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THE PHYSICAL AND BIOLOGFCAL FACTORS THAT INFLUENCE THE ISOMERIZATION OF LYCOPENE · 2010-12-10 ·...
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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
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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