homogenized cassava roots of Clone 03-15 variety as described above. The extracts were analyzed by HPLC-MS/MS.

Abbreviations are 13one, β-apo-13-carotenone; 14′AL, β-apo-14′-carotenal;

12′AL, β-apo-12′-carotenal; 10′AL, β-apo-10′-carotenal; 8′AL, β-apo-8′-carotenal.

Figure 2.9. Determination of β-apocarotenoids content of raw, boiled, and fried roots of IAC 265-97 cassava variety

Carotenoids and β-apocarotenoids were extracted from a known weight (approx.

1 gram) of homogenized cassava roots of IAC 265-97 variety as described above. The extracts were analyzed by HPLC-MS/MS.

Abbreviations are 13one, β-apo-13-carotenone; 14′AL, β-apo-14′-carotenal;

12′AL, β-apo-12′-carotenal; 10′AL, β-apo-10′-carotenal; 8′AL, β-apo-8′-carotenal.

Furthermore, we examined the effect of boiling and frying on the levels of

the different β-apocarotenoids in non-biofortified cassava. Figure 2.10 and Figure

2.11 show the content of individual β-apocarotenoids analyzed in raw, boiled and

fried roots of non-biofortified cassava varieties (Saracura and IAC 576-70). β-

Apo-13-carotenone and β-apo-10′-carotenal were the only β-apocarotenoids detected in raw, boiled and fried roots of IAC 576-70 cassava variety. Boiling or frying did not appear to have any effect on the levels of β-apo-13-carotenone but both processing methods were associated with a decrease in the concentration of β-apo-10′-carotenal. We detected only β-apo-13-carotenone in raw roots of the non-biofortified Saracura variety. As shown previously in Figure 2.6, this variety also had very low quantities of β-carotene. However, we did not detect any β- apocarotenoids in either boiled or fried roots of this variety.

Overall, biofortified cassava varieties had a higher relative content of β-

apocarotenoids as compared to non-biofortified varieties. There are inconsistent

results on the impact of boiling or frying on the β-apocarotenoids content in these

four cassava varieties. Again, it is important to remember that we only

determined levels of β-apocarotenoids in cassava varieties from a single sample

set.

Figure 2.10. Determination of β-apocarotenoids content of raw, boiled, and

fried roots of IAC 576-70 cassava variety

Carotenoids and β-apocarotenoids were extracted from a known weight (approx.

1 gram) of homogenized cassava roots of IAC 576-70 variety as described above. The extracts were analyzed by HPLC-MS/MS.

Abbreviations are 13one, β-apo-13-carotenone; 14′AL, β-apo-14′-carotenal;

12′AL, β-apo-12′-carotenal; 10′AL, β-apo-10′-carotenal; 8′AL, β-apo-8′-carotenal.

Figure 2.11. Determination of β-apocarotenoids content of raw, boiled, and

fried roots of Saracura cassava variety

Carotenoids and β-apocarotenoids were extracted from a known weight (approx.

1 gram) of homogenized cassava roots of Saracura variety as described above.

The extracts were analyzed by HPLC-MS/MS.

Abbreviations are 13one, β-apo-13-carotenone; 14′AL, β-apo-14′-carotenal;

12′AL, β-apo-12′-carotenal; 10′AL, β-apo-10′-carotenal; 8′AL, β-apo-8′-carotenal.

2.5 Discussion

According to our knowledge, this study is the first time that β-apo-13- carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and

β-apo-8′-carotenal have been identified and quantified in cassava varieties biofortified with β-carotene. Moreover, our laboratory has also previously detected these β-apocarotenoids in cantaloupe melons and orange-fleshed honeydew using improved LC-MS techniques [2]. This study found that the total

β-apocarotenoids content are about 1.5% of the amounts of β-carotene in these fruits. The concentrations of total β-carotene in these melons are similar to those of the two biofortified cassava varieties. Clone 03-15 cassava variety had the highest levels of total β-carotene and β-apocarotenoids (Figure 2.6 and Figure

2.7).

The main findings of the present study include the following. We found that the total levels of β-apocarotenoids relative to β-carotene in raw roots of

Saracura, Clone 03-15, IAC 576-70 and IAC 265-97 was 0.8%, 0.3%, 0.2%, and

0.5% respectively (Table 2.3). The total concentrations of β-apocarotenoids in the cassava varieties biofortified with β-carotene were higher than non-biofortified varieties (Figure 2.7). Boiling and frying of cassava roots was associated with a decrease in the total β-apocarotenoids content in IAC 265-97, IAC 576-70, and

Saracura. However, in Clone 03-15 cassava variety, boiling and frying did not have any effect on the levels of total β-apocarotenoids (Figure 2.7). These two

processing methods appeared to increase the amounts of total β-apocarotenoids in this variety.

Boiling and frying are two common household methods for the preparation of cassava dishes [31]. The inconsistency in the results of the impact of boiling and frying on the levels of total β-apocarotenoids can be rationalized in two ways.

First, we hypothesize that the decrease in the total amounts of β-apocarotenoids could be as a result of degradation from exposure to high temperatures such as those observed in these two processing methods. Previous studies have reported the degradation of β-carotene by heat during processing and this is often associated with a reduction in the β-carotene content of the final processed product [25, 26].

Therefore, this concept could possibly explain the decreased total content of β-apocarotenoids in boiled and fried roots of IAC 265-97, IAC 576-70, and

Saracura variety. Second, we hypothesize that the increased β-apocarotenoids levels in Clone 03-15 variety could be explained by the formation of these compounds during boiling or frying. Several studies have shown that processing methods involving heat can cause the oxidation of full length carotenoids to produce metabolites such as β-apocarotenoids [41]. A previous study reported formation of 13-β-apo-carotenone and 12′-apo-β-carotenal from the oxidation of

β-carotene at 110°C for 30, 60, and 90 minutes [42].

2.6 Conclusion

This preliminary study is the first attempt to quantify β-apocarotenoids content of biofortified foods. We have demonstrated the presence of all possible long-chain β-apocarotenoids in conventionally bred cassava biofortified with β- carotene. We observed inconsistencies in the impact of boiling and frying on the levels of β-apocarotenoids in all cassava varieties. These discrepancies in the effect of boiling and frying on the quantities of β-apocarotenoids are not fully understood and require further investigation. A major limitation of our study is that analysis of β-carotene and β-apocarotenoids in all cassava varieties were carried out on a single sample (i.e. no replicates).

2.7 Acknowledgements

We thank Dr. Steven J. Schwartz for their expert technical assistance and collaboration on this research project. We also thank Dr. Ken M. Riedl and Dr.

Jessica L. Cooperstone for assistance with HPLC-MS/MS method development and analysis of β-apocarotenoids in cassava samples. We are also grateful to Dr.

Carlo dela Sena for his assistance in HPLC training and method development.

Furthermore, we thank Dr. Mark L. Failla for the raw and processed roots of different cassava varieties used in this project.

Chapter 3. Kinetics of uptake and metabolism of β-

apo-8′-carotenal and β-apo-13-carotenone by Caco-2

human intestinal cells

3.1 Abstract

Recently, we detected low but significant quantities of β-apocarotenoids in

high β-carotene foods [2]. These cleavage products of β-carotene are formed by

chemical and enzymatic oxidations [18]. However, little is known about their

bioavailability and intestinal absorption. This research aims to determine kinetics

of absorption, transport and metabolism of these compounds using Caco-2

intestinal cells as a model. Pure β-apocarotenoids were delivered to fully

differentiated monolayers of Caco-2 cells using tween-40 micelles as previously

described [53]. Cell pellets were re-dissolved in phosphate buffered saline pH 7.4

(PBS) before extraction. The hexanes extracts were analyzed by HPLC. There

was rapid uptake of β-apo-8′-carotenal into cells. We detected two unidentified

metabolites (X and Y) of β-apo-8′-carotenal. The formation of compound X increased with time corresponding with a decrease in β-apo-8′-carotenal. This suggests that β-apo-8′-carotenal was a precursor for compound X. Also, cellular uptake of β-apo-13-carotenone was rapid and this compound was extensively degraded over time. Understanding the mechanisms of absorption and metabolism of β-apocarotenoids relative to their quantities in foods is critical in exploring the functions of these metabolites, some of which have been shown to be potent antagonists of vitamin A [20].

Keywords: cell uptake; β-apocarotenoids; carotenoids metabolism; kinetics

3.2 Introduction

Humans obtain carotenoids from the diet by consuming fruits and

vegetables such as carrots, kale and orange-fleshed honeydew [8, 54]. The

central cleavage of provitamin A carotenoids such as β-carotene yields retinal

(vitamin A) but studies have shown that there is eccentric cleavage of provitamin

A and non-provitamin A carotenoids to yield β-apocarotenoids (Figure 3.1) [18].

The exact mechanisms of formation and the physiological role(s) of these eccentric cleavage products of carotenoids are not fully understood [18].

However, these cleavage products are naturally occurring in the diet and may be directly absorbed in the intestine, or be formed as a result of enzymatic or non- enzymatic oxidative cleavage of the parent carotenoids [18]. Previous studies have demonstrated that these β-apocarotenoids may have unique biological activities that involve antagonizing vitamin A signaling [20]. However, little is known about the bioavailability and intestinal absorption of these β- apocarotenoids.

The absorption of carotenoids involves the following steps: (1) disruption of food matrix to release carotenoids, (2) incorporation of carotenoids into mixed micelles, (3) uptake of carotenoids by intestinal cells, (4) packaging of absorbed carotenoids into chylomicrons, and (5) secretion of chylomicron-packaged carotenoids into the lymph [8, 19]. The factors that affect the intestinal absorption and transport of carotenoids include food matrix, food processing methods, consumption of dietary lipids, other carotenoids, concentration of β-carotene, and 59 single nucleotide polymorphisms (SNPs) [37]. The uptake of β-carotene at the apical membrane (Figure 3.2) occurs through facilitated transport involving scavenger receptor class B type 1 (SRB1), cluster determinant 36 (CD36) and

Niemann-Pick disease type C1 gene-like 1 (NPC1L1) although some β-carotene can be absorbed through passive diffusion at supraphysiological doses [7, 35,

36]. After uptake, β-carotene is either converted to retinol by β, β-carotene 15,

15′ oxygenase 1 (BCO1) and retinal reductase or absorbed intact. The retinol or intact β-carotene are then incorporated into chylomicrons and secreted into the lymph [7]. The chylomicrons then undergo further degradation and the remnants are absorbed by the liver where retinyl esters are stored until its metabolites are required by other tissues. The retinyl esters are hydrolyzed into retinol which could be utilized by hepatocytes or re-esterified by lecithin:retinol acyltransferase

(LRAT) and stored until further required [18].

The study of absorption and metabolism of provitamin A carotenoids in humans is important in populations with major intake of provitamin A carotenoids

[19]. Rodents are not a very good animal model to study human carotenoids uptake because they have the ability to efficiently convert most of β-carotene in their intestine to vitamin A [8, 53]. In vitro cell culture systems represent a simple alternative model for studying mechanisms of intestinal absorption and metabolism of carotenoids at the molecular level [8, 53]. The human intestinal

Caco-2 cell line is a well-established in vitro model that has been used in most carotenoids absorption studies [8]. When cultures of Caco-2 cells are post-

confluent, they become differentiated and have morphological and functional

features comparable to normal enterocytes [55, 56].

In the present study, we have used the Caco-2 human intestinal cell line

as a model to investigate the kinetics of intestinal absorption and transport of

pure compounds of two naturally occurring β-apocarotenoids (β-apo-8′-carotenal and β-apo-13-carotenone). In addition, we demonstrate the rapid metabolism of each compound after cellular uptake.

Figure 3.1. Products of eccentric cleavage pathway of β-carotene

The main product of the eccentric cleavage of β-carotene is β-apo-10′-carotenal and this reaction is catalyzed by β-carotene 9′10′-oxygenase 2 (BCO2). In addition, β-carotene can undergo chemical and enzymatic oxidations in vivo and in foods to yield other long-chain and short chain β-apocarotenoids. The exact mechanism and enzymes responsible for these other products are largely unknown [7].

LUMEN ENTEROCYTE LYMPH/BLOOD

Chylomicrons

Retinyl Esters apoB β-carotene

BCO1

β-CAROTENE RETINAL SRB1 β-CAROTENE β-CAROTENE

Figure 3.2. Overview of intestinal absorption of β-carotene

incorporated into chylomicrons. Also, β-carotene can be absorbed intact and incorporated into chylomicrons. These chylomicrons containing retinyl esters and intact β-carotene are then secreted into the lymph [7].

3.3 Materials and Methods

3.3.1 Chemicals and Supplies

Unless otherwise stated, all chemicals and supplies were purchased from

Sigma-Aldrich (St. Louis, MO, U.S.A) or Fisher Scientific (Pittsburgh, PA, U.S.A).

Cell culture reagents were obtained from Gibco® Life Technologies Corp (Grand

Island, NY, U.S.A). β-Apo-13-carotenone was synthesized by Dr. Robert Curley

Jr. at The Ohio State University (Columbus, OH, U.S.A).

3.3.2 Cell culture conditions

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

(Rockville, MD). Cells were grown and maintained as described previously [1].

Briefly, 75 cm2 flasks were seeded with 0.4 × 106 cells and grown in Dulbecco’s

minimum essential media (DMEM) containing 25 mM glucose, 15% heat-

inactivated fetal bovine serum (FBS), 1% L-glutamine (200mM), 1% penicillin-

streptomycin, 1% non-essential amino acids (NEAA) and 1% fungizone. Cell lines were incubated at 37°C in an atmosphere of air/CO2 (95:5 v/v), and cells

were passaged every 7 days or when cells were at 75 – 80% confluency.

3.3.3 Preparation of Tween-40 micelles

We prepared tween-40 micelles of β-apocarotenoids according to a

previously described method [53] and delivered them in serum-free media to

cells (see below). Briefly, carotenoids were solubilized in hexane or ethanol to

prepare a stock solution of the pure compounds. The required volume of β-

carotene and β-apocarotenoids in appropriate solvents, 22.5 µL of 10% v/v

Tween 40 (in ethanol) and 400 µL of acetone were introduced into a 4 mL glass

vial. The resulting mixture was dried under stream of nitrogen gas at 30 – 40°C,

re-solubilized in 200 µL of acetone and dried again. Dried residues of

micellarized carotenoids were then solubilized in 1 mL serum-free media,

vortexed briefly and sonicated for 1 minute. The resulting mixture was then

dissolved in the appropriate volume of serum-free media to yield the desired test

concentrations.

3.3.4 Stability of β-apocarotenoids during incubation in cell culture

conditions

Previous studies have shown that carotenoids and xanthophylls are

susceptible to spontaneous chemical oxidation and degradation during

incubation in cell culture environment [57, 58]. Therefore, we investigated the

stability of the β-apocarotenoids in the same cell culture conditions as were used for the project. Stability of β-apocarotenoids was examined by incubation for 8 hours in six-well plates devoid of cells. The method for preparing tween micelles of β-apocarotenoids has been discussed above [53]. Dried residues of micellarized β-apocarotenoids were then solubilized in 1 mL serum-free media, vortexed briefly and sonicated for 1 minute. The resulting mixture was then dissolved in the appropriate volume of serum-free media to yield the desired test concentrations. 2 mL of serum-free media containing β-apo-8′-carotenal or β- 65

apo-13-carotenone was then added to wells without Caco-2 cells and incubated

at 37°C. Aliquots of media at 0, 1, 4, and 8 hours were collected and stored at -

80°C before extraction and HPLC analysis.

3.3.5 Uptake of β-apocarotenoids by Caco-2 cells

For experiments, six-well plates were seeded with 0.15 × 106 cells and

maintained in cell culture conditions described above until a confluent monolayer

was achieved. Once post-confluent, the amount of FBS used in media was

decreased to 7.5%. The media was regularly replaced after 2 days. Cell cultures

used for experiments were between passages 22 − 40 when monolayers were 10

− 14 days post-confluent (i.e. once well differentiated). At the beginning of each

experiment, cell monolayers were washed once with basal DMEM. 2 mL of

serum-free media containing β-apo-8′-carotenal or β-apo-13-carotenone was added to each well and incubated at 37°C.

After incubation, the integrity of the monolayer was examined by phase contrast microscopy and media containing test compound was collected into a 15 mL centrifuge tube. There were no observed changes in morphology of cell monolayers for treatment of β-apo-8′-carotenal or β-apo-13-carotenone. Cell monolayers were washed once with 2 mL of ice-cold PBS containing 2 g/L bovine serum albumin and twice with 2 mL of ice-cold PBS to remove extracellular carotenoids. 2mL of ice-cold PBS was added to wells, and cells were harvested into 15 mL centrifuge tubes using a plastic scraper. Tubes were then centrifuged at 1000 x g for 10 minutes at 4 °C. Supernatant was discarded, 66 and cell pellets were stored under nitrogen gas at -80 °C before extraction and

HPLC analysis of carotenoids [31].

3.3.6 Extraction of β-apocarotenoids

Extraction from media

Cell culture media samples before and after incubation were extracted according to the method of. 1 mL of media and 1 mL of acetone were placed in a

15 mL centrifuge tube and vortexed for 1 minute. 4 mL of hexanes was added and the mixture was vortexed again for 1 minute. The resulting mixture was then centrifuged at 3000 x g for 10 minutes at 4°C. The upper layer was transferred into an 11 mL screw cap glass vial and solvent was dried under a stream of nitrogen gas at 30 – 40°C. Extracts were stored at -80°C before HPLC analysis.

Extraction from cell monolayer

Cell pellets were re-suspended in 1 mL of PBS and probe sonicated for 20 seconds. 1 mL of acetone was added and the tube and vortexed for 1 minute. 4 mL of hexanes was added and the mixture was vortexed again for 1 minute. The resulting solution was then centrifuged at 3000 x g for 10 minutes at 4°C. The upper layer was transferred into an 11 mL screw cap glass vial and solvent was dried under a stream of nitrogen gas at 30 – 40°C. Extracts were stored at -80°C before analysis.

3.3.7 HPLC Analysis

Method 1: Analysis of β–carotene and β–apo-8′-carotenal

Carotenoids were analyzed using HPLC method according to Ferruzzi et al [59] and Seo et al [60] with various modifications. Hexane extracts were solubilized in 200 µL of methanol/methyl tert-butyl ether (1:1 v/v), syringe filtered

(0.22 µm pores; Millipore) and analyzed. Separation of carotenoids was achieved using an Agilent 1200 series HPLC system and a reverse-phase YMC C30 column (4.6 µm x 150 mm, 5 µm; Waters, Ireland) coupled to a UV/VIS detector.

The following elution profile was used with 98:2 v/v methanol–1M ammonium acetate pH 4.6 (solvent A) and 100% methyl tert – butyl ether (solvent B): gradient from 85% solvent A to 60% solvent A over 10 minutes, 60% solvent A for 10 minutes, gradient from 60% solvent A to 85% solvent A over 6 seconds, and 85% solvent A for 4 minutes. The flow rate used was 0.8 mL/minute and elution was monitored at 450 nm for β–carotene and monitored at 380 nm, 450 nm and 460 nm for β–apo-8′-carotenal. The total runtime for this method was 24 minutes. Carotenoids in samples were quantified by comparing peak area against a standard curve prepared with known concentrations of standard compounds [60].

Method 2: Analysis of β–apo-13-carotenone

Dried extracts were dissolved in 200 µL of methanol/LC-MS grade water

(1:1 v/v), syringe filtered (0.22 µm pores; Millipore) and analyzed. The HPLC

68 system and column used for this analysis were the same as those discussed earlier. The following elution profile was used with methanol (solvent A) and 0.22

µm filtered LC-MS grade water (solvent B): 85% solvent A was run isocratically for 15 minutes. Elution was monitored at 345 nm and the flow rate used was 0.8 mL/minute. Levels of β-apo-13-carotenone in media and cells were quantified by comparing peak area against a standard curve prepared with known concentrations of their respective standard compounds.

Statistical analysis of Data

All data are presented as mean and standard deviations. Means and standard deviations were performed using Microsoft Excel 2013 (Microsoft

Corporation, Redmond, WA). Graphs were produced using GraphPad statistical software version 4.0 (GraphPad Software Inc., La Jolla, CA).

3.4 Results

3.4.1 Stability of β-apo-8′-carotenal and β-apo-13-carotenone

In order to ensure that β-apocarotenoids did not undergo spontaneous chemical oxidation and degradation during incubation in cell culture environment, we investigated the stability of these compounds in the same conditions as will be used for the project. Serum-free media containing 1 µM or 5 µM of β-apo-8′- carotenal or β-apo-13-carotenone were incubated in a cell-free system for 1 – 8

hours. The percentage recovery of β-apocarotenoids in incubated media was calculated relative to their concentration of β-apocarotenoids in serum-free media at the start of the stability experiment (i.e. media at 0 hours).

Figure 3.3 and Table 3.1 show the results of the stability of β-apo-8′- carotenal during incubation in cell culture conditions. We observed that the recovery of β-apo-8′-carotenal from media decreased with increasing incubation time. The calculated % recovery of 1 or 5 µM of β-apo-8′-carotenal in basal

DMEM was greater than 85% after 8 hours of incubation. Figure 3.4 and Table

3.2 summarizes the results of the stability of β-apo-13-carotenone throughout the incubation period in cell culture conditions. Also, as seen with β-apo-8′-carotenal, we observed that there was loss of β-apo-13-carotenone in serum-free media following incubation. The calculated % recovery of 1 or 5 µM of β-apo-13- carotenone in serum-free media was more than 76% after 8 hours of incubation.

As shown in Table 3.1, the extent of loss in media after 8 hours of incubation for 1 or 5 µM of β-apo-8′-carotenal was minimal and similar for both initial concentrations. In contrast, we observed that the rate of loss in basal media after 8 hours of incubation for 1 µM of β-apo-13-carotenone was 10% more than that of 5 µM of β-apo-13-carotenone (Table 3.2). Based on the above observations, we chose 1 µM of β-apo-8′-carotenal and 5 µM of β-apo-13- carotenone as the initial concentrations for the time dependent uptake experiments.

Figure 3.3. Stability of β-apo-8′-carotenal in cell culture conditions

Serum-free media containing tween micelles of 1 or 5 µM of β-apo-8′-carotenal

were incubated at 37°C for 1, 4, and 8 hours. After incubation, the extracts from media were analyzed by HPLC.

Sample name Time of Concentration of β- % Recovery incubation apo-8′-carotenal in (hours) media (pmol/well) 1 µM of β-apo-8′- 0 1555 ± 28 carotenal 1 1522 ± 4 97.9 4 1421 ± 20 91.4 8 1339 ± 18 86.1

5 µM of β-apo-8′- 0 7800 ± 179 carotenal 1 7278 ± 133 93.3 4 7132 ± 106 91.4 8 6828 ± 135 87.5

Table 3.1. Stability of β-apo-8′-carotenal in cell culture environment

Serum-free media containing tween micelles of 1 or 5 µM of β-apo-8′-carotenal

were incubated at 37°C for 1, 4, and 8 hours. After incubation, the extracts from media were analyzed by HPLC. % Recovery was calculated relative to the concentration of β-apo-8′-carotenal in media at 0 hours.

Figure 3.4. Stability of β-apo-13-carotenone in cell culture conditions

Serum-free media containing tween micelles of 1 or 5 µM of β-apo-13-

carotenone were incubated at 37°C for 1, 4, and 8 hours. After incubation, the extracts from media were analyzed by HPLC.

Sample name Time of Concentration of β- % Recovery incubation apo-13-carotenone (hours) in media (pmol/well) 1 µM of β-apo-13- 0 672 ± 3 carotenone 1 613 ± 49 91.2 4 544 ± 8 80.9 8 514 ± 40 76.5

5 µM of β-apo-13- 0 3818 ± 164 carotenone 1 3587 ± 222 93.9 4 3507 ± 143 91.8 8 3312 ± 48 86.8

Table 3.2. Stability of β-apo-13-carotenone in cell culture environment

Serum-free media containing tween micelles of 1 or 5 µM of β-apo-13- carotenone were incubated at 37°C for 1, 4, and 8 hours. After incubation, the extracts from media were analyzed by HPLC. % Recovery was calculated relative to the concentration of β-apo-13-carotenone in media at 0 hours.

3.4.2 Kinetics of uptake of β-carotene: effects of incubation time and

concentration

We studied the uptake of β-carotene as function of incubation time and concentration. Fully differentiated monolayers of Caco-2 cells were incubated with 1 µM of β-carotene for 30 minutes – 8 hours. We observed that there was

accumulation of β-carotene in cells at 30 minutes and the cellular β-carotene

content increased with incubation time up to 8 hours (Figure 3.5; Figure 3.6). We

also investigated the effect of concentration on β-carotene uptake. When

monolayers of differentiated cells were incubated with β-carotene (1 – 5 µM) for 4

hours, the amount of β-carotene in cells increased with increasing initial

concentration of β-carotene (Figure 3.7).

In addition, we determined the % recovery of β-carotene after experiments

by adding the concentration of β-carotene in cells and media after incubation and

comparing this total value to the amount of β-carotene in the media at the start of

the experiment (Table 3.3). The recovery of β-carotene was greater than 60% in

both time and concentration dependent uptake experiments. Furthermore,

previous studies have shown BCO2 can convert β-carotene to β-apocarotenoids.

This could potentially create a result bias in our uptake studies of β-

apocarotenoids. However, we did not detect any β-apocarotenoids in cells

incubated with β-carotene as a function of incubation time or increasing

concentration.

carotene - β

UV/VIS spectrum of β-carotene

Figure 3.5. Representative chromatogram of time dependent uptake of β- carotene in Caco-2 cells

A fully differentiated monolayer of Caco-2 cells was incubated with serum-free media containing 1 µM of β-carotene at 37°C for 2 hours. After incubation, the extracts from media and cells were analyzed by HPLC.

Figure 3.6. Time course of uptake of β-carotene in Caco-2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free

media containing 1 µM of β-carotene at 37°C for 30 minutes, 1, 2, 4, and 8 hours.

After incubation, the extracts from media and cells were analyzed by HPLC.

Figure 3.7. Dose dependent uptake of β-carotene in Caco-2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 1, 2, 3, 4 or 5 µM of β-carotene at 37°C for 4 hours. After incubation, the extracts from media and cells were analyzed by HPLC.

β-carotene β-carotene β-carotene in % Recovery recovered in recovered media at 0 media in cells hours (pmol/well) (pmol/well) (pmol/well) Time dependent uptake Time of incubation (hours) 0.5 982 144 1327 84.9 1 692 185 ” 66.1 4 499 362 ” 64.9 8 300 538 ” 62.6

Concentration dependent uptake (Incubation time = 4 hours) Initial concentration (µM) 1 723 311 1401 73.9 2 1281 563 2176 84.8 3 1992 709 3265 82.7 4 3245 818 3876 104.8 5 3183 907 5396 75.8

Table 3.3. Recovery of β-carotene during cell culture experiment

3.4.3 Effects of concentration of β-apo-8′-carotenal and incubation

time on the uptake and metabolism of β-apo-8′-carotenal

In this study, we investigated the kinetics of uptake of β-apo-8′-carotenal as a function of incubation time and concentration. Figures 3.8 – 3.13 summarize the results of the experiments for the kinetics of time course and dose response of β-apo-8′-carotenal. In preliminary experiments, we observed that there was rapid uptake of β-apo-8′-carotenal into cells but we could only account for about

10% of the parent compound in media and cells after 8 hours of incubation (data

not shown). Thus, we decreased our maximum incubation time to 8 hours.

Figure 3.8 shows a representative chromatogram of the uptake of β-apo-

8′-carotenal in Caco-2 cells. When cells were incubated with β-apo-8′-carotenal for 1 hour, compound X (an unidentified metabolite) was the major peak detected in cells with another unknown metabolite (compound Y) and β-apo-8′-carotenal present in small amounts. Our HPLC conditions enabled us to resolve both metabolites from the parent compound. We also “quantified” cellular content of X and Y based on the assumption that these metabolites have the same extinction coefficient as β-apo-8′-carotenal. Hence, the “concentration” of X and Y in cells

are arbitrary.

Figure 3.9 shows the kinetics of time uptake of β-apo-8′-carotenal for an individual experiment. Monolayers of 10 – 14 days post-confluent Caco-2 cells were incubated with 1 µM of β-apo-8′-carotenal for 30 minutes – 8 hours. As

mentioned previously, we observed the rapid accumulation of cellular content of

β-apo-8′-carotenal and detected two unknown metabolites (X and Y). Based on

our arbitrary quantification, we observed that the formation of compound X in

cells increased as a function of incubation time and this corresponds with a

decrease in β-apo-8′-carotenal (Figure 3.9). Figure 3.10 and Figure 3.11 summarize the results of different experiments for the time dependent uptake of

β-apo-8′-carotenal in Caco-2 cells. The % recovery of β-apo-8′-carotenal during time course was high after 30 minutes of incubation (approximately 92%) but this decreased as a function of incubation time and was about 15% after 8 hours of incubation (Table 3.4).

In addition, we studied metabolic conversion of β-apo-8′-carotenal as a function of concentration. Figure 3.12 and Figure 3.13 describe the kinetics of dose response of β-apo-8′-carotenal in Caco-2 cells. When differentiated monolayers were incubated with β-apo-8′-carotenal (1 – 5 µM) for 4 hours, we observed that the amount of β-apo-8′-carotenal in cells increased with increasing initial concentration of β-apo-8′-carotenal (Figure 3.12). Furthermore, we observed that the formation of compound X in cells increased linearly (Figure

3.13). However, from our recovery calculations, we could only account for about an average of 35% of β-apo-8′-carotenal after 8 hours of incubation (Table 3.4).

carotenal - ′ 8 - apo - Compound X Compound β Compound Y Compound

UV/VIS spectrum UV/VIS of Compound X spectrum of Compound Y

UV/VIS spectrum of β-apo-8′- carotenal

Figure 3.8. Representative chromatogram of time dependent uptake of β- apo-8′-carotenal in Caco-2 cells

A fully differentiated monolayer of Caco-2 cells was incubated with serum-free media containing 1 µM of β-apo-8′-carotenal at 37°C for 1 hour. After incubation, the extracts from media and cells were analyzed by HPLC 82

Figure 3.9. Kinetics of uptake and conversion of β-apo-8′-carotenal in Caco-

2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free

media containing 1 µM of β-apo-8′-carotenal at 37°C for 30 minutes, 1, 4, and 8 hours. After incubation, the extracts from media and cells were analyzed by

HPLC. The results shown represent one experiment and the points are means ± standard deviations of 3 separate wells or more.

Figure 3.10. Time course of uptake of β-apo-8′-carotenal in Caco-2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free

media containing 1 µM of β-apo-8′-carotenal at 37°C for 30 minutes, 1, 2, 4, and

8 hours. After incubation, the extracts from media and cells were analyzed by

HPLC.

Figure 3.11. Time course of conversion of β-apo-8′-carotenal into an

unknown metabolite (compound X)

Differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 1 µM of β-apo-8′-carotenal at 37°C for 30 minutes, 1, 2, 4, and 8 hours. After incubation, the extracts from media and cells were analyzed by

HPLC.

Figure 3.12. Dose dependent uptake of β-apo-8′-carotenal in Caco-2 cells

Differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 1, 2, 3, 4, or 5 µM of β-apo-8′-carotenal at 37°C for 4 hours. After incubation, the extracts from media and cells were analyzed by HPLC.

Figure 3.13. Dose dependent conversion of β-apo-8′-carotenal into an unknown metabolite (compound X)

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 1, 2, 3, 4 or 5 µM of β-apo-8′-carotenal at 37°C for 4 hours.

After incubation, the extracts from media and cells were analyzed by HPLC.

β-apo-8′AL β-apo-8′AL β-apo-8′AL in % Recovery recovered in recovered media at 0 media in cells hours (pmol/well) (pmol/well) Time dependent uptake Time of incubation (hours) 0.5 1255 56 1431 91.6 1 1016 50 ” 74.5 4 457 17 ” 33.1 8 208 11 ” 15.3

Concentration dependent uptake (Incubation time = 4 hours) Initial concentration (µM) 1 413 11 1400 30.3 2 980 27 3035 33.2 3 1600 47 4554 36.2 4 2220 66 6710 34.1 5 3118 83 7620 42.0

Table 3.4. Recovery of β-apo-8′-carotenal during cell culture experiment

Abbreviation is 8′AL, β-apo-8′-carotenal.

3.4.4 Kinetics of uptake of β-apo-13-carotenone: effects of

concentration and incubation time

We studied the kinetics of time and concentration dependent uptake of β-

apo-13-carotenone. We also report a newly developed HPLC method to measure

the levels of β-apo-13-carotenone in media and cell samples. We identified the

peak as β-apo-13-carotenone by comparing the retention time and UV/VIS

spectrum to that of known concentration of the pure compound. Figure 3.14

shows a representative chromatogram of the uptake of β-apo-13-carotenone in

Caco-2 cells.

Figure 3.15 and Figure 3.16 summarize the kinetics of uptake of β-apo-13-

carotenone as a function of incubation time or concentration. Post-confluent cell

monolayers were incubated with 5 µM of β-apo-13-carotenone for 15 minutes – 8

hours. We observed rapid uptake of β-apo-13-carotenone in cells but subsequent

extensive degradation with increasing incubation time. In addition, differentiated

Caco-2 cells were incubated for 30 minutes or 4 hours with β-apo-13-carotenone

(1 – 5 µM). We observed that cellular accumulation of β-apo-13-carotenone

increased with increasing concentration.

Furthermore, we determined the recovery of β-apo-13-carotenone. Initial experiments had shown that β-apo-13-carotenone recovery in cells after 4 hours of incubation was less than 5% (data not shown). Therefore, we reduced our

89 maximum incubation time to 1 hour. The % recovery of β-apo-13-carotenone ranged from 49 – 76% for all experiments within 1 hour (Table 3.5).

carotenone - 13 - apo - β

UV/VIS spectrum of β-apo-13-carotenone

Figure 3.14. Representative chromatogram of uptake of β-apo-13- carotenone in Caco-2 cells

Differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 5 µM of β-apo-13-carotenone at 37°C for 30 minutes. After incubation, the extracts from media and cells were analyzed by HPLC.

Figure 3.15. Time course of uptake of β-apo-13-carotenone in Caco-2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 5 µM of β-apo-13-carotenone at 37°C for 15, 30, 45 minutes, 1,

4 and 8 hours. After incubation, the extracts from media and cells were analyzed by HPLC. The inset shows experiment 2 on an expanded time scale.

Figure 3.16. Dose dependent uptake of β-apo-13-carotenone in Caco-2 cells

Fully differentiated monolayers of Caco-2 cells were incubated with serum-free media containing 1, 2, 3, 4, or 5 µM of β-apo-13-carotenone at 37°C for 4 hours

(Experiment 1) and 30 minutes (Experiment 2 and 3). After incubation, the extracts from media and cells were analyzed by HPLC.

β-apo-13 in β-apo-13 in β-apo-13 in % Recovery incubated cells media at 0 media (pmol/well) hours (pmol/well) (pmol/well) Time dependent uptake Time of incubation (minutes) 15 1872 556 3910 62.1 30 1588 727 ” 59.2 45 1197 879 ” 53.1 60 1015 925 ” 49.6

Concentration dependent uptake (Incubation time = 30 minutes) Initial concentration (µM) 1 479 80 735 76.0 2 750 203 1724 55.3 3 1024 400 2610 54.6 4 1580 592 3660 59.4 5 1925 875 4304 65.0

Table 3.5. Recovery of β-apo-13-carotenone during cell culture experiment

3.5 Discussion

The Caco-2 human intestinal cell line is a well-established in vitro model

used in most studies of absorption and metabolism of carotenoids at the

molecular level [8, 53]. To our knowledge, this present preliminary study is the

first to investigate the kinetics of uptake and metabolism of pure compounds of β-

apo-8′-carotenal and β-apo-13-carotenone using Caco-2 cells as a model. We

chose β-apo-8′-carotenal because this is the longest β-apocarotenoid that could

result from the oxidative cleavage of β-carotene and we selected β-apo-13-

carotenone because previous studies had shown that it is a potent antagonist of

vitamin A signaling [20].

The key outcomes of the present research study include the following. We

studied the uptake of β-carotene in order to show that we could replicate

previous kinetics data. We observed that there was accumulation of β-carotene

in cells at 30 minutes and the cellular β-carotene content increased with

incubation time up to 8 hours (Figure 3.5; Figure 3.6). We also investigated the

effect of concentration on β-carotene uptake and found that the amount of β-

carotene in cells increased with increasing initial concentration of β-carotene.

These results are similar to those observed by During et al [53].

Also, when cells were incubated with β-apo-8′-carotenal, we observed the

rapid uptake of cellular content of β-apo-8′-carotenal but subsequent disappearance with increasing incubation time. This corresponded with the

formation of two unknown metabolites (X and Y). We refined a previous HPLC

method and this enabled us to separate both metabolites from the parent

compound. Compound X was the major peak detected in cells with the other

unknown metabolite (compound Y) and β-apo-8′-carotenal present in small amounts (Figure 3.8). In addition, we made arbitrary quantifications of these metabolites by making the assumption that these metabolites have the same extinction coefficient as β-apo-8′-carotenal. Based on our arbitrary quantifications, we observed that the formation of compound X in cells increased as a function of incubation time (Figure 3.9). Collectively, these results suggest that β-apo-8′-carotenal was a precursor for compound X.

In addition, we studied metabolic conversion of β-apo-8′-carotenal as a function of concentration (Figure 3.12; Figure 3.13). When monolayers of differentiated Caco-2 cells were incubated with β-apo-8′-carotenal (1 – 5 µM) for

4 hours, we observed that the amount of β-apo-8′-carotenal in cells increased with increasing initial concentration of β-apo-8′-carotenal (Figure 3.12). We also found that the formation of compound X in cells increased in a roughly linear manner (Figure 3.13). These results are in contrast to uptake of β-carotene which observed that the accumulation and secretion of β-carotene are saturable processes [53].

Ongoing work is focused on identification of the metabolites of β-apo-8′- carotenal observed in this study. We have attempted to speculate the structures of X and Y based on HPLC retention times and UV/VIS spectrum. Compound X

has a retention time of 7.3 minutes while compound Y elutes at 5.1 minutes. This

indicates both compounds are more hydrophilic than β-apo-8′-carotenal

(retention time = 8.8 minutes).

Also, there was a clear hypsochromic shift in the UV/VIS absorption spectra of both metabolites (β-apo-8′-carotenal λmax = 460 nm). Compound X has

a λmax of 450 nm, which is 10 nm lower than β-apo-8′-carotenal. Previous in vitro studies have shown that long chain carotenoids (e.g. β-carotene and lycopene) can undergo chemical oxidations to yield numerous mono- and di-epoxide derivatives [39, 41, 61]. The 5,6-epoxy-β-carotene and 5,6:5′,6′-diepoxy-β- carotene have λmax about 6 – 7 nm and 12 – 14 nm lower, respectively, than that

of β-carotene [39, 41]. Thus, we suggest that compound X could be an epoxide

derivative of β-apo-8′-carotenal at either the 5,6 or 5,6:5′,6′ positions.

On the other hand, compound Y absorbs maximally at 380 nm similar to

retinal. However, the UV/VIS spectrum of compound Y, which has 2 peaks, is

distinct from retinal. In addition, we analyzed several long-chain β-

apocarotenoids in the same HPLC conditions used to resolve β-apo-8′-carotenal

and its metabolites (data not shown). The standard compounds examined were

β-apo-13-carotenone, retinal, β-apo-14′-carotenal, β-apo-12′-carotenal, and β-

apo-10′-carotenal. We found that none of these β-apocarotenoids eluted at the

same retention time as X and Y.

Furthermore, we investigated the effects of incubation time and

concentration on the kinetics of uptake of β-apo-13-carotenone. When post- 97

confluent monolayers were incubated with β-apo-13-carotenone, we found that there was rapid uptake of β-apo-13-carotenone in cells but subsequent disappearance with increasing incubation time. We have attempted to explain these results in two ways. First we theorize that there might be metabolism of β-

apo-13-carotenone in the cells. However, we are unable to this identify any

metabolic products at this time because there is limitation in our detection

technology. There is the need to couple our uptake experiments to more

sensitive detection methods such as HPLC-MS/MS. Second, we hypothesize that

the extensive degradation following cell uptake is due to the detoxification of β-

apo-13-carotenone by phase 1 and 2 enzymes. Further studies are required to

verify this assumption.

In addition, monolayers of differentiated Caco-2 cells were incubated with

β-apo-13-carotenone (1 – 5 µM) for 1 hour. We observed that cellular

accumulation of β-apo-13-carotenone increased with increasing concentration.

These results are also in contrast to the uptake of β-carotene and might suggest

that the intestinal absorption of β-apo-13-carotenone is not facilitated by a

specific membrane transporter.

3.6 Conclusion

We investigated the kinetics of uptake and metabolism of β-apo-8′-

carotenal and β-apo-13-carotenone in Caco-2 cells. Previous studies have

shown that β-apocarotenoids are present in fruits, vegetables, and animal diets

containing β-carotene; as well as mouse tissues and human plasma. Therefore,

we hypothesized that these cleavage products are directly absorbed from the diet similarly to β-carotene. Our findings suggest that β-apocarotenoids might not be directly absorbed from the diet because there is extensive metabolism or degradation in Caco-2 intestinal cells.

3.7 Acknowledgements

We thank Dr. Mark L. Failla for his expert technical assistance and

contributions to the successful completion of this research project. We also thank

Dr. Chureeporn Chitchumroonchokchai (Julie C) for laboratory training in cell

culture techniques and assistance with carotenoid extraction methodology. We

also express our gratitude to Dr. Robert W. Curley Jr. for the synthesis of β-apo-

13-carotenone.

Chapter 4. Epilogue

100

The previous chapters are exploratory studies that provide a foundation

for further investigation of the extent of formation of β-apocarotenoids in biofortified staple crops and bioavailability of these compounds with a view to

understand the biological role(s) of β-apocarotenoids, some of which are antagonists of vitamin A. Key findings from this research project include the presence of all long-chain β-apocarotenoids in biofortified cassava and rapid uptake and metabolism of β-apo-8′-carotenal and β-apo-13-carotenone in vitro.

The first study, Chapter 2, focused on the analyses of the β-carotene and

β-apocarotenoids content of conventionally bred cassava biofortified with β- carotene. Cassava (Manihot esculanta, Crantz) is a daily staple in many households in developing countries with an estimated 70 million people consuming at least 500 Calories per day [26, 28]. However, commercial cultivars of this crop have minimal nutritional value as indicated by the very low amounts of provitamin A carotenoids, iron, zinc, and protein. Previous studies have only investigated the retention and bioavailability of β-carotene after processing or storage [1, 28, 31].

In this study, we measured the levels of β-carotene and β-apocarotenoids in raw and processed cassava roots using a previously described high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. We found that the total levels of β-apocarotenoids relative to β-carotene in raw roots of Saracura, Clone 03-15, IAC 576-70 and IAC 265-97 was 0.8%,

0.3%, 0.2%, and 0.5% respectively. The total concentrations of β-apocarotenoids

101

in the cassava varieties biofortified with β-carotene were higher than non-

biofortified varieties. Boiling and frying of cassava roots was associated with a

decrease in the total β-apocarotenoids content in IAC 265-97, IAC 576-70, and

Saracura. However, in Clone 03-15 cassava variety, boiling and frying did not have any effect on the levels of total β-apocarotenoids. These two processing methods appeared to increase the amounts of total β-apocarotenoids in this variety.

The second study, Chapter 3 focuses on the kinetics of cell uptake and metabolism of β-apo-8′-carotenal and β-apo-13-carotenone using Caco-2 intestinal cells as a model. We were able to replicate previous kinetics data of the uptake of β-carotene. We observed that there was accumulation of β-carotene in cells at 30 minutes and the cellular β-carotene content increased with incubation time up to 8 hours. We also investigated the effect of concentration on β- carotene uptake and found that the amount of β-carotene in cells increased with increasing initial concentration of β-carotene. These results are similar to those observed by During et al [53]. Also, we did not detect any β-apocarotenoids in cells incubated with β-carotene as a function of incubation time or increasing concentration.

When cells were incubated with β-apo-8′-carotenal, we observed the rapid uptake of cellular content of β-apo-8′-carotenal but subsequent disappearance with increasing incubation time. This corresponded with the formation of two unknown metabolites (X and Y). We refined a previous HPLC method and this

102

enabled us to separate both metabolites from the parent compound. Compound

X was the major peak detected in cells with the other unknown metabolite

(compound Y) and β-apo-8′-carotenal present in small amounts. In addition, we made arbitrary quantifications of these metabolites by making the assumption that these metabolites have the same extinction coefficient as β-apo-8′-carotenal.

Based on our arbitrary quantifications, we observed that the formation of compound X in cells increased as a function of incubation time. Collectively, these results suggest that β-apo-8′-carotenal was a precursor for compound X.

Furthermore, we investigated the kinetics of uptake of β-apo-13- carotenone. When post-confluent monolayers were incubated with β-apo-13- carotenone, we found that there was rapid uptake of β-apo-13-carotenone in cells but subsequent disappearance with increasing incubation time. We have not identified any metabolic products of β-apo-13-carotenone because of limitation in our detection technology. There is the need to couple our uptake experiments to more sensitive detection methods such as HPLC-MS/MS. The possibility of the detoxification by phase 1 and 2 enzymes leading to extensive degradation of β-apo-13-carotenone is also worth exploring.

The goal of biofortification is to increase the amounts of β-carotene in staple crops consumed by VAD populations to enable these individuals meet the requirements of vitamin A. One major concern regarding biofortification with β- carotene is that it might increase the levels of β-apocarotenoids present in staple crops. Therefore, what would be the consequences of people consuming more β-

103

apocarotenoids? In our analysis of cassava biofortified with β-carotene, we found

the levels of β-apocarotenoids relative to β-carotene to be below 1% in the biofortified cassava varieties studied. These levels are way below those previously quantified in fruits and vegetable rich in β-carotene. Thus, there is no indication that biofortification of staple crops with β-carotene leads to greater amounts of β-apocarotenoids than is already naturally occurring in plants.

It has often been assumed that supplementation with β-carotene is the safest way to supplement vitamin A in deficient populations because it does not lead to vitamin A toxicity as seen in supplementation using preformed vitamin A.

However, recent studies have suggested that β-apocarotenoid levels are increased in β-carotene supplements and this might be responsible for negative effects seen in trials that involve supplementation of vitamin A as β-carotene. The rationale was that β-apocarotenoids are readily absorbed and higher amounts of very reactive β-apocarotenals might have deleterious effects. We studied the uptake of pure β-apo-8′-carotenal and β-apo-13-carotenone and demonstrate the extensive metabolism and degradation of these compounds in Caco-2 cells.

In conclusion, there are several aspects of this research topic that still need to be addressed. The bioavailability and metabolism of β-apocarotenoids and their impact on the bioactivity of these compounds with relevance in humans remain poorly understood. We suggest that individuals will consume β- apocarotenoids in the same quantity as any other foods that contain β-carotene.

Hence, it is important to understand the intestinal absorption of β-apocarotenoids

104 after consumption of a meal rich in β-carotene. Studies elucidating the transporters and enzymes involved in the uptake and metabolism of β- apocarotenoids are required. Furthermore, there is the need to conduct studies that involve feeding human subjects with different concentrations of β-carotene from natural sources to determine the in vivo production of β-apocarotenoids relative to the amount of β-carotene absorbed.

105

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Appendix A. Additional figures for Chapter 2

113

Figure A.1. Profile of β-apocarotenoids in boiled roots of Clone 03-15 variety

Carotenoids were extracted from boiled root of Clone 03-15 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

114

Figure A.2. Profile of β-apocarotenoids in fried roots of Clone 03-15 variety

Carotenoids were extracted from fried root of Clone 03-15 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

115

Figure A.3. Profile of β-apocarotenoids in raw roots of IAC 265-97 variety

Carotenoids were extracted from raw root of IAC 265-97 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

116

Figure A.4. Profile of β-apocarotenoids in boiled roots of IAC 265-97 variety

Carotenoids were extracted from boiled root of IAC 265-97 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

117

Figure A.5. Profile of β-apocarotenoids in fried roots of IAC 265-97 variety

Carotenoids were extracted from fried root of IAC 265-97 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

118

Figure A.6. Profile of β-apocarotenoids in raw roots of IAC 576-70 variety

Carotenoids were extracted from raw root of IAC 576-70 cassava variety as

described above. The extracts were analyzed by HPLC-MS/MS.

119

Figure A.7. Profile of β-apocarotenoids in boiled roots of IAC 576-70 variety

Carotenoids were extracted from boiled root of IAC 576-70 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

120

Figure A.8. Profile of β-apocarotenoids in fried roots of IAC 576-70 variety

Carotenoids were extracted from fried root of IAC 576-70 cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

121

Figure A.9. Profile of β-apocarotenoids in boiled roots of Saracura variety

Carotenoids were extracted from boiled root of Saracura cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

122

Figure A.10. Profile of β-apocarotenoids in fried roots of Saracura variety

Carotenoids were extracted from fried root of Saracura cassava variety as described above. The extracts were analyzed by HPLC-MS/MS.

123

1. β-apo-13-carotenone standard curve 1.6E+6

1.4E+6 y = 4.4149E+17x R² = 9.9920E-01 1.2E+6 1.0E+6 8.0E+5 6.0E+5 Area Peak 4.0E+5 2.0E+5 0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

2. Retinal standard curve 9.0E+6 y = 2.4299E+18x 8.0E+6 R² = 9.9874E-01 7.0E+6 6.0E+6 5.0E+6 4.0E+6

Area Peak 3.0E+6 2.0E+6 1.0E+6 0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

Figure A.11. Standard curves of pure β-apocarotenoids

Top panel: LC-MS standard curve for β-apo-13-carotenone. Bottom panel: LC-

MS standard curve for retinal.

124

3. β-apo-14′-carotenal standard curve 3.5E+6 y = 8.5814E+17x 3.0E+6 R² = 9.9961E-01 2.5E+6

2.0E+6

1.5E+6 Area Peak 1.0E+6 5.0E+5 0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

4. β-apo-12′-carotenal standard curve 3.0E+6

y = 8.0674E+17x 2.5E+6 R² = 9.9728E-01 2.0E+6

1.5E+6

Area Peak 1.0E+6

5.0E+5 0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

Figure A.12. Standard curves of pure β-apocarotenoids

Top panel: LC-MS standard curve for β-apo-14′-carotenal. Bottom panel: LC-MS standard curve for β-apo-12′-carotenal.

125

5. β-apo-8′-carotenal standard curve 2.5E+6 y = 6.8143E+17x 2.0E+6 R² = 9.9876E-01

1.5E+6

1.0E+6 Area Peak

5.0E+5

0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

6. β-apo-10′-carotenal standard curve 3.5E+6 y = 8.6254E+17x 3.0E+6 R² = 9.9987E-01 2.5E+6

2.0E+6 1.5E+6 Area Peak 1.0E+6 5.0E+5 0.0E+0 0 1E-12 2E-12 3E-12 4E-12

moles injected

Figure A.13. Standard curves of pure β-apocarotenoids

Top panel: LC-MS standard curve for β-apo-8′-carotenal. Bottom panel: LC-MS standard curve for β-apo-10′-carotenal.

126

7. β-carotene standard curve 9.0E+5 8.0E+5 y = 1.6545E+16x + 3.6148E+03 R² = 9.9523E-01 7.0E+5 6.0E+5 5.0E+5 4.0E+5

Area Peak 3.0E+5 2.0E+5 1.0E+5 0.0E+0 0 1E-11 2E-11 3E-11 4E-11 5E-11 6E-11

moles injected

Figure A.14. Standard curves of pure β-carotene

LC-MS standard curve for β-carotene.

127

Appendix B. Additional figures for Chapter 3

128

β-apo-8′-carotenal standard curve 900 800 y = 7.8175x - 0.7211 700 R² = 0.9999 600 500 400

300 Area Peak 200 100 0 0 20 40 60 80 100 120 -100 ρmol/injection

β-apo-13-carotenone standard curve 160

140 y = 1.4809x - 0.0924 120 R² = 0.9988 100 80 60 Area Peak 40 20 0 0 20 40 60 80 100 120 -20 ρmol/injection

Figure B.1. Standard curves of pure β-apocarotenoids

Top panel: HPLC standard curve for β-apo-8′-carotenal. Bottom panel: HPLC standard curve for β-apo-13-carotenone.

129

all-trans-β-carotene standard curve 1000 y = 9.2599x - 3.5408 800 R² = 0.9975

600

400

Area Peak 200

0 0 20 40 60 80 100 120 -200 ρmol/injection

Figure B.2. Standard curves of pure β-carotene

HPLC standard curve for β-carotene.

130