THE PHYSICAL AND BIOLOGFCAL FACTORS THAT INFLUENCE THE ISOMERIZATION OF LYCOPENE

Sujatha Chakravarthi

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Nutritional Sciences

University of Toronto

O Copyright by Sujatha Chakravarthi 200 1

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TEE PENSICAL AND BIOLOGICAL FACTORS THAT INFLUENCE THE ISOMERIZATION OF LYCOPENE

Master of Science Sujatha Chakravarthi

Graduate Department of Nutritional Sciences University of Toronto

ABSTRACT

Epidemiological and dietary intervention studies demonstrate an inverse relationship

between the consumption of tomato and tomato products and the incidence of cancer. The

overall aim of this thesis was to evaluate the effect of physical and biological factors that

influence lycopene isomerization. Following results were observed: 1) AU-trans iycopene

was the predominant isomer in tomatoes, tomato products and supplement. 2) Heating

tomato juice in the preseace of olive oïl enhanced cis isomerization of lycopene. 3)

Tissue and serum samples had higher levels of cis- isomers. 4) Peak lymph concentration

of lycopene was observed 6 hours pst-administration. These results suggest that cis-

isomers of lycopene may be responsible for the protective effect in the prevention of

cancer and cardiovascular diseases.

My humble obeisance at the Lotus Feet of Bhagawan Sri Sathya Saï and without whose

blessings this thesis would not have taken a tangible shape.

This thesis is dedicated to my son Shashank, my bundle of joy.

To my husband Anand, for his patience, support, love and friendship throughout the

graduate program. To my mother and father for al1 their efforts with special recognition

to my mother Meera, without whose help during my pregnancy days, this thesis would

not have seen its day.

I offer my deep gratitude to Dr. A.V. Rao, for his steadfast support, guidance, throughout

my research work. Thanks to Dr. S. Agarwal for his valuabIe inputs.

Thanks to Dr. David Yeung and Dr. Reinhold Vieth for their guidance as members of my

advisory cornmittee.

Special thanks to Voula Philips, Emilia D'souza Thomas and Mrs. Hardy for helping me

sort out office matters.

TABLE OF CONTENTS

Aclmowledgernents

List of Abbreviations

List of Tables

List of Figures

Publication arising fkom thesis research

1 . Introduction

1.1 Hypothesis and Objective

1.1.1 Rationale

1 -1 -2 Overall Hypothesis

1.1.3 Overall Objective

1.1.4 Specific Objectives

2 . Review Of Literature

2.1 Lycopene

2.1.1 Chemistry

2.1.2 Occurrence

2.1.3 Bioavailability and Tissue Distribution of Lycopene

2.1.4 Antioxidant Properties of Lycopene: in vitro and in vivo

2.2 Lycopene and Cancer

2.3 Lycopene and Cardiovascula. Disease

2.4 Lycopene and Other Diseases

iv

Page

* * *

111

vii

*.* w 1

ix

X

3 . Isomeric Forms of Lycopene in Tomato and Tomato Products and the Effoct of

Heating on Isomerization 32

3.1 Introduction 33

3.2 MaterïalsandMethods 34

3 -2.1 Heating with Different Fatty Acids 34

3 -2.2 Analysis of Total and Isomeric Forms of Lycopene 35

3.2.3 Instrumentation and Chromatography 36

3 -2.4 S tatistical Anaiysis 36

3.3 Results 36

3.4 Discussion 40

4 . lsomeric Forms of Lycopene in Rat Tissues and Human Semm 43

4.1 Introduction 44

4.2 Materials and Methods 45

4.2.1 Animal Study 45

4.2.2 HumanStudy 46

4.2.3 Lycopene Isomer Estimation 48

4.2.4 Statisticai Analysis 49

4.3 Results 5 1

4 -4 Discussion 57

5 . Absorption of Lycopene Using a Rat Cannulation Model 60

5.1 Introduction 61

5.2 Materials and Methods 63

5.2.1 Animals 63

5 -2.2 Surgical procedure

5.2.3 Sample Analyses

5-2.4 Statistical Analysis

5.3 Results

5-4 Discussion

6 . General Discussion and Future Studies

6.1 Discussion

6.2 Future Investigations

7 . References

8. Appendices

Appendix A: Tomato product agenda

Appendix B: Rodent diet composition

Appendix C: Tomato oleoresin composition

LIST OF ABBREVIATIONS

TBARS

LDL

VLDL

HDL

ROS

DNA

SFA

MTBE

HPLC

BHT

Lambda Max 1 Exponentiai Coefficient

Thiobarbituric Acid Reactive Substances

Low-Density Lipoprotein

Very-Low Density Lipoprotein

High-Density Lipoprotein

Reactive Oxygen Species

Deoxy Ribonucleic Acid

Age-related macula degeneration

Alpha

Beta

Gamma

Odds Ratio

Confidence Interval

Pol yunsaturated Fatty Acid

Monounsaturated Fatty Acid

Saturated Fatty Acid

High Performance Liquid Chromatography

Butylated Hydroxy Toluene

Ionization Energy

vii

LIST OF TABLES

Description Page

Table 2.1. Lycopene isornenzation studies at various time points 14

Table 2.2. Lycopene content in tomatoes and tomato products 17

Table 2.3. Lycopene content in various foods 18

Table 3 -1. Effect of heating t o m juice in the presence of heat and lipids on 39

lycopene isomers

Table 4.1. Relative proportion of trans to cis lycopene isomers in rat serum 53

and tissues

Table 4.2. Cis and @ans isomers of lycopene in human sera at the start and 55

end of the study

Table 4.3. Profile of total lycopene cis isomers and 5 4 s isomer at the 56

beginning and end of study

Table 5.1. Ratio of lycopene isomers in rat semm over tirne 70

LIST OF FIGURES

Figure 1.1.

Figure 1.2,

Figure 2.1 .

Figure 2.2-

Figure 2.3.

Figure 2-4.

Figure 2.5.

Figure 3.1.

Figure 4.1.

Figure 4.2.

Figure 4-3-

Fi,pre S. 1.

Figure 5.2.

Description

Structure of all-tram lycopene

Lycopene content of common h i t s and vegetables

Schema of lycopene absorption and transport

Geometrical isomers of lycopene

Formation of various fiee radicals fiom rnolecular oxygen

ROS and antioxidants in chronic disease process

Lycopene and human health

Relative amounts of total cis and total tram lycopene isomers in

tomatoes and tomato products

Study Design

a) Animal 50

b) Human 50

Cis and trans lycopene isomers in rat senim and tissues 52

Percentage of different cis isomers of lycopene in rat serum and tissues 54

Time course for lycopene absorption in mesenteric lymph duct 68

cannulated rats

Ratio of alI-h-andtotal cis lycopene in rat semm over t h e

Page

3

5

12

13

22

28

30

38

PUBLICATION ARISXNG FROM THESIS RESEARCH

S Chalcravarthi and AV. Rao. Isomeric forms of lycopene in tomato and tomato products

and the effect o f heating on isomerization. Int J Food Sci Nutr (manuscript to be

submitted) (Chapter 3).

S Chalcravarthi and AV. Rao. Isomeric forms of lycopene in rat tissues and human sera.

Nutr Cancer. (manuscript to be submitted) (Chapter 4).

1 . INTRODUCTION

Recent studies have shown that people consuming tomatoes and tomato products are at a

significantly lower nsk of several chronic diseases including cancer and cardiovascular

diseases (Steinmetz and Potter, 1996; Giovannucci, 1999). Lycopene has been identified

as being responsible for the beneficial effects of tomatoes. Lycopene is a natural pigment

present in some plants and microorganisms. It facilitates the absorption of light during

photosynthesis and also provides protection against photosensitization (Adams et al.,

1996). Being a mernber of the carotenoid family, lycopene is made up of two tetraterpene

units joined by a tail-to-tail bonding. It has a straight open chain hydrocarbon structure

with thirteen double bonds out of which L 1 are conjugated. In plants, lycopene is present

in a thermodynamically stable form, the dl-ms form (Figure 1.1 .), but may also be

present in various cis-forrns.

Humans cannot synthesize lycopene and its presence in the senun indicates dietary

intake. Lycopene in human serurn exists in an isomeric mixture in which 50% of total

lycopene is present as ris isomers. Unlike other carotenoids, lycopene lacks the beta [BI-

ionone ring structure and hence lacks pro-vitamin A activity. The molecular formula of

lycopene is C40H56 and its molecular weight is 536.85 daltons (ansky, 1998; Nguyen

and Schwartz, 1999). It is a lipophilic hydrocarbon molecule and insolubIe in aqueous

solutions. Lycopene is a red pigment that absorbs light in the visible range. A petroleum

ether solution of lycopene has lambda max [A-] 472 nm and extinction coefficient CE%]

3450 (Clinton, 1998). Lycopene acts as an antioxidant as it is a potent singlet oxygen

quencher.

Figure 1 . 1 . Structure of all-n-an<; lycopene (Clinton, 1998). Lycopene has a molecular formula of C43H56 and has eight isoprenic units. It bas 1 I conjugated and 2 non-conjugated double bonds.

Although lycopene is present in many h i t s and vegetables, the main source in the

western diet is fkom tomatoes and tomato products (Figure 1.2.). The amount of lycopene

present in tomatoes can Vary with the variety, degree of ripeness of tomatoes and other

climatic conditions and agricultural practices. Lycopene, ingested in its natural all-tram

form found in tomatoes is poorly absorbed (Stahl and Sies, 1992). Bioavailability can be

defined as a measurement of the amount o f the compound absorbed into the bloodstrearn.

Studies have shown that heat processing of tomatoes and tomato products, induces

isomerization of lycopene to predominantly the cis fonn, thus increasing its

bioavailability (Stahl and Sies, 1992; Gartner et al., 1997). Giovannucci et al. (1 995)

suggested that only the intake of processed tomato products was related to a lower risk of

prostate cancer. This is probably because processed tomato products contain a higher

level of lycopene in the cis fom.

Tissue culture studies in animals and humans have provided evidence that lycopene

reduces the risk developing cancer. In a meta-analysis of 72 studies, over half of these

studies (57) reported an inverse association between tornato intake or circulahg

lycopene and the risk of several types of cancer [cancers of prostate, breast, lung, colon

etc]. 35 studies were statistically significant. None of these studies have reported any

adverse effects of high tomato intake or high circulating lycopene levels (Giovannucci,

1999). In another study, lycopene was found to be the only micronutrient present in the

human serum associated with a decreased risk for breast cancer (Dorgan et al., 1998).

Majority of the studies to date have focused on the effect of lycopene on cancer

prevention with a

Tom ato Tom a to Tomato Watermelon Guam (pink) Grapefruit (raw ) juice Sauce (pin k)

Food Source

Figure 1 -2. Lycopene content of selected £bits and vegetables (Stahl and Sies, 1996). Tomato juice and tomato sauce contain the highest levels of lycopene followed by pink guava, watermelon, raw tomatoes and pink grapehit.

particular emphasis on the development of prostate cancer. There is limited information

regarding the factors that influence the absorption of lycopene and its isomerization.

I t is postulated (Giovannucci et al., 1995; Sakamoto et al., 1994) that isomerization of

lycopene may occur in the body. It is therefore hypothesized that alh-ans lycopene may

be converted into a more bioavdable form czk fonn in the body and thereby exert its

protective effect against the devetopment of specific cancers [colon cancer, breast cancer,

lung cancers etc]. The location of pans to cis isomerization in the body, and the chernical

and biological properties of isomerization of dl-tram lycopene is not yet clear. The

relative biological significance of various geometric isomers is yet to be established. The

overall aim of this thesis is to investigate the physical and biological factors that

influence the isomerization of dl-trtlns lycopene to its cis foms and the subsequent

absorption of lycopene in rats.

1.1 Hypothesis and Objective

1.1. I Rationale

The observation that the majority of lycopene in tissues and sera is present in the cis-

isomenc fonn while lycopene is present in its dl-trans isomeric fonn in foods and

supplements, suggests that isomerization is an important step in the biological role of

lycopene in vivo. Furthemore, finding that the ratio of the all-truns to cis foms of

lycopene in fiesh tomatoes is significantly diffaent fiom the ratio in human blood and

tissues after the consurnption of tomato and tomato products, suggests that isomerization

6

of lycopene occurs in vivo. This has drawn considerable attention towards the structural

features between the two isomeric forms. Some of the differences that can be highlighted

are:

The difference in molecular shape of the cr's-isomers of lycopene compared to the

d l -mns form may alter the ability of ck-isomers to incorporate into lipoproteins

or subcellular lipid structures and its interaction with various proteins.

Cis-isomers are less likely to crystallize or aggregate and may therefore be

solubilized more efficiently in lipophilic solutions and consequently more readily

transported within cells or between tissues. This property could perhaps reflect the

participation of lycopene in vivo in specific biologic reactions (Clinton, 1998).

Processing has been shown to increase the bioavailability of lycopene and higher

amounts of cis-lycopene have also been observed in human and animal tissues

and semm suggesting that perhaps the cis-isomers of lycopene are packaged in the

chylomicrons more efficiently than the dl-tram form (Stahl and Sies, 1992).

Schierle et al. (1997) observed that tomato-based foodstuffs and rneals had the dl-trans

form of lycopene but in the human plasma, the isomeric ratio of lycopene favoured the

cis-group with 5-cis lycopene as the predominant isomer.

The relationship between consumption of lycopene and the incidence of specific diseases

particularly cancer have been studied. However, very little is known about the

isomerization of lycopene and its role in disease prevention or pathogenesis. In order to

understand the role of lycopene in vivo, the isomerization of lycopene warrants fürther

research.

Lycopene has a straight chain hydrocarbon structure with thirteen double bonds out of

which 1 L are conjugated and 2 non-conjugated. It is predominantly present in its all-trans

form in nature- Humans cannot synthesize lycopene therefore its presence in the human

serum and tissues would indicate dietary consumption. Majority of lycopene in tissues is

present in its cis-isomeric form while in food it is present in its dl-pans form.

The presence of unsatwated double bonds in the structure of dl-tram lycopene allows it

to be isomerised to its cis forms. This isomerization is influenced by several physical and

biological factors.

1.1.3 Overall Objective

To study the physical and biological factors that influence the isomerization of lycopene.

1.1.4 Speczjic Objectives

1) To determine total, dl-tram and cis-isomers of lycopene in comrnercially

processed tomato products.

2) To evaluate the effects of heat and types of lipids on lycopene isomerization in

tomato juice.

3) To determine total and isomeric forms of lycopene in rat serum and human senim.

4) To assess absorption and isomerization of lycopene in rats by lymph duct

cannulation.

Lycopene and J3-carotene have emerged as effeçtive antioxidants in various research

findings. The consumption of lycopene and p-carotene is associated with a reduction in

the risk of developing various types of cancer (Steinmetz and Potier, 1996; Giovannucci,

1999). Absorption of lycopene occurs in the gastrointestinal tract via chylomicrons.

Observations fkom Stahl and Sies (1 992) suggest that the cis-isomers of lycopene

probably due to their kinked shapes are packaged in the chylomicrons more efficïently

than the all-tram fom. Lycopene is then transported fiom the liver to the blood strearn

aft er incorporation into the low-density lipoprotein FDL] cholesterol fiaction (Figure

2.1 .).

Various geometrical isomers of lycopene occur either naturally in k i t s and vegetables,

or are formed following mechanical and thermal food processing (Chandler and

Schwartz, 1988) (Figure 2.2.). Several isomerization studies at various time points have

been camed out (Table 2.1 .). The bioavailability of lycopene fiom tomatoes and tomato

products has been s h o w to increase with food processing (Stahl and Sies, 1992; Gartner

et al., 1997). This could perhaps be due to an increased formation of cis-isomers of

lycopene that are more bioavailable. Supportïng this statement, Boileu et al. (1999) have

reported cis-isomers of lycopene to be more bioavailable in vilro than its dl-hum

counterpart. At present, the significance of the conversion of mns- to -cis foms of

lycopene with respect to its absorption and biological efficiency is not well understood

and requires investigation. .

- Lycopene Lycopene and other Carotenoids and other

Micelles

Mucosal Side

Carotenoids Caro tenoids

met abolites

Cleavuge Oxidation Isomerriation

Metabolites

Serosal Side

s P.,., @

.DL @

VLDL @

Circulation

Figure 2.1. Schema of lycopene absorption and transport (Johnson, 1998) Lycopene and other carotenoids are taken up by bile acid micelles and via passive diffision enter the serosd side of the intestine. Here they undergo several reactions and lycopene and the metabolites are taken up by chylomicrons and transported to the lymphatic and portai system. Lycopene and other carotenoids are seen to be present in LDL, VLDL and HDL fkactions in the circulation.

Figure 2.2. Geometncal isomers of lycopene (Nguyen and Schwartz, 1999) All-tram lycopene (A) is the predominant fom of lycopene in food products and undergoes isomerization to give a series of cis-isomers. The above are some of the comrnonly identified cis-foms of lycopene (B-H).

Table 2.1, Lycopene isomerization studies at various time points

(Adapted and modified fiom Nguyen et al., 1 999)

2.1 Lycopene

Lycopene is a natural pigment synthesized only by plants and microorganisms. It is a

member of the carotenoid family and is, an acyclic isomer of p-carotene. Lycopene does

not have provitamin A activity since it lacks the p-ionone ring. Its molecular formula is

and molecular weight is 536.85 daltons. It is one of the most potent antioxidants

with a singlet oxygen quenching ability that is twice that of f3-carotene and 10 times that

of alpha [a]-tocopherol (Krinsky, 1998; Nguyen and Schwartz, 1999). Lycopene exists in

several geometric foms (Figure 2.2.). In foods, lycopene is seen to be present

predominantly in its all-fians form (approximately 95.4% of total lycopene content)

whereas in serum and tissues it is mostly present as cis isomers (Krhsky, 1998; Nguyen

and Schwartz, 1998; Stahl and Sies, 1992). Lycopene is a highly unsaturated hydrocarbon

molecule having 1 1 conjugated and 2 non-conjugated double bonds. It can undergo light,

thermal energy or chernical reactions resuiting in a cis-pans isomerization that gives rise

to various geometric isomerk forms (Figure2.2.). All-trans, bis, 9-ci., 13-cis and 15-cis

lycopene are the most commonly identified forms of lycopene (Nguyen and Schwartz,

1999; Stahl and Sies, 1996).

2.1.2 Occurrence

Tomatoes and tomato products are the major sources of dietary lycopene and are

important contributors of carotenoids in the diet (Table 2.2.). Other sources of lycopene

15

include red fniits and vegetables, like watermelon, pink grapefkuit, and puik guavas

(Table 2.3 .) (Shi and Maguer, 2000; Nguyen and Schwartz, 1999).

Al1 processed tomato products such as tomato juice, ketchup, paste, sauce and soup, are

rich sources of lycopene (Table 2.2.). Rao et al. (1999), using a food fiequency

questionnaire have estimated the average daily dietary lycopene intake levels to be 25

mg/day, with processed tomato products accounting for 50% of the total intake.

Lycopene fiom processed tomatoes is reported to be more bioavailable than fiom fkesh

tomatoes. (Stahl and Sies, 1992). This may be attributed to the release of lycopene fiom

the food matrix due to food processing, the presence of lipids in the heated tomato juice

and/or heat, which induces a trans to cir isomerization (Rao and Agarwal, 1999).

Isomerization of dl-@ans lycopene to its cis isomers is enhanced by the acidic

environment in the gastric milieu. Furthermore it has been implicated that the pH, as well

as the food matrix influences the level of isomerization of lycopene (Re et al., 2000).

2.1.3 Bioavailability and Tissue Distribution of Lycopene

Several factors influence the bioavailability of lycopene fiom tomato and tomato

products, including isomerization, thermal processing and the presence of lipids.

Carotenoids, due to a high nurnber of conjugated double bonds, can undergo truns-to-cis

isomerization as a result of phytochemical or thermal processes to give an array of mono-

or poly-cis isomers. This phenomenon of cis-pans isomerization is also called

stereomutation (Stahl and Sies, 1994). Lycopene undergoes stereomutation to give a

series of cis-isomers (Figure 2.2.).

16

Table 2.2. Lycopene content in tomatoes and tomato products (Rao and Agarwal, 1999)

Tomato Product Lycopene Content

( W g weigbt)

Fresh Tomatoes 8.842

C

Cooked Tomatoes 37

Tomato Sauce 62

Tomato Paste 54- 1500

Tomato Soup (condensed) 79.9

Tomato Powder 1 126.3-1264.9

1

Tomato powder and tomato paste have the highest lycopene concentrations than fresh and cooked tomatoes and tomato products.

Tomato Juice

Pizza Sauce

Ketchup

50-1 16

127.1

99- 134.4

Table 2.3. Lycopene content in various foods (Shi and Maguer, 2000)

Food Source Lycopene Content

(mg(1OOg wet basis)

Fresh Tomato Fruit 0.72-20

Watermelon 2.3-7.2

Guava (Pink) 5.23-5.50

Grapefruit (Pink) 0.35-3.36

Papaya O. 1 1-5.3

I Rosehip Puree

Sweet Potato

Fresh tomato fiuit has the highest lycopene content, followed by watennelon and pink guava. Other fniits have lycopene content ranging fkom 0.01-3.36mg/lûûg of wet h i t .

0.68-0.7 1

0.02-0.1 1

Apple Pulp f

0.1 1-0.18

Lycopene is present predominantly in its all-hnns form in food products but many cis-

isomers of lycopene and other carotenoids have been identified in human and animal

serum and tissues (Jensen et al., 1987; Chandler and Schwartz, 1987; Sowell et al., 1988).

Arnong the cis-isomers, 5-cis, 9-cis, 13-ch and 15-cis are the most comrnonly identified

forms of lycopene (Nguyen and Schwartz, 1999). It is suggested that cis-isomers of

lycopene might be more efficiently absorbai than their all-wans counterpart due to the

greater solubility of the former in mked micelles and the lower tendency of cis-isomers

to aggregate (Britten, 1995; Stahl and Sies, 1992). Cis-isomers of lycopene are more

bioavailable than tram-isomers in vitro, using bile acid micelles prepareci fiom

crystalline lycopene. Similarly, cr's-isomers of lycopene are more bioavailable than pans-

isomers of lycopene in vivo, using cannulated ferrets (Boileu et al., 1999).

Each carotenoid differs greatly in its dynamics of absorption, distribution and utilisation.

Carotenoids are tightly bound to macromolecules in most foods, causing problems in

absorption. Processing methods in m a b g of tomato products helps to breakdown the

cellular matrix of foods releasing lycopene fiom its bound fom (Rao and Agarwal, 1999;

Johnson, 1998; Williams et al., 1998). Studies have also shown that heating can fûrther

improve the absorption of carotenoids. Apart fiom the processing in the industry, tomato

products may also undergo various cooking treatments before consumption. This cooking

with oil would enhance the isomerization of lycopene fiirther to produce more

bioavailable cis-isomers. Lycopene was shown to be more bioavailable when tomato

juice was boiled for 1 hr with 1 percent corn oil (Stahl and Sies, 1992).

Lipids play a major role in carotenoid dissoiution and absorption. Lycopene was shown

to be present in al1 lipoprotein fractions, with the majority beïng present in the LDL

fiaction. The solubilized lycopene is incorporated into micelles (formed fiom lipids and

bile acids) via passive transport. Once in the intestine, lycopene is packed into

chylomicrons and released into the lyrnphatic system to be transported to the liver (Figure

2.2.). Lipoproteins transport lycopene into the plasma for its distribution to different

organs (Rao and Agarwal, 1999; Stahl and Sies, 1996).

Carotenoids including lycopene are found in chylomicrons and very low-density

lipoproteins W D L ] post-prandially. The concentration of carotenoids in LDL and hi&-

density lipoprotein P L ] bas been seen to increase with tirne and reach peak

concentrations 24-48 hrs afier consumption (Sies and Stahl, 1998). Non-polar lipophilic

carotenoids like lycopene and p-carotene are transported primarily by LDL

(approximately 75%) with the rernauiing 25% transported by HDL and VLDL (Johnson,

1998). The polar carotenoids are carrieci equally by LDL and HDL (Johnson, 1998).

Serum lycopene, due to its lipophilic characteristics is speculated to be situated towards

the centre of the LDL molecule. This would explain its slower disappearance rate than

serum p-carotene (Clinton, 1998; Stahl and Sies, 1996). In humans, lycopene makes up

2 1-43% of the total plasma carotenoid (Stahl and Sies, 1996). Lycopene has a half-life of

2-3 days in humans (Rao and Agarwal, 1999). Large differences in lycopene levels in

different body tissues have been reported suggesting that perhaps there is more efficient

transport of lycopene to some tissues (Nguyen and Schwartz, 1999).

2.1.4 Antioxidant Properties of Lycopene: in vitro and in vivo

In vitro systems provide much of the evidence in support of the antioxidant fbnctions of

dietary lycopene. Dietary lycopene rnay increase the lycopene in the body and, acting as

an antioxidant, may trap reactive oxygen species (Figure 2.3.), increase the overall

antioxidant potential or reduce the oxidative damage to lipoproteins, membrane lipids,

proteins (important enzymes) and DNA LDeoxy Rïbonucleic Acid] (genetic materiai),

thereby lowering oxidative stress, Reduction in oxidative stress may lead to reduced risk

for cancer and cardiovascular diseases. 0th benefits of increased lycopene status in the

body are to regulate gene hctions, improve inter ce11 communication, etc thus lowering

cancer risk. These mechanisms may also be interrelated and operate simultaneously to

provide health benefits. The conjugated double bonds in lycopene enable it to act as an

efficient antioxidant. The sïnglet oxygen quenching abilities of various carotenoids, a-

tocopherol, bile acids and retinoic acid were compared and lycopene emerged as the most

efficient quencher. Lycopene had a two fold higher quenching capacity when cornpared

to p-car0 tene (Di Mascio et al., 1 989). Similarly, when the radical scavenging capacity of

lycopene was compared with vitamin E, it was found to be three times more potent than

vitamin E (Miller et al., 1996). The conjugated double bond system was concluded to be

responsible for this effect. Mortensen et al. (1997) did a similar cornparison of the

scavenging ability of various carotenoids against different radicals and determined

lycopene to be an efficient antioxidant. Lycopene used different scavenging mechanisms

(electron transfer or adduct formation) in order to neutralize different types of radicals.

2 "OH

Figure 2.3. Formation of various fiee radicals fiom molecula. oxygen. Reactive oxygen species are chemical species w*th one or more unpaired electrons in their outer orbit and with the ability to exist independently. These are generated endogenously fiom normal cellular metabolic processes such as mitochondrial respiration, fatty acid metabolism, cytochrome p450 reactions, respiratory burst of phogocytic cells. Exogenous factors include ozone, tobacco smoke, ultraviolet light, fatty acids in foods and more (Stahl a d Sies, 1997).

As lycopene is highiy hydrophobic, its scavenging ability can be seen to be efficient in

lipophilic envkonments (Gerster, 1997; Stahl and Sies, 1996). Although there is not

much research published on the in vivo oxidative effect of lycopene, the oxidation

products of l ycopene (5,6-dih ydroxy-5,6-dihydro-l ycopene, 1 ycopene epoxide) in the

serum suggests the utilization of lycopene as an antioxidant in vivo (Williams et al.,

1998). When subjects were supplemented with different dietary sources of lycopene, the

semm thiobarbituric acid reactive substances [TE3ARS] a biomarker for Iipid

peroxidation, was seen to be significantly reduced (Rao and Agarwal, 1998a). When

lycopene was withdrawn fiom the diet for two weeks, the serum lycopene levels were

reduced by 50% and increased the serum TBARS by 25%. Among, smokers s e m

lycopene levels decreased by 40% and s e m TBARS was increased by 40% afier

smoking 3 cigarettes (Rao and Agarwal, I998b). Ingesting tomato products such as

tomato juice, spaghetti sauce and oleoresin for one week significantly increased the

serum lycopene levels. This increase in serum lycopene was accompanied by lower levels

of TBARS indicating a reduction in s e m lycopene peroxidation. Serum protein and

DNA oxidation were also shown to be reduced although not significantly. The lack of

significance was attributed to the short treatment time (one week) (Agarwal and Rao,

1998). Lycopene has been suggested as being important in preventing oxidation of lipid

membranes and preserving the integrity of cellular membranes.

Previous studies (Nguyen and Schwartz, 1998; Boileu et al., 1999; Clinton et al., 1996)

have led to the assumption that the higher percentage of lycopene cis- isomers in human

and animal biological samples is in part due to the consumption of heat-treated tomato

products containing cis-isomers of lycopene- It has been shown that there is no change in

the isomer profile of various lycopene dietary sources irrespective of product type,

moisture content, variety of tomatoes, the container type, and the severity of heat

treatrnent (Nguyen and Schwartz, 1998)- Hence, it is suggested that a high ratio of cis- to-

tram lycopene in human biological samples is formed during digestion, absorption, or

uptake into the blood Stream.

The biological significance of cis- isomerization of lycopene, its bioavailability, and

antioxidant properties in the prevention of cancer and cardiovascdar diseases is not well

understood. Therefore, studies have to be put forth to gain fùrther insight on the

isomerization of Iycopene and the biological effects of cis-mns isomerization of

Iycopene

2.2 Lycopene and Cancer

Research in the ânticarcinogenic properties of lycopene is accumulating. Findings fkom

animal, epidemiological, and tissue culture studies provide evidence of the effect of

Iycopene in lowering the development of cancer. Giovannucci et al. (1995) have reported

an inverse relationship between the consumption of tomatoes and tomato products and

the nsk of prostate cancer. in a meta-analysis of 72, epidemiological studies, over half of

the studies have reported inverse associations between tomato or lycopene consumption

or blood lycopene levels and risk of several types of cancer [cancers of prostate, breast,

lung, colon etc]. 35 of these inverse associations were statistically significant

(Giovannucci, 1999).

The remaining 15 were inconclusive. A recent case-control study of 65 prostate cancer

patients and 132 cancer-fiee controls, investigating the effects of plasma lycopene, other

carotenoids, and retinol, showed signifïcant inverse associations of prostate cancer with

plasma concentrations of lycopene [odds ratio (OR), 0.17; 95% confidence interval (CI),

0.04-0-78; P for trend, 0.00521 and zeaxanthin [(OR), 0.22; 95% CI, 0.06-0-83; P for

trend, 0.00281. Lutein and B-cryptoxanthin had borderline associations and no obvious

associations were seen for a- and B- carotenes, retinol and a- and gamma [y]-tocopherols

(Lu et al-, 2001).

Even though there is limited data, it is clear that carotenoids are not uniformly and

equally distributed in tissues. Several geometrical configurations of lycopene in human

plasma and tissue samples have been recorded, with the cis-isomer content ranging fiom

50% - 88% of the total lycopene level (Krinsky et al., 1990; Stahl et al., 1992, 1993).

Comparison of the major carotenoid levels of normal vs rnalignant human prostrate tissue

showed the rnalignant prostrate tissue to have higher concentrations of lycopene than

other carotenoids (Clinton et al., 1996). Several case control studies of digestive tract

cancers revealed that individuals with the highest intake of tomatoes were protected fkom

digestive tract cancer, stomach, colon and rectal cancers when compared to individuals

with the Iower intake (Franceschi et al., 1994). A ceMcal dysplasia study of black

women suggested that higher lycopene intakes and high serum lycopene levels might

protect against dysplasia (Kanetsky et al., 1998). Kim et al. (2000) recently assessed the

chemopreventive potential of lycopene in a multiorgan carcinogenesis modeI. The

incidences and multiplicities of lung adenornas plus carcinomas in male mice receiving

50 ppm lycopene were significantly reduced. These fhdiogs suggest that lycopene might

have a potential as a chemopreventive agent against carcinogenesis.

2.3 Lycopene and Cardiovascular Disease

Two leading causes of mortality in North America are cardiovascular diseases and stroke.

Carotenoid intake has often been associated with a reduced risk of these diseases

(Canfield et al., 1993; Mayne 1 996). Low-density lipoproteins serve as cholesterol

caniers into the blood stream. Oxidation of LDL plays a role in the initiation and

promotion of atherosclerosis wbich is the underlying disorder for heart attacks and

ischemic strokes (Witzum, 1994; Parthasarathy et al., 1992; Heller et al., 1998) Nutrients

with antioxidant potential, have been seen to reduce the progression of atherosclerosis

because of their potential to stop the oxidative process (Figure 2.3.) which is one root

cause for the disease Weller et al., 1998; Parthasarathy, 1998; Morris et al., 1994).

Oxidative stress is when the balance between reactive oxygen species (ROS) and

antioxidants is disrupted where there is accumulation of ROS in the body creating

potential for celluIar darnage (Trilling and Jaber, 1996). ROS are reactive chernical

species with one or more unpaired electrons in their outer orbit with the ability to exist

independently (Gutteridge, 1995). The high reactivity of ROS causes thern to initiate the

oxidative destruction of important biomolecules such as DNA, lipid and protein, which if

lefi uncorrected can lead to cellular damage and disturbances in physiologicd functions

(Beckrnan and Ames, 1998; Benzie, 1996). Lycopene has been shown to have a strong

interaction with active oxygen species, (Hydrogen peroxide) which can generate the

hydroxyl radicals. The conjugated double bonds act as capture points for the reactive

26

species thereby rendering them inactive, This interaction of lycopene (Figure 2.4.) and

other carotenoids with singlet oxygen and other reactive species results in the generation

of short-lived reaction products (mostly apocarotenals or apocarotenones, epoxides)

thereby helping in the prevention of several chronic diseases including cancer (Gerster,

1997).

Vitamin E has been proven both clinically and epidemiologically to have protective

antioxidant properties (Rimm et al., 1993; Hodis et al., 1995; Paolisso et al., 1995).

However, neither a-tocopherol nor B-carotene have given conclusive results in dietary

intervention trials (Mayne, 1996; Omem et al., 1996; Hennekens et al., 1996). Rissanen

et al. (2001) in a Kuopio Ischaemic heart disease risk factor study, have shown that low

levels of serum lycopene is associated with increased risk of acute coronary events and

stroke in middle-aged men previously fiee of chronic heart diseases and stroke.

Lycopene lowers the oxidation of LDL-cholesterol and may thereby d u c e the risk for

cardiovascular disease (Diaz et al., 1997; Stahi and Sies, 1996; Cademi et al., 1997;

Gerster, 1997; Gartner et al-, 1 997). Several studies have shown a direct correlation

between tomato and tomato product consumption and a reduced risk of cardiovascular

diseases (Aganval and Rao, 1998; Rao and Agarwal, 1998; Kucuk et al., 1999;

Parthasarathy, 1998) (Figure 2.5.). Linseisen et al, (1 998) reported that a single dose of

lycopene along with B-carotene, lutein, canthaxanthin and vitamin E prevented the ex

vivo oxidation of isolated LDL-

1 Endogenous Metabolic

( Activïty OR Exogenous

I I

Lipid, Protein & DNA f l r - l

Chronic Diseases

Figure 2.4. ROS and antioxidants in chronic disease process (Agarwal and Rao, 2000). Lycopene has been shown to have a strong interaction with active oxygen species, (Hydrogen peroxide) which can generate the hydroxyl radicais. The conjugated double bonds act as capture points for the reactive species thereby rendering hem inactive. This interaction of lycopene and other carotenoids with singlet oxygen and other reactive species gives rise to short-lived reaction products (mostly apocarotenals or apocarotenones, epoxides) [Gerster, 1997).

Recently, Agarwal and Rao (1998) conducted a study on healthy human adults by

providing them with a diet containing lycopene fiom tornato products in one or two

servings per day for one week, An increase in senun lycopene level was seen to

significantly lower the levels of oxidized LDL suggesting that lycopene is an important

antioxidant in tomato and tomato products might help to reduce the risk for heart disease.

2.4 Lycopene and Other Diseases

The protective effect of lycopene has also been shown to extend to other diseases (Figure

2.5.). Age-related rnacular degeneration (ARMD) is partly promoted by oxidative stress

due to exposure of retinal pigment of the eye to light and oxygen. The hwnan retina has

two pigments lutein and zeaxanthin and very little or no lycopene but low levels of serum

lycopene were found to be related to a hi& risk of ARMD (Mares-Perlman et al., 1995).

Cataracts result fiom gradual opacification of the lens with aging arising partly fiom

oxidative stress. Although intake of carotenoids, vitamins C and E have been associated

with reduced risk of cataract, there is little evidence identimg the beneficial effect of

lycopene (Mayne, 1996; Schalch and Weber, 1994). HIV infection results fiom the

destruction of T-helper cells (CD4) thereby impainng the immune response of the

individual. Two studies showed that both H N positive women and children had low

s e n i m lycopene levels when compared to their controls (Coodley et al., 1995; Periquet et

al., 1995). Schmidt et al. (1997) in an Austrian stroke prevention study showed that the

risk for microangiopathy-related cerebral damage, a risk factor for cerebrovascular

disease in the elderly was related to low s e m lycopene and a-tocopherol levels.

29

Figure 2.5. Lycopene and Human Health. Elevations in blood and tissue levels of lycopene may lead to a reduction in chronic diseases via different mechanisms.

Homnick et al. (1993) showed that the children with cystic fibrosis had low serum

lycopene levels than their controls. In summary, lycopene appears to have wide ranging

benefits against chronic diseases, Detennining the mechanisms by which lycopene may

have health benefits awaits fûrther investigation.

3 . ISOMERIC FORMS OF LYCOPENE IN TOMATO AND

TOMATO PRODUCTS AND THE EFFECT OF HEATING

3.1 Introduction

Tomatoes are an important agricultural crop worldwide and also constitute an integral

part of the human diet (Stienmetz and Potter, 1996; Giovannucci, 1999). Recent studies

have shown that people consuming tomatoes and tomato products are at a significantly

Iower risk of several chronic diseases includïng cancer and cardiovascular diseases (Rao

and Agarwal 1998; Giovannucci, 1999; Nguyen and Schwartz, 1999). Approximately 80

% of tomato consumption in the westem diet comes fiom processed tomato products such

as tomato juice, paste, puree, ketchup and sauce (Gould, 1992). Lycopene, a major

carotenoid present in tomatoes, has been identified as the active component responsible

for the beneficial health effect (Stahl and Sies, 1996). Recent studies have indicated

potential health benefits of a diet nch in tomato and tornato products. Consumption of

tornato and tomato products have been to shown to have and inverse relationship to risk

of several cancers and cardiovascular diseases (Rao and Agamal 1998; Agarwal and

Rao, 1998).

Lycopene is a straight chain hydrocarbon containing 1 1 conjugated and 2 unconjugated

double bonds. It can exist in its all-mns isomeric form or be isomerized to its cis

configurations (Nguyen and Schwartz, 1998). Recent studies have shown increased levels

of cis-lycopene in processed tornato products- Lycopene was shown to be absorbed more

efficiently fiom processed tomatoes compared to raw tomatoes (Stahl and Sies, 1992).

Increased absorption from processed tomato products can be associated with increased

levels of cis-lycopene. However, the levels of lycopene isomers in commercially

available tomato products and the effects of different oils used during the

33

processing/cooking of these products on lycopene isomerization upon heating have not

been well studied- The aim of this study was to measure the levels of total lycopene, ail-

trans isomer and cis isomer in commercially available tomato products and a lycopene

supplement. Also, the effect of heating tomato juice in the presence of different lïpids on

the isomerization of lycopene was studied.

3.2 Materials and Methods

The standard for dl-tram lycopene was obtained fiom (Sigma Chernical Co. St-Louis,

MO). Al1 extraction and HPLC solvents (Fisher Scientific, Colk Fairlawn, NJ) were

certified HPLC or ACS grade, Tomatoes and the oils were purchased from the local

markets and the tomato products were provided by H.J Heinz Company of Canada.

Lycored Company of Israel provided the lycopene supplement in the form of oleoresin

soft gel capsules.

3.2.1 Heating with D@ierent Fatty Acids

The selection of lipid samples was based on the degree of unsaturation. Tomato juice was

heated at 100" C continuously for 1 hour with the addition of 5 % corn oil

[polyunsaturated fatty acid (PUFA)], olive oiI [monounsaturated fatty acid (MUFA)] or

butter [saturated fatty acid (SFA)]. The heated tomato juice was cooled, and the volumes

standardized to their pre-heated levels and refiigerated (4' C). The tomato juice samples

were placed in vials and protected from light by covering with aluminum foi1 until

analyzed. Previous storage studies in our laboratory (unpublished) showed no change in

lycopene isomer profile on storage at - 7OC. Lycopene procedures (section 3.2.2) were

standardized and used to analyze the lycopene isomers in the products.

3.2.2 Analysis of Total and Isomeric Foms of Lycopene

About 500ml of tomato product samples were taken in a tube. Raw tomato was

homogenized in a blender without any water and weighed - 500mg (equivalent to 500~1)

and taken. Triplicate extractions of lycopene fiom oleoresin, homogenized tomatoes and

tomato products using hexane, methanol, acetone (2:l: l), containing 2.5% bromo

hydroxy toluene [BHT] (to prevent oxidation of lycopene) was carried out. Lycopene was

extracted by vortexing vigorously and vent shaken for 15 minutes on a rotator- 5 ml water

was added vortexed and vent shaken again for 5 minutes. The extracts were allowed to

stand for 5 minutes to separate the layers. 1 mi of the upper layer was aspirated in a g l a s

tube. The aspirated extracts were dried by flushing with nitrogen, dissolved in the mobile

phase (methano1: methyl-t-butyl ether W E I ) and then analyzed by reverse phase high

performance Iiquid chromatography [HPLC] using a C30 polymeric column (YMC, hc.,

NY, U.S.A.) at a flow rate of 1.0 ml/min. The peaks were eluted with methanol: MTBE

(62:38) and monitored at 460 nrn using an absorbance detector (Clinton et al., 1996). The

total and isomeric amounts of lycopene in the samples were calculated using the peak

areas. Triplicate extractions of the heated tomato juice samples were also camed out in a

similar manner.

The HPLC system consisted of Waters 450 system. Separations of geometric isomers of

lycopene were achieved using polymeric C30 reverse phase HPLC colurnn (250 x 4-6

mm) (YMC, Inc, NY, U-%A)- The mobile phase was 38 % MTBE in methanol, at a flow

rate of 1 .O ml/min. Column effluent was measured at 460 nm.

3.2.4 Statisticul Analysis

The statistical analysis was done using a one-way ANOVA (Sigma Stat Software, Jandel

Scientific). The pst-test was done using Tukeys test. P values l e s than 0.05 were

considered statistically significant, Results are expresseci as mean fr SEM.

3.3 Results

Relative amounts of cis and tram isomers in oleoresin, raw tomato and various tomato

products are s h o w in Figure 3.1. The isomeric content of these products ranged from 5

to 10 % cis-isomers and 90 - 95 % ail-tram-isorners. Amounts of all-tmns and different

cis isomers in tornato juice heated in the presence of various oils are presented in Table

3.1. In general, the relative percent of cis isomers increased upon heating in the presence

of al1 lipids. The amount of dl-trans lycopene isomer was reduced in olive oil when

compared to that present in corn oil and butter (6672.01 mM/L vs. 1 1666.98 mM/L and

13964.26 rnM/L) (Table 3.1).

Tomato juice heated with olive oïl had a significantly higher percentage of cis-isomers

(23.26 + 0.3; P<0.05) and lower percentage of tram (76.74 t 0.3; P<O.OS) when

compared to the other two groups (Table 3.1 .). There was no significant difference

between the profile of tram and cis isomers of corn oil and butter treated tomato juice

([Tram] 83.82 + 1.8 1 vs- 84.53 f 1-674 & [Cis] 16-18 + 1.8 1 vs. 15.47 t 1.67) (Table

3.1 .). There was a 12 - 20% decrease in the tram isomer percentage of tomato juice

heated in the oils when compared to the unheated juice. The cis isomers increased 200 - 360% on heating with the oils fkom the value of the unheated tomato juice. Heating with

olive oïl had a highest percent increase in cis isomer percentage (365.2%) when

compared to that in corn oil(223.6 %) and butter (209.4%). These results suggest that the

type of fatty acid is an important consideration in cis-isomerization of lycopene upon

heating.

Raw Oleoresin Tomato Tomato Tomato Tomato Tomato Tomato Juice Soup Sauce Paste Ketchup

Products

Figure 3.1. Relative amounts of total cis and total tram lycopene. Values expressed are means k SEM, n = 3

Table 3.1. Effect of heating tomato juice in the presence of lipids on lycopene isomers.

.yco pene

(n = 3)

% Trans

% cis

Corn Oil Olive Oil Butter

[mean t SEM]

Values w ith di fferent superscripts are significantly different, P<O.OS (One-way ANOVA and Tukeys Test).

Lycopene

(n = 3)

All - Trans

Cis I I I I I

Corn Oil

[mM/L]

1 1666.98 a

2266.65 a

Olive Oil

[mM/L]

10681.51

3258.48

Butter

[mM/L]

11765.81"

2167.1 9 a

3.4 Discussion

Although lycopene is present in its dl-tram isomeric fom in raw tomatoes and tomato

products, it is also present as cis isomers in animal and human serum and tissue samples

(Krinsky, 19%; Nguyen and Schwartz, 1998; Stahl and Sies, 1992). In this study

oleoresin, raw tomatoes and processed tomato products were shown to have 5 - 10% cis

isomers and 90 - 95 % tram isomers of lycopene (Figure 3.1 .). Thermal processing has

been seen to enhance the bioavialability of lycopene (Sies and Stahl, 1 992; Gardner et al.,

1997). This ùnproved lycopene bioavailability fiom processed foods is assumed in part to

be due to the mechanical and thermal processing which ruptures the plant cells thus

releasing 1 ycopene and also due to heat induced pans to cis isomerization (Gartner, 1997;

Stahl and Sies, 1992). Amounts of dl-pans and different cis isomers in tomato juice

heated in the presence of various oils are shown in Table 3.1. In general, the relative

percent of cis isomers increased upon heating in the presence of al1 lipids. The amount of

all-pans lycopene isomer was reduced in olive oil when compared to that present in corn

oil and butter (6672.0 1 mM/L vs. 1 1 666.98 mM/L and 1 3 964.26 mM/L) (Table 3.1 .)

Since lycopene is a fat-soluble compound, the presence of lipids in the diet cm also

influence its absorption fkom food. The arnount of fat in the diet required to enhance

carotenoid absorption is researched to be 3 - 5 g/meal, although the ingested carotenoids

would differ in their physiochernical characteristics of absorption (van het hof et al.,

2000). Previous studies in our laboratory have evaluated the effect of heating tomato

juice in the presence of 10 % corn oil. Heating for one hour showed the presence of 30 %

of lycopene as cis isomers in tomato juice compared to only 5% in unheated juice

40

(unpublished). Heating with either 5 % or 10% oil for one h o u made no difference in the

percentage of cis isomers (unpublished). There has been no study reported so far showing

the effect of different fatty acids and heating on the isomerization of Iycopene. In

addition, since tomato products are subjected to heating by several normal cooking

procedures with different types of fat, the isomerization pattern in the presence of three

different types of oils, namely corn oil (PUFA), olive oil (MUFA), and butter (SFA),

were chosen for this study. In this study, when tomato juice was heated with olive oil

there was significant hi* percentage of cis-isomers (P<0.05) when compared to the

other two types of oil (Table 3.1 .). These results clearly indicate that the type of fatty acid

can significantly influence isomerization of lycopene during heating.

Clark et al. (2000) has recently shown that the type of oïl can influence the absorption of

the carotenoid consumed and concluded that both astaxanthin and lycopene were

significantly (P<O.OS) better absorbed in olive oil than in corn oil. The absorption

efficiency in olive oil can be partially attriiuted due to the foxmation of cis isomers

shown in this study. The mechanisrn responsible for the observed increase in lycopene

cis-isomers when tomato juice is heated with monounsaturated fat m a i n s to be

elucidated. The possibilities include: isomerization of the all-trans isomer following

thermal treatrnent with reactions that are related to the structures of the different fatty

acids with dl-tram lycopene. The dl-pans isomer which is the stable form is probably

unstable in mono unsaturated fats in the presence of heat leading to the formation of the

kinked cis isomeric forms.

In summary, the findings Çom tbis study suggest the need for fürther investigation of the

biochemistry behind the structural reactions of different oils or fatty acids with lycopene,

and their biological significance- A study can be undertaken where lycopene supplernent

and other tomato products are heated with the three different types of oils and analyzed

for lycopene isomers. It would give an idea about the interaction of the different oils with

different tomato products.

4 . ISOMERIC FORMS OF LYCOPENE IN RAT TISSUES

AND HUMAN SERUM

4.1 Introduction

There are a number of epidemiological (Giovanucci et al., 1 995; Franceschi et al., 1 994)

and clinical (Clinton et al., 1996) studies relating tomato consumption to its anticancer

properties in humans. A number of plausible rnechanisms for the anticancer effects of

lycopene have been suggested including its efficient singlet oxygen quenching capacity,

its ability to induce ce11 to ce11 communication (Zang et al., 199 1), and modulation of

hormonal, immune systems and other metabolic pathways (Fuhramn et al., 1997;

DiMascio et al., 1989; Boehm et al., 1995; Levy et al., 1995; Bertram et al., 1995).

Lycopene fiom natural plant sources seems to exist predominantly in its all-tram form,

which is considered to be the most thermodynamically stable form (Krinsky, 1990;

Nguyen and Schwartz? 1999). However, upon consumption of the dl-hwns lycopene, it is

shown to be present in an isomeric mixture in the human plasma, containing 50% of the

total lycopene a s cis-isomers. Several commoniy identified fonns of lycopene include al1

tram, 5-cis, 9-cis, 1 3-cis, and 1 5 4 s fonns (Nguyen and Schwartz, 1 999).

A limited number of studies have shown cis isomer of lycopene to be the predominant

form in the semm and tissues of individuals fed tomato products containing dl-tram

lycopene (Stahl et al-, 1992; Schierle et al., 1997; Holloway et al., 1999)- It has been

observed that the ratio of lycopene c i s - m s geometrical isomers in biological fluids and

tissues like the prostate gland is different fkom that of raw tomatoes (Clinton et al., 1996).

The exact site of lycopene isomerization and their biological significance is not well

understood. Use of animal models might provide a better understanding of lycopene

isomerization and its role in cancer nsk reduction (Clark et d., 1998).

The purpose of this study was to investigate the changes in the levels of the distriiution

of different isomeric forms of lycopene in rat tissues and hurnan sera after consuming

diets containing lycopene to better understand lycopene isomerization at various tissues

to provide insight into the site of cis-tram isomerization reactions of lycopene in the

body.

4.2 Materials and Methods

4.2.1 Animal Study

Three male Fischer rats (F344) at six weeks of age weighing approximately 185 grams

purchased fiom HarIan Sprague Dawley, Inc. Indianapolis, Indiana were used in the

study. Animals were housed singly in plastic cages with comcob bedding under

controlled temperature (22' C) and humidity (50%) conditions. Animals were maintained

under a 12-hour light and dark cycle spanning fkom 7 am to 7 pm. Al1 rats ate and drank

ad libitum. At the end of the experiment, rats were killed by cervical dislocation. Animals

were cared for according to the guidelines under the Canadian Council on Animal Care.

The Ethics Cornmittee for the use of animals, University of Toronto, approved the

protocol.

4.2.1.1 Study Design

Following an acclimatization period of two weeks starting with laboratory Purina chow

for one week followed by the ATN93M diet for the second week al1 the animals were

given the AiN93M diet (Appendix B) supplernented with 10 ppm lycopene (Appendix

45

C). The study perîod lasted for a total of 8 weeks. At the end of the experiment, the

animals were sacrificed and blood and tissues were coilected (Figure 4.1 a).

4.2.1.2 Blood and Tissue Collection

Blood samples were collected at the end of the experiment by cardiac puncture and

processed to obtain the serum. The blood samples were centrifüged at 2000 rpm for 10

minutes to ob tain the serum. The top layer was aspirated, labeled and stored at - 70°C

until analyzed. Animals were dissected and the lung, prostate gland, heart, spleen, and

liver were collected. Al1 tissues were washed with 0.9% saline, blot dried, weighed and

homogenized. The samples were then storeà in vials at -70° C until they were analyzed

after about 10 months.

4.2.1.3 Instrumentation and Chromatography

S e m and tissue samples were analyzed using a HPLC system that consisted of Waters

7 17 plus Auto sampler and UV-Vis 2487 detector fiom Waters. Separations of geometric

isomers were achieved using polymeric C30 reversed phase HPLC column (250 x 4.6

mm) (YMC, Inc, NY, U.S.A). The mobile phase was 38 % MTBE in methanol, at a flow

rate of 1.1 ml/min. Colurnn effluent was rnonitored at 460 nm.

4 . 2 . 2 Human Study

4.2.2.1 Study Design

Seventeen healthy human subjects (10 male and 7 fernale) aged 25 to 35 years, with

normal body mass index (1 8.5 - 24.9), non-smokers and not on medication were

46

recx-uited- Seven different diets with combinations of different tomato products were

designed, each providing 30 mg lycopene/day. Diets were also standardized for fat and

calories (Appendix A).

Subjects undenvent a 2 week washout period during which they maintained theu regular

diet but without any tomato or tomato products and also were told to avoid any food

source of lycopene (Figure 4.1 b). After the washout phase, the subjects were asked to

consume their regular diet along with the given test tomato products (Treatment Phase).

During the treatment phase, the subjects consurned each of the seven test diets, one each

day in a prescribed random order. Afier the f b t seven days, the treatments were repeated

until the end of the treatment phase. Diet records were maintained fiom the start until the

end of the study. Fasting blood samples were collected at the beginning and end of the

treatment phase. The Human Subjects Review Cornmittee, University of Toronto,

approved the procedure used in this study.

4.2.2.2 BIood Collection

Fasting blood sarnples were collected and processed immediately for senun. Blood was

collected without the anticoagulant in order to coagulate the blood. The blood was

centrifuged immediately at 2000 rpm for 10 minutes at 4°C. The upper layer, the s e m

was aspirated and collected for analysis. The sera were labeled and stored at -70°C until

analyzed after 6 months.

4.2.3 Lycopene isomer Estimation

4.2.3.1 Serum Analysis

The lycopene was extracted fkom rat and human s e m according to the method

previously published (Rao and Agarwal, l998a). Lycopene isomers were analyzed using

reverse phase HPLC using VYDAC 201HS54 column and an absorbance detector at 472

nm with a flow rate of ImVrnin. An extemal lycopene standard was used to identiQ and

quanti@ lycopene peaks (Rao and Agamal, 1998a; Stahl et al., 1992).

4.2.3.2 Tissue Analysis

Rat tissue samples were also processed and analyzed according to the methods previously

published with minor modifications (Stahl et al., 1992). Rat tissue samples were washed

in saline to remove blood and blot drïed on a filter paper. The samples were stored at -

70°C until analyzed. For analysis, the tissue samples were minced, weighed (300 mg- 10.0 -

g) and transferred to a glass container. Samples were saponified by adding saturated

sodium hydroxide solution and BHT in ethanol, followed by ovemight incubation at

37OC. The incubated mixture was then centrifiiged and the upper layer aspirated and dned

under nitrogen to prevent oxidation of lycopene. The dried aspirate was made up with the

mobile phase h4TBE in methanol and injectai in a C30 reverse phase column at a flow

rate of 1 -0 rnlhin. The column effluent was monitored at 460 nrn.

4.2.3.3 Instrumentation and Chromatography

Human serum samples were analyzed by a HPLC system consisting of a Hewlett Packard

1 100 automated systern. Separations of geometric isomers were achieved using

48

poiymeric C30 reversai phase HPLC column (250 x 4.6 mm) W C , Inc, N.Y, W3.A).

HP Chem station data acquisition software (Version 8.01) was used to analyze the data.

The mobile phase was 38 % MTBE in methanol, and the samples were analyzed at the

flow rate of 1 .O mVmin. Column effluent was monitored at 460 nm.

4.2.4 Statistical Analysis

Descriptive analysis Mean, Standard Deviation, Standard Error of Mean] of the

different cis and tram isomers in different tissues and sera was cornputed. Al1 statistical

evaluations paired t-test] were completed using Sigma Stat 2.0 (Jandel Scientific). All

values are expressed as mean +- SEM.

a- Animal Study

WeekO Week 1 Week 8 *

Week 2

Rat chow AIN93M Diet AIN93M + lOppm Lycopene Diet

Experimental Period Acclimitization P e n d

* Al1 animals killed and semm and tissues collected.

b. Euman Study \

O Week znd Week * tith Week *

Washout Phase Treatment Phase

* Fasting Blood Collection for S e m

Figure 4.1. Study Design

4.3 Results

Rats fed a diet containing 10ppm lycopene for 6 weeks had a higher level of cis isomers

(36 - 75%) in the tissues and serum compared to the oleoresin used in the diet (7.74%).

Overall, this represents approximately a 75 % increase in cis isomer over oleoresin

(Figure 4.2.). Similarly when healthy human subjects consumed tomato products, the

serum levels showed a significantly higher percentage of czk lycopene at the end of 4

weeks (77.93 % total lycopene vs 45.68 % total lycopene, P<0.000 1 ) compared to its

level in the beginning of the study (Table 4.2.).

A diverse may of 1 ycopene cis isomers was detected by HPLC analysis of rat sera and

tissues, (Figure 4.3.) and human serum (Table 4.3.). Previous reports have indicated the

presence of various geometric forms of lycopene in plasma and tissues. Four peaks were

separated which were tentatively identified by their retention times as dl-pans, 9-cis, 13-

cis, and 15-cis isomers (Schmitz et al., 199 1; Stahl et al., 1992; Stahl and Sies, 199 1).

Similar peaks comparable to the retention times reporteà above were observed in this

study. This would prove that no autolysis or auto isomerization occurred during storage at

-70' C. Five different peaks were detected in this study and are represented according to

their retention times in Figure 4.3 and Table 4.3. Also, this study found the Iiver, heart

and prostate to have higher trans: cis lycopene ratios when compared to the other organs

(Table 4.1 .).

Oleaesin Spleen Liw Semm Prostate Heart

Rat Tbsues and Serun

Figure 4.2. CLÎ and Trans lycopene isomers in rat serum and tissues Values expressed are mean + SEM, n = 3.

Table 4.1. Relative proportion of tram to cis lycopene isomers in rat semm and tissues.

Truns : Cis

Spleen 1.61 : 1

Liver 0.77 : 1

Serum 0.59 : 1

Prostate 0.48 : 1

L

Heart 0.33 : 1

, :.18min; 12.01 11.61 10.86 1 13.48 10.48 , 10.21 11.59

=22min, 36.51 / 36.79 1 30.95 , 26.5 j 21.89 i 30.76 ' 20.61

;a36 min 1 36.35 21 -5 21.74 1 11.36 13.27 14.51 14.47

5-CÏS ' 26.77 34.72 i 39.2 51.47 43.04 48.39

Figure 4.3. Percentage of different cis isomers of lycopene in rat serum and tissues. AI1 bars are expressed as mean t SEM, n = 3. Only 5-cis lycopene has been identified so far, dl other cis isomers are expressed according to their retention time.

Table 4.2. Cis and Tram isomers of lycopene in human sera at the start and end of the study

Lycopene

Isomers

Values with letter ' b ' are significantly higher p<0.0001] than the values with letter ' a' at the 2nd week with a paired t-test. Values expresseci are mean + SEM, n = 17

Beginning of Treatment

Period 12"~ Week]

End of Treatment Period

16" Weekl

% Total

Lycopene

77.93 t 0.75 b

22.07 t 0.75 b

aM/L

8 10.6 a

688-62 b

AM-Tram

Cis

nM/L

2572.88 b

8932.10 b

% Total

Lycopene

45.68 f 7.07 a

54.32 + 7.3 a

Table 4.3. Profile o f total lycopene cis isomers and 5-cis isomer at beguining and end of study-

Cis Isomers

-

14 min

18 min

22 min

36 min

Except for 5-cis lycopene al1 other cis isomers are expressed according to their retention time. Values expressed are mean f SEM, n = 17.

At Zna week

meginning of Treatment Period]

{% Total Cis)

At tirn week

[End of Treatment Period]

( % Total Cis)

4.4 Discussion

Consumption of lycopene supplement in the form of oleoresin and tomato and tomato

products having 90 - 95 % dl-@ans lycopene increased the percentage of cis-isomer in

the body of both rats and humans, Previous animal studies that measurd lycopene in

tissues found cis-lycopene levels in the liver to be higher than levels in other tissues a k

lycopene supplernentation (Boileu et al., 1999; Ferreira et al., 2000). This study also

found the liver to be one of the three organs with high ci.-lycopene levels dong with

heart and prostate (Table 4.1 .). Since b e r is the f i s t organ in the body to receive

nutrients and also the major organ for fat storage, lycopene, which is transported via

chylomicrons afier their absorption fiom the gut, was observed to have the highest level

in the liver. Higher levels of cis-lycopene in the extrahepatic tissues suggest possible de

novo isomerization of lycopene transported fiom the liver. Very little information is

available about the levels of lycopene isomers in other tissues. In the present study the

Ievels of cis isomers observed were 55.7 k 0.10 and 66.6 i 2.14 % of total lycopene in

the liver and prostate tissues, in rats fed lOppm lycopene. These differences in the isomer

levels couid be due to differences in species of animals used, arnount of lycopene

administered, method of administration, medium of lycopene used, and duration of the

treatment.

The presence of different levels of cis-lycopene isomers in the tissues suggests either a

selective uptake of the carotenoid or the involvernent of tissue isomerase(s). Camtenoids

are carried in the blood by lipoproteins, particularly LDL (Erdman et al., 1993). It is

conceivable that the cis foms of lycopene are compactly packed ïnto the chylomicrons to

be transported to the portal and lymphatic circulation and tissues high in LDL receptors.

So far the pattern of tram to cis isomer of lycopene in the rat tissues has not been

identified. In this study, 5 different types of lycopene cis-isomers were observecl on the

basis of their retention times, with 5 4 s lycopene being the most predominant of them d l .

There is no information regarding the biological activity of 5-cis lycopene comparing it

to the other isomenc forms.

Similarly, the human study also demonstrates that conswnption of dl-tram lycopene

containing tomato and tomato products significantly increased the percentage of cis-

lycopene in the serum after 4 weeks. Other sîudies (Holloway et al., 2000; Sakamoto et

al., 1994) have also obsewed elevated plasma total lycopene and cis-isomers in human

subjects conswning processed tomato products.

Seven geometrical isomers of lycopene were partidy separated and identified in the

human tissues (Stahl et al., 1993). Similarly, four to five geometrical lycopene isomers

were identified in human plasma samples after consumption of tomato products (Krinsky

et al., 1990). In this study, five (5-cis, 14min, 18min, 22 min, 36 min) different cis-

isomers of lycopene were observed in tbe human serum. Research has shown that the

ratio of cis-trans isomers in biological fluids such as plasma and in tissues such as the

prostate gland, differ greatly fiom the ratios in fiesh tomatoes. In the human plasma the

percentage of d l -mns lycopene averaged only 4 1 % of total lycopene, 5-cis lycopene

reached 28%, 13-cis and 15-cis lycopene together amounted to 12% and other cis-

isomers werel6% (Schierle et al., 1997). Results from this study also showed a shift in

favor of the cis lycopene in the semm and tissue sarnples compared to tomato and tomato

products and the oleoresin. The predominant form of cir lycopene observed was the 5-cis

lycopene (Figure 4.3 and Table 4.3). The mechanism for the transformation of all-tram to

the cis lycopene in vivo and the resulting biological significance remain unknown.

Although lycopene clearance rates fiom the body have not been defined (Boehm and

Bitsch, 1999; Agarwd and Rao, 1998, Mueller et al., 1999) showed the biological half-

life of lycopene to be approximately 2-3 days (Stahl and Sies, 1996). Statistical analysis

was not done on the data-

In conclusion, this study demonstrated that intake of tomato and tomato products nch in

all-trans lycopene, significantly ïncreases the percentage of cis isomers in the body. The

mechanisms and site of lycopene isomerïzation and the biological significance are not

clear, further studies should be undertaken to address the role of lycopene in human

health.

5 . ABSORPTION OF LYCOPENE USlNG A RAT

CANNULATION MODEL

5.1 Introduction

There is considerable interest in carotenoids and their roles in the prevention and

treatment of several chronic diseases such as cardiovascular diseases, cancer, and other

age-related degenerative diseases. However, carotenoids are known to differ fiom each

other in metabolism, transportation, and tissue distribution, and are thought to be

specifically associated with certain diseases (Mayne 1996; Gaziano 1996); Dipiock

199 1). Both human epidemiologic (Giovannucci et al., 1995; Franceschi et al., 1994) and

clinical (Clinton et al., 1996) studies have suggested the possibility that the ïntake of

lycopene present in tomatoes d u c e the risk of cancer and cardio-vascular diseases.

Giovannucci et al. (1 995) demonstrated an inverse relationship between the dietary intake

of lycopene and the incidence of cancer of several sites including the prostate gland.

Furthermore, the presence of geometnc-isomeric forrns of lycopene in the diet is

rnarkedly different fiom that found in animal and human tissues and serum prompting

investigation to study isomerïzation during absorption in rats. Severai researchers have

also demonstrated differences in the patterns of lycopene isomers in the diet (tomatoes

and tomato products) and in human biological sarnples (Clinton et al., 1996; Schierle et

al., 1997).

The higher percentage of cis-isomers of lycopene in the tissues and semm is assumed to

be partly due to the consumption of heat treated tomato products where heating is seen to

increase the cis-isomers of lycopene (Stahl and Sies, 1992; Gartner et ai., 1997; Schierle

et al., 1997; van het Hof et al., 1998). We have also seen a similar increase in the

percentage of cis-isomers on heating (Chapter 3). These findings would suggest that the

61

hi& ratio of cis to tmns lycopene isomers in the human and animai biological samples

might have resulted during digestion or absorption. It is known that dietary lycopene is

mostly in the all-pans form but serum and tissues have high levels of cis-lycopene.

Studies have also shown that dl-pans lycopene is absorbed into the body. However,

whether lycopene is first isomerized to its cis form prior to its absorption is not yet

established.

There are several studies that have investigated the absorption and metabolism of B-

carotene (Wang et al., 1992; Hollander et al., 1978; Bloomstrand et ai., 1967; Gugger et

al., 1992) but very few regarding the absorption of lycopene. Clark et al. (1 998) have

dernonstrated that the rat is an appropriate animal model to study the absorption of

carotenoids without provitamin A activity. They have also shown that lycopene seemed

to reach steady-state-concentrations in the lymph by six hours and that its absorption was

not affected by the presence of canthaxanthin, another non-provitamin A carotenoid.

There are very few studies looking at the time course for the absorption of lycopene.

Lycopene being fat soluble, has a similar intestinal absorption pattern as that of fats. Bile

acids and fat help to solubilize the lycopene released form the food matrix. After

absorption into the intestinal cells via micelles, lycopene is transported into the plasma by

lipoproteins and chylomicrons fiom the intestinal mucosa to the blood Stream via the

lymphatics (Johnson, 1998).

The biologicai significance of lycopene isomerization is not well understood. The lymph

duct cannulation rat model has been used to study lipid and B-carotene absorption and the

conversion of carotenoids to vitamin A (Huang et al., 1965; Thompson et al., 1950).

62

Although Clark et al. (1998) have demonstrated that rats may not be a good model to

study carotenoids with pro-vitamui A activity because rats cleave them more efficiently

during absorption than humans, they are proven to be a good model to study non- pro-

vitamin A carotenoids such as lycopene that are absorbed intact. Lycopene reached

steady state concentrations in the lymph by 6 hours and the efnciency of the carotenoid

was not affected by the concentration infuseci. Lycopene and canthaxanthin did not affect

each other's absorption. However, the objective of this study was to establish the

absorption peak of lycopene in lymph-cannulated rats and to measure lycopene

isomerization.

5.2 Materials and Methods

Three fernale rats with a mean weight of 300 g at the time of the surgery were obtained

fiom Harlan S prague Dawley (Indianapolis, IN). The animals were housed individually

and were allowed fiee access to water and Pwina rat chow (Ralston Purina Company, St.

Louis, MO). All procedures involving the use of animals were according to the Canadian

Council on Animai Care. The Ethics Cornmittee for the use of animals, University of

Toronto, approved the protocol.

5.2.2 Surgical procedure

The rats were fasted ovemight (approx. 12 hrs) before the administration of lycopene.

Oleoresin, having 98% dl-tram iycopene, was dissolved in corn oil was given to the rats.

Rats consumed a diet containing LOOppm ( 100ppm is equal to 100 pg of lycopene/gm of

diet). The level of lycopene administration was selected based on previous studies in our

laboratory (Jaïn et al., 1999), and the mal1 amounts of lyrnph that can be collected from

the cannulated rat. Lycopene was gavaged to the rats and the surgical procedures started

at 2 hrs (timed according to pnor standardization procedures) afier gavaging the lycopene

dose. The rats were anesthetized with a gas inhalant, (Isoflurane 5 % induction) and

maintained at 2 % to allow preparation of the surgical site. Rats do not metabolize

isoflurane and therefore they recover quickly fiom the anestheticThe surgical procedures

for the mesenteric lymph duct cannulation were slightly rnodified as described by Hauss

et al. (1998). Isoflurane was used to maintain anesthesia (maintenance 2 %) because of

the early recovery of the rat following surgery. The mesenteric lymph duct was

catheterized using a 0.96 mm outside diameter x 0.58 inside diameter clear vinyl tubing

(Dural Plastics and Engineering, Australia) and secured with a small amount of surgical

glue (Vetbond, 3M Animal Care products). Just before cannulation, the cannula was

nnsed with heparin to prevent clot formation (10,0000 unitslml). During surgery, the

exposed intestines were covered with gauze soaked in saline to prevent drying. After

surgery, the rats were put into a plastic restrainer. Lymph was collected by &ravi& into a

glass test tube with EDTA every half hour for 8 '/z hours. The lymph was collected on ice

and stored at -70' C until analyzed. The tubing and the lymph were covered with a large

sheet of foi1 to prevent light induced isomerization. A red light was used to maintain the

64

body temperature d u ~ g the procedure. At the end of 8 1/2 hours, the rats were given

isoflurane and kïlled by exsanguination.

5.2.3.1 Total Lycopene analysis of lymph to determine the peak absorption time

Triplicâte lymph sarnples were analyzed fiom each time collection. To 100 pl lymph, 100

pl of 2-propanol (to denature the proteuis) was added in g l a s tubes and vortexed. About

500 p1 of extraction mixture (hexane:methylene chlonde, 5: 1, v/v) with 0.015% BHT was

then added to the tubes, vortexed and allowed to incubate for one hour in the dark at

room temperature. Dunng the one-hour incubation penod, the tubes were vortexed twice.

Tubes were then centrifuged at 4 O C for 10 minutes at approxirnately 2000 rpm. 400 pl of

the upper layer was aspirated and dried by flushing with nitrogen gas, dissolved in 100 pl

mobile phase containing Methano1:Acetonitrile:Methylene Ch1oride:Water

(700: 700 :200: 1 6) and 1 00 pI was injected. Total 1 ycopene peaks were analyzed using

reverse phase Waters HPLC using a ~ 1 ' 8 column and an absorbance detector at 470 nm

with a flow rate of Imlhin. An extemal lycopene standard was used to identiw and

quanti@ lycopene peaks (Rao and Agarwal, 1998a; Stahl et al., 1992).

5.2.3.2 Analysis of Lycopene Isomers in the Serum

The blood collected at the end of the experiment was centrifbged at 2000 rpm for 15 min

and the sera aspirated and stored at -20' C until analyzed the next day. S e m sample (5

ml) was extracted in glass tube with the addition of propanol. About 500 pl of extraction

mixture (hexane:methylene chloride, 5: 1, v/v) with 0.0 15% BHT was then added to the

65

tubes, vortexed and allowed to incubate for one hour in the dark at room temperature.

During the one-hour incubation period, the tubes were vortexed twice. Tubes were then

centrifüged at 4 O C for 10 minutes at approximately 2000 rpm. 400 pl of the upper layer

was aspirated and dried by fiushing with nitrogen gas, dissolved in 100 pl mobile phase

containing methanol and MTBE in the ratio of 62:38 v/v. Lycopene isomers were

analyzed using reverse phase HPLC using a C30 VYDAC 201HS54 column and an

absorbance detector at 472 nm with a flow rate of I d m i n . The lycopene isomers were

identified by their elution tirne (Clinton et al., 1996). However only semm from only one

rat could be used for isomer determination due to canndation surgery difficulties.

Statistica E Analysis

Statistical analysis of total lycopene peaks in the lymph and the different cis and tram

isomers in the senun were computed. Al1 statistical evaluations were completed using

Sigma Stat 2.0 (Jandel Scientific). AI1 values are expressed as mean f SEM-

5.3 Results

Lipid soluble carotenoids are released fiom the food matrix, emulsified into mixed bile

sa1 ts micelles, incorporated into chylomicrons and then into Iipoproteins which transport

them to the blood and tissues (Furr et al., 1997; Parker 1996; Olson 1994). The use of a

whoIe animal systern is necessary to assess the overall process of absorption of

carotenoids. Rat has been proven to be a good mode1 to study the absorption of non-

provitamin A carotenoids (Clark et al., 1998).

The current study uses oleoresin (an dl-wans lycopene) (Appendix C) dissolved in

tomato oïl to investigate its absorption, Previous animal and human experiments have

s h o w that when oleoresin con ta ihg 98 % dl-pans lycopene is fed, it results in a higher

percentage of cis-isomers in the blood and tissues of animals and also in hurnan serum.

Following a single dose administration of dl-pans lycopene, the highest concentration in

the lymph was observed between 6 to 6 $4 hours (Figure 5.1 .). GIise et al. (1998)

determined the kinetics of accumulation of B-carotene and Iycopene when separately

given in OF1 mice at a single dose of lûmgkg body weight or in combination. When

given a single injection of P-carotene serum peak reached at t = 2 hrs, detected in the

liver after 0.75 hours and in the spleen after 5 hours, with peak values of 1 0.46 +/- 0.62

and 134 +/- 6 pg/g tissue.

Serum was collected fiom the cannulated rat at five hours and at the end of the study. The

ratio of al1 tram - cis isomer profile of the rat serum after lycopene ingestion decreased

towards the end of the study (8 hours) when compareci to that seen in the middle of the

study (5 hours) (Al1 tram -cis, 0.58: 1 Vs 2.25: 1) (Table 5.1 .). Figure 5.2 shows the

decrease in a11-tram lycopene over time. The ratio of dl-mans to cis isomer of lycopene

was seen to decrease in the senim from the fifth hour to the eighth hour (Table 5.1 .).

O 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Time after Cannulation [Hl

Figure 5.1. Time course for lycopene absorption in mesenteric lymph duct cannulated rats. Al1 values are mean f SEM n = 3.

Tirne (H)

Figure 5.2. Ratio of dl-hansltotal cis-lycopene in rat senun over time. Values are fiom serum of one rat.

Table 5.1. Ratio o f lycopene isomers in rat semm over time

1 Time [Hours] 1 All-Tram : Cis

Values are fkom semm of one rat.

5.4 Discussion

Carotenoids and their role in the prevention and treatment of several illnesses have been

studied extensively in recent years. Different carotenoids have different metabolism,

transport, and tissue distribution characteristics, and are thought to be specifically

associated with certain diseases ((Mayne 1996; Gaziano 1996; Diplock 1 99 1). Using a

rat mode1 we observed peak lycopene concentration in the lymph between 6 - 6 % hours

after its administration of lycopene. Similar observation was reported by Clark et al.

(1 998) when lycopene was giving continuously to rats by intraduodenal infusion. Boileu

et al. (1 999) reported an increase in total lycopene in ferrets dong with an increase in the

cis-isomer percentage at 2 hours in the lymph when compared to the first hour afier

feeding lycopene (40 mgKg body wt). Majority of lycopene in tomatoes and tomato

products is present in its dl-tram geometric f o m (Krinsky, 1998; Nguyen and Schwartz,

1998) in contrast to the profile seen in biological tissues (CIinton et al., 1996; Stahl and

Sies, 1992).

Digestion and absorption processes disrupt the food matrix and allow lycopene to be

incorporated into micelles prior to absorption. Studies have suggested ck-lycopene to be

more bioavailable than the pans fonn (Boileu et aI., 1999). Lycopene levels in the blood

were shown not to be statistically different between men and women (Brady et al., 1997;

Olmedilla et al., 1 994). So far there is no data showing the difference in absorption of

lycopene between the sexes. There is very little data reporting the peak absorption time of

lycopene in rats. There was dso a significant (P<O.01) increase in the percentage of cis-

isomer in the lymph than in the original dose as well as stomach, and intestinal contents

71

of ferrets. Results fiom this study suggest difference in absorption of lycopene compared

with the observations of Boileu et al. (1999). This may be due to the efficiency of

lycopene isomerization and other factors related to the absorption of fat-soluble dietary

components. Factors other than lycopene isomerization can also a u e n c e its absorption.

Presence of lipids and other soluble cornpounds such as other carotenoids, vitamin E and

cholesterol, bile acids secretion and the nature of lipoproteins can al1 be important

factors,

It is significant to note that humans do not synthesize lycopene and the only source is

dietary. Animals also do not synthesize lycopene. A recently completed study in our

laboratory (unpublished) has shown the possible existence of a homeostatic mechanisrn

by which serum lycopene levels are maintauied at a steady-state level. Ingesting lycopene

on a daily basis will only contribute towards enhanceci in vivo antioxidant potential but

not result in any adverse effects.

Glise et al. (1998) due to mode of administration, was not able to detect s e m lycopene

in mice 0-24 hours after a single lycopene intraperitonial dose (1 Omg/kg body wt). In

contrast, Stahl and Sies (1992) have reporteci that humans reach peak serum

concentrations (>300 nmoVL) 24 and 48 hours after consuming heated tomato juice. In

the present study, the trans:cis ratio of lycopene in the semm is seen to change over time

after a single dose of lycopene. The transxis ratio decreased at the end of the study (8

hours) (2.22: 1 ) when compared to 5 hours afier gavaging (0.58: 1) (Figure 5.2.). This

decrease in the pans ratio may be due to the increased levels of the cis isomerk fonns.

However, since lycopene was collected in the lymph, it is possible that less amount of

lycopene reached the portal circulation. Lycopene is transported by chylomicrons into the

lymphatic and portal systems. Further studies are required to elucidate this point. The

peak level of radioactivity of lycopene in rat plasma has been reported to occur between 4

and 8 hours and between 8 and 48 hours in monkeys after oral administration of a single

dose of [14c] lycopene in olive oil. The liver was found to be one of the major depots of

lycopene in monkeys (Mathews-Roth et al., 1990).

In summary, the data presented established the peak absorption of lycopene to be around

6 - 6 % hours. The isomenc composition of the lymph could not be analyzed due to

insufficient lymph collection and surgical difficulties. It is possible that if the rat was

cannulated and then gavaged, the peak absorption would have been earlier as the rat was

fasted overnight and fasting increases the efficiency of absorption. It is possible that the

time taken for the cannulation procedure [about an hour] could have slowed down the

absorption of lycopene in the rat. However, the tram:cis ratio of lycopene in the semm

decreased in the end of the study although the oleoresin gavaged to the rat had 98% all-

tram lycopene. This is rnost likely because of enhanced solubility of cis-lycopene in bile

acid micelles and preferential incorporation into chylomicrons and efficient transport into

the portal circulation. It is also possible that lycopene exists in both human and animal

tissues as - 50% cis-lycopene because this mixture is the most stable and represents an

equilibrium between trans-lycopene and its isomers.

6 . GENERAL DISCUSSION AND FUTURE STUDIES

6.1 Discussion

The overall airn of this study was to evaluate the effects of physical and biological

factors influencing lycopene isomerization and to assess absorption. The overall

hypothesis is that dl-tram lycopene is isomerized to a certain degree to its cis isomers

under the conditions of processing and digestion and thereby facilitates the absorption

of lycopene. To accomplish the objective of this thesis, the following specific

objectives were undertaken.

3 To determine total, dl-pans and cis-isomers of lycopene in commercially

processed tomato products.

3 To evaluate the effects of heat and types of lipids on lycopene isomerization in

tomato juice.

3 To determine total and isomeric forms of lycopene in rat serum and human

serum.

3 To assess absorption and isomerization of lycopene in rats by !ymph duct

cannulation.

It is important to know about the bioIogicaI activity of Iycopene isorners. With this

understanding, tomato processing might be adopted to enhance those isomers that are

preferentially absorbed and utilized. in this study, the lycopene isomers were analyzed

in various tomato products and the lycopene supplement. The various tomato products

analyzed were obtained fkom the local supexmarkets. Previous study pubLished in our

lab show that lycopene is the main carotenoid in tomatoes and tomato products (Rao et

75

al., 1 998). Schierle et al. (1 997) reported dl-tram lycopene to be the predominant

geometrical isomer varying from 96 - 35 % of total lycopene, in a nurnber of tomato-

based products and meals. Similarly, the results of this study show that dl-pans

lycopene is the most predominant lycopene isomer in tomatoes and tomato products.

Lycored, the lycopene supplement and raw tomato and the tomato products had 5 - 10% cis isomers and 90 - 95% all-pans isomen. Several studies show that processing

affects the isomerization of lycopene (Lovric et al., 1970; Padula and Rodriguez-

Amaya, 1987; Godoy and Rodriguez-Amaya, 199 1; Schierle et al., 1997).

The effects of heat and oil on isomerization of lycopene in tomato juice revealed that

heating along with the type of fat used decreased the dl-tram isomer of lycopene.

Tomato products are normally subjected to M e r normal cooking processes dong

with oil and fat apart from the processing done in the industry. Tomato juice was

chosen because it has less fat used during canning than other tomato products. Heat

treatment with corn oil (PUFA) and butter (SFA) did not influence the trczns to cis

isomerization of lycopene to the same extent as olive oil (MFA). Studies show that

lycopene is better absorbed in the presence of olive oil than corn oil (Clark et al.,

2000). Similarly, in this study, the translcis isomerization of lycopene was

significantly greater on heating with olive oil than with other types of fat. Previous

studies reporting better absorption and bioavailability of lycopene with olive oil could

be due to the increase in the lycopene cis isomer percentage. The reason for the

increase in lycopene cis isomers on heating with olive oil remains to be elucidated.

The plausible reason could be the influence of structural differences of the different

oils on lycopene. The source of lycopene supplement used in this thesis was tomato

oleoresin containing 6% lycopene by weight. Lycopene, in tomato oleoresin, which is

a tornato extract, is suspended in oil. There are compounds other than lycopene present

in the oleoresin. For this reason, the possibility of the effects of other substances

causing a synergistic effect on isomerization cannot be excluded. Similarly, raw

tomato and tomato products may also contain other substances causing a synergistic

effect with lycopene.

The animal and the human study conducted in this thesis was part of a bigger study

examinhg the antioxidant properties of lycopene. A lycopene concentration of 10 ppm

was chosen for the animal study as it represents a dietary dose of two senings of

tornatoes or tornato products per day. This concentration of lycopene if consumed

daily would be equivalent to consuming 14 servings of tomato products per week.

Moreover, lycopene at this dosage was found to be bioavailable and distrïbuted to

various tissues in the body without any adverse effects. The levels of lycopene in

animal tissues observed in different studies seem to v a . depending on the amount of

lycopene given, method of administration and duration of treatment of lycopene.

There are a limited number of animal studies reported in the Iiterature to elucidate the

biological effects of lycopene partly due to a lack of knowledge concerning

absorption, metabolism and tissue distribution of lycopene. Animal studies that have

measured lycopene in tissues have found higher cis-lycopene levels in the liver than

levels in other tissues afier lycopene supplementation (Boileu et al., 2000; Ferreira et

al., 2000). Similarl y, this study has also found the liver to be one of the three tissues

with hi& cis-lycopene levels (heart and prostate being the other tissues). The liver had

the highest percentage of 5-cis lycopene than other tissues. The liver is the first organ

in the body to receive nutrients and also has a greater capacity to store nutrients

especially those that are lipid soluble. As most of the carotenoids including lycopene

is transported in the blood through lipoproteins, (Erdman et al., 1993; Parker, 1996)

the high lycopene levels in al1 the tissues in rats would indicate that there is effective

transfer of lycopene from plasma lipoproteins to tissues. It is conceivable that the cis

forms of lycopene are compactly packed into the chylomicrons to be transported to the

portal and lymphatic circulation and tissues high in LDL receptors (such as the heart

and liver) selectively accumulate cis-lycopene. Within a species, the serum and tissue

profiles are very similar. For instance in humans, just greater than 50% of semm

lycopene is in its cis form, the hurnan prostate has approximately 80% and the liver

has approximately 60% of cis lycopene (Clinton et al., 1996).

The different levels of cis-lycopene isorner in the different tissues would also indicate

that probably there is a selective uptake of the carotenoid and that perhaps a tissue

specific mechanism is involved or the presence of isomerase enzymes in certain

tissues causing the isomerization of lycopene. So far no study has evaluated the

presence of the cis-isomer patterns of lycopene in the rat tissues. This study shows the

presence of 5 different types of lycopene cis-isomers, with 5-cis lycopene being the

most predorninant of them all. There are no known standards so far for the detection of

cis-isomers of lycopene therefore the cis peaks are identified according to their

chromatography retention times. In this study, the cis-isomers in tomato products, rat

tissues, serum and human serum are identified according to their chromatography

elution times. More cis-pans lycopene isomer tissue distribution studies must be

perfonned before amving at standard tissue values.

Humans also tend to accumulate a wide array of cis-lycopene isomers in senun and

tissues even though the dietary lycopene is rnainly in the all-trans form (Clinton,

1998). The hurnan study in this thesis used a two-week washout phase for the subjects,

which was sufficient to reduce any lycopene present previously in the body to baseline

levels. The half-life of lycopene has been reported to be approximately 2-3 days (Stahl

and Sies, 1996). The lycopene dose for this study was chosen according to the dietary

survey conducted in our lab, which indicated that on average, Canadians consume

about 2Smg of lycopene per day (Rao et al., 1998). Previous studies in our lab

(unpublished) have shown little increase in total blood lycopene when 3(hng/day of

lycopene containing tomato and tomato products were consumed for two weeks. This

could be because the addition ofjust 5 mg of lycopene/day from the usual

consumption level of 25 mg/day rnight not have been sufficient to significantly raise

the blood lycopene levels. The treatrnent penod in this study was therefore modified to

4 weeks. In this study, the ratio of trans-cis isomers favoured the cis forms in the

serum. These results are consistent with the values reported by several researchen

(Schierle et al., 1997; Stahl et al., 1993; Stahl et al., 1992; Krinsky et al., 1990).

Recalling that tomatoes contain less than 10% cis-lycopene and more than 90% all-

tram lycopene, raises several questions regarding the stability of trans-lycopene in

foods but conversion to its cis form in the body. Why the prostate contains 80% cis-

lycopene while the liver has only 60% cis-Iycopene remains to be elucidated? The

mechanism for the transformation of dl-tram to the cis lycopene in the body and the

resulting biological significance rernain unknown.

The rats in the cannulation study in this thesis were gavaged with oleoresin having 6

% lycopene and the mesenteric lymph duct of the rat was cannulated and the Iymph

collected at regular intervals. The semm was also collected at mid point of lycopene

absorption and at the end of the study in order to analyze the trans:cis ratio of

lycopene isomers. The rat cannulation model was chosen because; rats have shown to

be a usefùl model to study the absorption of non-provitamin A carotenoids like

lycopene (Clark et al., 1998). Lycopene peak absorption was seen to reach around 6 to

6 K hours in the present study. This agreed with the results reported by Clark et al.

(1998). The trans: cis ratio decreased over time in the senun of rats analyzed in this

study (2.22: 1 at 5 hours vs 0.58: 1 at 8 hours). This decrease in the tram ratio may be

attributed to the increased formation of the cis isomerk foms. Because of surgical

difficulties, only one rat could be used for this analysis. Therefore more studies are

needed to elucidate this point. 1t would be interesting to examine the trnns: cis isomer

profile of the Iymph.

It is clear £iom the present studies that heat induces isomerization, and heating with

olive oil significantly inmeases the cis-isomers more than with other oils. This

increase in cis-isomer percent could be attriiuted to the increased bioavailability of

lycopene in the body in tomato products heated with olive oïl. Furthemore, the

presence of higher percentage of cis fonns of lycopene than the trans form in animal

tissues and human sera on tomato and tornato product consumption leads to an

assumption that the cis forms are more bioavailable than the tram forms and cis

isomerization occurs somewhere in the body. The assumption that cis isomers due to

their kinked forms may be better transporteci in the body cannot be ruled out. It is well

accepted that carotenoids are subjected to metabolism at various sites in the body,

including the liver and the intestine. Beyond this basic fact, the fate of certain

carotenoids once they have been absorbed is not clear.

6.2 Future Investigations

Several studies indicate the anti carcinogenic effects of lycopene. A recent study

(unpublished) conducted in our lab in CO-ordination with other departments showed

that al1 the lycopene cis-isomers have Iower ionization energy than the dl-trans form

even though each one of them have a different ionization energy (IE) value. Ionization

is the simplest form of oxidation and the resuIts suggest that cis-isomers are more

easily oxidized (in the thermodynamic sense) than the dl-pans counterpart implying

that cis-isomers are better antioxidants than the dl-trans fonn (unpublished).

However, M e r research is essential to elucidate the purpose of lycopene

isomerization and its biological significance in vivo.

Several questions regarding the bioavailability and nutritional significance of dietary

Iycopene arise out of the observations in this thesis. Does cis isomerization occur

dunng the chylomicron-mediated absorption of lycopene? Are cis isomers formed

8 1

because they can be more easily packed into the chylornicrons than the pans isomer

for transport? Are there any isomerase enzymes present in the gastrointestinal tract,

which induce the tram to cis isomerization? Why does the prostate giand have higher

cis isomers (80% cis-lycopene) of lycopene than the liver (60% cis-lycopene)? 1s there

a tissue specific uptake of cis isomers? Are there any tissue specific enzymes present?

Some projects of interest ire listed below.

3 It would be worthwhile to determine the effect of feeding rats with lycopene

containing the different fatty acids used to study the effect of fat and heat

treatment on lycopene, in this thesis. FoUowing a perïod sufficient for tissue

accumulation (at least 2 weeks), the lycopene isomer profile could be

determined. This could elucidate the point of the difference in absorption with

different fats.

P A lyrnph-cannulated mode1 could address the aspect of fatty acid profile and

lycopene isomer absorption. Analysis of the stomach and intestinal contents

would give a better idea on the site(s) of isomerization of the dietary dl-tram

1 ycopene.

P Tissue specificity studies of the protein (enzyme) responsïble for

isomerization, its isolation and identification. Tissue specific sites of lycopene

isomerization can also be studied.

P Synthesizing higher cis-isomers of dietary lycopene and evaluating its

biological significance by feeding it to rats.

P uicubating trans-lycopene with various tissue homogenates and looking at the

isomerization.

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APPENDIX A

Tomato Product Agenda

+3 The following chart indicates the tomato source you must have for each day

D ~ Y

Wednesday

Friday

Saturday

Sunday

Monday

Tuesday

2 cans of Ready-to-serve soup at lunch

&

12 pouches of ketchup at dinner

1 c m of tomato sauce and 2 pouches of

ketchup at dinner

1 bag of tomato puree at dinner

8 pouches of Ketchup at breakfast

&

2 Tbsp. of Tomato paste at lunch & 2

Tbsp. of Tomato paste at Dimer (discard

the rest)

O For your convenience, we have

provided you with a Tbsp. for

measunng the tomato product.

1 can of tomato juice at breakfast

1 c m of Tornato sauce and 2 pouches of

ketchup at dimer

1 bag of Spaghetti sauce at dinner

*:* The recipes are provided on each day schedule.

APPENDIX B

AIN-93M Purified rodent diet

1 Ingredient 1 GramslKg 1

I

Cornstarch DYETROSE Sucrose Cellulose Soybean oil

choline bitartrate I

1 2.5 1

I 465-7 155-0 100.0 50.0 40.0

1

AIN-93M Mineral mix (35- diet)

Vitamin mix L-Cy stine

10-0 1.8

Ingredien t

[ Manganous carbonate 1 0.63 1

Grams/Kg

Calcium carbonate Potassium phosphate, monobasic Potassium citrate Sodium chloride Potassium sulphate Magnesium oxide Ferric citrate, U.S.P Zinc carbonate

1 Cupric carbonate 1 0.3 1

357.0 250.0 28 .O 74.0 46.6 24.0 60.6 1.65

' ~okssium iodate Sodium selenate Ammonium pannolybdate. 4H20 Sodium metasilicate. 9H20 Chromium potassium sulfate. 12H20 Lithium chlonde Boric acid Sodium fluoride Nickel carbonate Ammonium vanadate Sucrose

0.0 1 0.0 1025 0.00795

1.45 0.275 0.0 174 0.08 15 0.063 5 0.03 18 0,0066 209.806

AIN-93-VX vitamin mir (10- diet)

Ingredient

Dyets Inc. Bethlehem, Pennsylvania.

Grams/Kg

Niacin Calcium pantothenate Pyridoxine HC1 Thiamine HC1 Ribo flavin Folic acid Biotin Vitamin E acetate (500 IU/gm) Vitamin B 12 (0.1 %) Vitamin A palmitate (500,000 IU/gm) Vitamin D3 (400,000 IU/gm) Vitamin K 1 /Dextrose mix (1 Omg/grn) Sucrose

1

I 3 .O 1.6 0.7 0.6 0.6 0.2 0.02 15.0 2.5 0.8 0.25 7.5

967.23

APPENDIX C

Tomato Oleoresin Composition

Lycopene Phytoene Phytofluene Tocopherol S ter01

Material Content (%)

Fatty Acids as glycerides Total unsaponifiable matter

Fatty Acid Profile

71.4 + 1.9

Water soluble matter Lactic acid Water content Phosphorus Phospholipids (estimated fiom phosphorus) Nitrogen Ash

1 Fatty Acids 1 % ofTotal Peak /

3.6 t 0.8 OS8 + o. 1 0.6 1 f; 0.2 0.43 +, 0.1 11.5 k2.1 0-21 kO.1 0-74 + O. 1

LycoRed Natural Products Industries Ltd.

97

Myristic Acid (14:O) Palmitic Acid (1 6:O) Stearic Acid (1 8:O) Oleic Acid (1 8: 1) LinoIeic Acid (1 8:2) Linolenic Acid (1 8:3) Arachidic Acid (20:O) Behenic Acid (220)

Area 0.52 + 0.02

22.65 ,+ 0.27 5.26 t 0.09 12.91 + 0.41 47.96 f 0.84 9.65 10.98 0.97 + 0.06

0.52

Sterol Contents

S tigmasterol Sitosterol

Carotenoids in Tomato OIeoresin

0.62 f 0-1 5 0.54 +r0.10

Campesterol Cholesterol

% of total OD at 1 47Znnr

0.54 t 0.1 5 0.23 + 0.07

Tocopherol Phytoene and Phytofluene Contents

Lycopene p-Carotene Oxidized products

96.5 10.6 1.8 + 0.5 1.8 k0.2

Compound "/O (w/w)

Phytoene Phytofluene Tocopherols

1 .O2 + 0.24 0.62 f 0.29 0.4 1 0.1