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EFFECTS OF THE CONSUMPTION OF FRESH AND PROCESSED TOMATO PRODUCTS ON BLOOD AND TISSUE LYCOPENE CONCENTRATIONS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Charlotte Moxley Allen, M.S.

*****

The Ohio State University 2000

Dissertation Committee: Approved by: Professor Steven J. Schwartz, Advisor

Professor John B. Allred

Professor Steven K. Clinton

Professor David B. Min

Professor Anne M. Smith UMI Number 9962371

UMI*

UMI Mlcroform9962371 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

The consumption of tomato products has been associated with a reduced risk of several cancers in epidemiologic studies. Lycopene, the predominant carotenoid in tomatoes, is hypothesized to be one component contributing to the health benefits of tomato products. This research has been designed to evaluate the effects of consuming various tomato products on the lycopene concentrations circulating in blood and in buccal mucosal cells (BMC) and/or human milk secretions.

Three distinct Clinical Studies were designed to evaluate the objective. The first compares blood and BMC lycopene after consumption of sauce made from two different tomato varieties; one predominantly 2\\~trans lycopene and the other predominantly tetra-cû lycopene. The second examined blood and BMC lycopene concentrations after standard, daily servings of three commercially available tomato products. The third investigated how blood, BMC, and human milk lycopene changed in a group of lactating mothers consuming either fresh tomatoes or processed tomato sauce.

The first study demonstrated an increase in total blood lycopene concentrations that was significant only when the tetra-cw lycopene sauce was consumed, although there was no change in BMC. We observed a 48% drop in total plasma lycopene during the lycopene free diet portion of the second study, and a significant increase over baseline o f90 -192% for all groups. BMC lycopene concentrations increased during intervention by 42 - 165%. Total plasma lycopene increased in the third study by 12% and 23% for fresh and processed groups, respectively, while the milk lycopene increase was significant for the processed group only. No change was observed in BMC.

This research demonstrates that daily servings of processed tomato products will increase blood lycopene to protective levels in less than 2 weeks. That increase can be observed in BMC tissues after 2 weeks. Cis lycopene isomers are absorbed from the diet and result in increased blood m-lycopene. Blood lycopene concentrations will increase rapidly in a group of lactating women when consiuning slightly higher than typical lycopene amounts in the diet. The transfer of lycopene from blood to breast tissue is rapid and is observed in milk secretions especially when processed tomato products are consumed.

til Dedicated to my parents Barbara and Lowell.

IV ACKNOWLEDGMENTS

I would like to express my sincere appreciation to my advisor, Dr. Steve

Schwartz, for the opportunity to develop a unique, non-traditional graduate program.

His flexibility in allowing me to tailor my graduate experience to my special circumstances has made the difference in my success. The members of my graduate committee. Dr. Anne Smith, Dr. John Allred, and Dr. David Min are acknowledged for their support and patience. I would like to extend special gratitude to Dr. Steve

Clinton who was always readily available to offer comment and support and whose editing input has been an invaluable leaming experience.

Special thanks to the members of the Haas Lab for their input and technical assistance. Moral support and motivation from Carla and Sari are especially appreciated, without them my experiment schedule would have been unbearable. My husband, Scott, has been a source of support and love through all the challenges of my graduate career as well as the first to celebrate my successes. To him, I am profoundly grateful. VITA

April 2,1969 ...... Bom - Aberdeen, MD

1987 ...... Graduated Aberdeen High School

1991 ...... B.S. Chemistry Virginia Commonwealth University Richmond, VA

1996 ...... M.S. Food Science and Technology Virginia Polytechnic Institute & State University, Blacksburg, VA

1996 - present ...... Graduate Research Associate The Ohio State University Columbus, OH

PUBLICATIONS

D. Wright, R. Caldwell, C. Moxley, and M. S. El-Shall. 1993. Homogeneous nucléation in supersaturated vapors of polar molecules: Acetonitrile, benzonitrile, nitromethane, and nitrobenzene. J. Chem. Phys. 98 (4): 3356-3368.

FIELDS OF STUDY

Major Field: Food Science and Nutrition

VI TABLE OF CONTENTS

PAGE ABSTRACT...... ii

DEDICATION...... iv

ACKNOWLEDGMENTS...... v

VITA...... vi

LIST OF TABLES...... x

LIST OF FIGURES...... xii

CHAPTER 1: LITERATURE REVIEW Carotenoid Introduction and Nomenclature...... 1 Biosynthesis ...... 2 cis-trans Isomerization...... 3 Analytical...... 7 Carotenoids in Food and Diet ...... II Metabolism...... 17 Potential Role in Human Disease...... 20 Antioxidant Mechanism and Action ...... 22 Animal Studies...... 24 Human Intervention...... 25 Objective...... 30 References...... 31

CHAPTER 2: Diets Rich in cû-Lycopene Increase Circulating cû-Lycopene Isomers in Humans...... 44 Abstract...... 45 Introduction...... 47 Subjects and Methods ...... 49 Results...... 54 vii Discussion...... 56 References...... 60

CHAPTER 3: Plasma Lycopene Concentrations Increase in Healthy Adults Consuming Standard Servings of Processed Tomato Products Daily ...... 70 Abstract...... 71 Introduction...... 73 Subjects and Methods ...... 76 Results...... 80 Discussion...... 84 References...... 92

CHAPTER 4: Blood and Milk Lycopene Isomer Concentrations Increase in Lactating Women Consuming Daily Servings of Processed or Fresh Tomato Products.. 102 Abstract...... 103 Introduction...... 105 Subjects and Methods ...... 107 Results...... 113 Discussion...... 116 References...... 122

DISSERTATION SUMMARY...... 129

APPENDIX A: Extraction Procedures...... 133 Lycopene Extraction Procedure from Tomato Fruit...... 134 Buccal Cell Procedure...... 135 Plasma Extraction...... 136 Milk Extraction Procedure...... 137

APPENDIX B: Supplements to Chapter 2 - Thngmne Study ...... 138 GCRC protocol ...... 142 IRB protocol ...... 153 Abstract for EB 1998 ...... 164

APPENDIX C: Supplements to Chapter 3 - Campbells Study ...... 166 Subject demographics ...... 168 BMI values...... 169 Buccal Proteins...... 170 Total Cholesterol Results...... 171 viii AICR Abstract 9/99 ...... 172 IRB protocol ...... 174 IRB revision 1/20/99 ...... 186 IRB revision 4/27/99 ...... 190 GCRC protocol ...... 192 Instructions for 3 day diet record book...... 203 Dietary schedule ...... 206 Intervention period - foods to avoid ...... 207 Washout period - foods to avoid ...... 209 Dietary Information Sheet ...... 210 Recipe Suggestions ...... 212 Consumption instructions Sauce...... 216 Soup...... 217 V8 Juice...... 218

APPENDIX D...... 220 Creamatocrit estimates of milk fat...... 222 Abstract EB 2000...... 225 OARDC grant ...... 227 IRB ammendments 4/16/98 ...... 239 6/18/98 ...... 240 8/12/99 ...... 241 Advertisement ...... 242 IRB protocol ...... 243

IX LIST OF TABLES

PAGE CHAPTER 2

Table 1. Peak maxima and retention time of select major carotenoids found in Roma and Tangerine sauces ...... 64

CHAPTER 3

Table I. Carotenoid Content of food Products as Consumed in the Study...... 96

Table 2. Mean Plasma Carotenoid Concentrations throughout the Study (pmol/L) ...... 98

Table 3. Spearman rank order correlations of lycopene concentrations in plasma and buccal mucosal cells for all treatment groups during the study ...... 101

CHAPTER4

Table 1. Spearman rank order correlations of lycopene concentrations in plasma and human milk after intervention for all treatment groups during the study ...... 128

APPENDIX B

Table 1. Plasma carotenoid concentrations before and after intervention...... 139 APPENDIX c

Table 1. Nutrient Intakes of Subject Population Based on diary Information...... 167

APPENDIX D

Table I. Plasma concentrations of major carotenoids in each treatment Groups...... 221

Table 2. Milk concentrations of major carotenoids in each treatment group expressed as nmol/L ...... 223

Table 3. Milk concentrations of major carotenoids in each treatment group expressed as nmol/g lipid ...... 224

XI LIST OF FIGURES

PAGE CHAPTER 2

Figure 1. C30 reversed phase separation of the Tangerine and Roma sauce extracts (tracings at 471 nm)...... 63

Figure 2. Representative structures of lycopene isomers...... 65

Figure 3. Tetra-cis lycopene before (top) and after (bottom) exposure to iodine and light ...... 66

Figure 4. Experimental design of the study ...... 67

Figure 5. Plasma lycopene changes after intervention ...... 68

Figure 6. Chromatograms of plasma extract at baseline (A) and after feeding Roma (B) and Tangerine (C) sauce in subject #693...... 69

CHAPTER 3

Figure 1. Representative Cig separation of plasma extract ...... 97

Figure 2. Total plasma lycopene concentrations during the course of the study ...... 99

Figure 3. Total buccal mucosal cell lycopene concentrations during the course of the study ...... 100

CHAPTER 4

Figure 1. Representative HPLC chromatogram (C30) illustrating the separation of carotenoids in human milk extract...... 125

XII Figure 2. Plasma lycopene changes after 3-day intervention with lycopene free diet (control), fresh tomatoes (fresh), or processed tomato sauce (processed)...... 126

Figure 3. Milk lycopene changes after 3-day intervention with lycopene free diet (control), fresh tomatoes (fresh), or processed tomato sauce (processed)...... 127

APPENDIX B

Figure 1. Tangerine tomato (LA 3002) extract at 437 nm (top) and a maxplot of 250-550 nm (bottom) ...... 140

Figure 2. Tangerine tomato sauce extract at 437 nm (top) and a maxplot of 250-550 nm (bottom) ...... 141

Xlll CHAPTER 1

LITERATURE REVIEW

Carotenoid Introduction and Nomenclature

The term ‘carotenoids’ refers to a group of pigments, yellow to red in color, which are widely distributed in nature. Carotenoids in a wide variety of plants and animals, including many fruits and vegetables, plumage of some birds, petals of flowers, and microorganisms are found in protein complexes, called carotenoproteins.

Carotenoids can be characterized as carotenes (hydrocarbon carotenoids) or xanthophylls (oxycarotenoids) (Isler, 1971). More than six hundred different carotenoids have been isolated from natural sources and characterized (Straub 1987).

In the plant kingdom, their presence is often masked by chlorophyll. They are responsible for the colors of many fruits such as: pineapple, citrus fhiits, tomatoes, paprika and rose hips. Normally, carotenoids occur in low concentrations, but this varies from source to source.

Carotenoid nomenclature begins with numbering of the basic C 40 structure at one end with 1 and continuing to the center with 15 (Isler, 1971). The opposite end numbering begins with 1 ’ continuing to the center with 15’. Methyl groups protruding from the backbone are numbered similarly 16 to 20 and 16’ to 20’. Oxygen containing groups vary among the xanthophylls but are commonly observed in the

3/3’ or 4/4’ positions.

Lycopene is the main carotenoid responsible for the red color of many tomato products and has been suggested as the main phytochemical responsible for the beneficial effects of tomatoes. It has no provitamin A activity. Lycopene is composed of isoprene subunits arranged in a system of 11 conjugated double bonds responsible for color (Stahl and Sies 1994). The absorption maximum for lycopene (472 nm, hexane) is dependent on the number of double bonds and on solvent influences.

Biosynthesis

Carotenoids are synthesized de novo by higher plants, some algae and bacteria

(Goodwin, 1971). Animals cannot synthesize carotenoids and obtain them by consuming other carotenoid-containing organisms. All carotenoids are biosynthesized from mevalonic acid to acyclic hydrocarbons, the predominant example is lycopene with 11 conjugated double bonds. A series of progressive desaturations, cyclizations, and hydroxylations in the case of the xanthophylls, yield the diverse array of carotenoid pigments (Eugster, 1989). The ability to absorb light in the visible region of the electromagnetic spectrum explains the diverse pigments. There are several unique characteristics common to all carotenoids: a polyisoprenoid structure, a long conjugated chain of double bonds in the central portion of the molecule, and near symmetry around the central double bond (Karrer and Jucker, 1950). They are extremely hydrophobic molecules with little or no solubility in water. As a result they are dependent on proteins and lipids for absorption, storage and transport in biological tissues (Erdman et al., 1993). The individual variations are derived from the parent

C40 carbon skeleton and can be modified by cyclization at one or both ends, by changes in hydrogenation level, and by addition of oxygen-containing frmctional groups.

The ripening of tomato fruit is a highly regulated process in which the color, flavor, aroma, and texture change. As the fruit ripens, the carotenoid content dramatically increases (Laval-Martin et al. 1975) by a massive accumulation of lycopene within the plastids and the disappearance of chlorophyll. Carotenoids play roles as essential photoprotectants in green tissues and as dispensable colorants in flowers and fruits. Chlorophyll can become excited in the photosynthetic system and react with oxygen to form singlet oxygen. The carotenoids prevent the formation of the damaging singlet oxygen by quenching the excited chlorophylls (Krinsky, 1979).

In fruit, carotenoids are also likely to aid in pollination by insects and animals that contribute to seed dispersal.

Cis-trans isomerization

Since carotenoids contain several carbon-carbon double bonds, a large number of geometric isomers (changes around the double bond) are possible. For example, a polyene with the formula R(CH=CH) 9 R’ can theoretically exist in five hundred twelve different cis-trans isomeric forms. The number observed in nature is considerably less, because methyl groups protruding from the polyene chain sterically hinder the

formation of many possible confirmations (Pauling, 1939). Although cis

configurations are generally unfavorable from a thermodynamic standpoint, di-, tri-,

and poly-cis carotenoids have been identified (Clough and Pattenden, 1979).

Isomerization can occur through exposure to light, heat, organic solvent, iodine and

other chemical compounds. The resulting mixtures of carotenoids are composed of

usually two or three predominant cis isomers. There are several common observations

of all the transformation products of natural tran^-carotenoids (Zechmeister, 1944).

• Color intensity decreases as a result of isomerization

• Isomerization products are more soluble than starting materials

• Melting points of cw-isomers are lower

• Isomerization products often revert to the /ran^-configuration on

crystallization.

• Cû-isomers always absorb at lower wavelengths.

• The cis- peak appears in the UV spectrum (320-360 nm range) and is

characteristic of the isomers.

• The c/5-peak is greater in magnitude the closer the cis bond gets to the

center of the molecule.

In nature, carotenoids are typically present in the al\-trans configuration.

However, there are many examples of cis carotenoid isomers in biological tissues.

4 Carotenoid cis isomers have been identified in human serum and in various organ tissues, including isomers of lutein (13-, 13’-, 9-, 9’-) (Krinsky et al., 1990), p- carotene (15-, 13-, 9-), and lycopene (Stahl et al., 1992).

There are a number of events that can bring about carotenoid isomerization.

Stability of P-carotene under different laboratory conditions was studied (Scita, 1992) and both UV and fluorescent lights were found to be damaging. Under dark conditions and mild thermal treatment an equilibrium mix of 66% all-trans, 8% 9-cis, and 25% 13-c/s was formed (Chandler and Schwartz, 1988; Pesek et al., 1990). This was due to the rate of formation of 13-cm being faster than that of 9-cis. The thermal and iodine catalyzed light isomerization of a- and p-carotene predominantly leads to formation of significant amounts of 13-cw isomer (Chen et al., 1994). The authors also demonstrated that the 13-C/5 isomer was preferentially formed during thermal treatment while the 9- and 15 cis isomers were formed at a faster rate during light induced isomerization.

For some time isomerization of fruit and vegetable carotenoids has been attributed to thermal treatment during food processing and preparation, p-carotene m-isomer content was reported to increase from 12.9% in firesh tomatoes to 31.2% in canned tomatoes (Lessin et al., 1997). Spaghetti sauce from canned tomatoes was reported to increase lycopene cis isomers (Schierle et al., 1997). An analysis of commercial tomato products, such as vegetable juice, tomato juice, tomato sauce, tomato soup, tomato paste, and pizza sauce showed that none had a total cis isomer content greater than 10.1% (Nguyen and Schwartz, 1998). Furthermore, the lycopene cis isomer content of the products did not reflect the amount of heat treatment received, which contrasted with the isomerization observed with P-carotene. The authors did demonstrate that lycopene isomerized readily in organic solvent and the amount ofcis isomers increased over time.

Improvements in analytical technology in recent years led to the discovery that lycopene in human blood and tissue is distributed among approximately 10-20 different isomers (Stahl et al., 1993). Interestingly, the ratio of lycopene cis-trans geometric isomers in biological fluids such as plasma and in tissues such as prostate differ from those isomer ratios in fresh tomatoes (Clinton et al., 1996). The typical

‘red’ tomato contains >95% all-trans lycopene, blood samples are approximately 60% cû-lycopene and 40% i[\-trans lycopene, while tissues such as prostate are as high as

80% cû-isomers. It has previously been assumed that the higher percentage of lycopene m-isomer in human biological samples is due in part to consumption of heat-treated tomato products containing cw-isomers of lycopene. It has been established that the majority of lycopene in tomato products is present as the all-trans form and is not substantially changed by food processing (Nguyen and Schwartz,

1998). An evaluation of tomato products of various moisture content, fat content, and container type, however, suggests that lycopene is remarkably stable to isomerization reactions under typical industrial thermal processing conditions (Nguyen and

Schwartz, 1998, 1999).

The vast majority of commercially available tomatoes are > 95% all-trans lycopene. Tangerine tomatoes {Tangella and Tangerine Golden Jubilee tomatoes) are a unique variety due to the biosynthesis of the tetra-m-isomer of lycopene and the absence of the 2\\-trans lycopene isomer. Prolycopene, tetra-cw lycopene, with four

(7,9,7’,9’) of its 11 double bonds in the cis conriguration, is the geometric isomer to d\\-trans lycopene (Zechmeister, 1962) and has an unique visible absorption spectra 35 nm shorter than the all-trans isomer. Also, it is the primary lycopene component in the Tangerine tomato having undergone a recessive mutation at the t locus giving it the orange color (Ulrich and MacKinney, 1968). Phytoene has been determined through stereochemical assignments to be the clear branch point for poly c/s-lycopene formation in the Tangerine tomato fruit (Clough and Pattenden, 1983). The stereochemical assignment given to prolycopene was confirmed through total synthesis of the pigment (Pattenden and Robson, 1987). It is not possible to distinguish prolycopene from all-trans lycopene with mass spectra, but the 'H and

NMR data of synthesized prolycopene have been identified (Mercadante et al., 1998;

Englert, 1995).

Analytical

Chromatography was discovered with the separation of plant chlorophylls and carotenoids (Tswett, 1906) and began to be widely used in 1931. Open column chromatographic separations are obtained using alumina, calcium hydroxide, or other adsorbents (Britton, 1985). Open columns are large in diameter and uniform packing is difficult to achieve, thus today the technique is most often used for purifying carotenoids (Britton, 1985). The current method of choice is high performance liquid chromatography (HPLC) in either the normal phase or reversed phase mode. HPLC separations have been reported with normal phase alumina (Vecchi et al., 1981), silica

(Rhodes et al., 1988), calcium hydroxide (Schmitz et al., 1995) and nitrile (Khachik et al., 1992b). By far, the reversed phase separations with C|g stationary phases are preferred (Craft, 1992). Maintenance of constant column temperature is reconunended to reduce variation in retention and selectivity with reversed phase separations (Craft, 1992).

Most recently, a polymeric C 30 stationary phase improves the reversed-phase

(RP) liquid chromatography separations of carotenoids and their geometric isomers

(Sander et al., 1994). Properties of the C30 column were specifically designed for the analysis of carotenoids, resulting in adequate retention of polar carotenoids and excellent selectivity toward both polar and non polar carotenoids, including structural and geometric isomers (Clinton et al., 1996; Craft, 1992; Emenhiser et al., 1995, 1996;

Sander et al., 1994). Geometric isomers of six conunon carotenoids were separated on the C30 column to demonstrate the especially good shape selectivity of this column over the Cis RP columns. Among the RP columns, the C30 is able to resolve the geometric isomers of asymmetrical carotenoids in which cis bonds are present at the same carbon number but at opposite ends of the molecule.

Carotenoid analyses are commonly performed by liquid chromatography with ultraviolet and visible absorbance (UV-vis) detectors operating at the maximum absorbable wavelength (Nollet, 1992). The wavelength of maximum absorbance is primarily dependent on the number of conjugated double bonds in the chromophore.

Tentative identifications can be established by comparison with the retention time of a standard and UV-visible absorption spectra since multiple spectroscopic tools are often unavailable. Diode array detectors can provide spectral information simultaneously for polar lipids, colorless carotenoids and colored carotenoids during a single HPLC run without interfering with the flow of solvent (de Leenheer and Nelis,

1992). This technology has proven extremely helpüil in compound identification and method development. A variety of methods for UV-visible detection of carotenoids in plant and animal tissue have been developed (Tonucci et al., 1995; Khachik et al.,

1991; Epier et al., 1993).

While diode array detectors are the most common tool used to tentatively identify carotenoids, mass spectrometry can be a useful addition to identify isomers of a particular carotenoid. Absorbance spectra often reveal the presence of a cis bond in carotenoid structures. This occurs as a result of increased absorbance in the near-UV region of the spectrum, approximately 142 nm below the absorbance maximum.

Asymmetric carotenoids with cis bonds at the same carbon number but on opposite ends of the molecule can not be distinguished by UV-visible absorption spectra.

Fractions that eluted from a C 30 column and assumed to be lycopene isomers were analyzed by electrospray mass spectrometry for molecular weights the same as all- trans lycopene and were found not to be oxygen-addition products (Emenhiser et al.

1996). To fully determine the structure of cis-trans carotenoid isomers ‘H-NMR is required to elucidate the presence and position of the cis bonds. The primary drawback is the large amount of purified sample needed for analysis. Eight cis- isomers of lycopene were synthesized and characterized by ‘H-NMR (Hengartner et al., 1992).

Interest in the analysis of carotenoids in biological tissues and serum has originated from research showing a possible link with intake of various carotenoid containing food and prevention of many types of cancer (Giovannucci et al., 1995,

1999; Clinton et al., 1996). Many studies have described methods for the separation and identification of carotenoids in blood components (Khachik et al., 1991; Khachik et al., 1992; Parker, 1993; Yeum et al,, 1996; Micozzi et al., 1992). Tissues that have been analyzed for carotenoid content include: adipose (Parker, 1993), prostate

(Clinton, et al., 1996), cervical tissue (Emenhiser et al., 1996), and buccal mucosal cells (Peng and Peng, 1992). While these methods of tissue analysis were primarily

HPLC-UV-vis detection methods, many were done on very large amounts of tissue or even cadaverous tissue. Analysis of microscale biopsies, necessary for subjects who are otherwise healthy, is essential to further understand the process of uptake of carotenoid nutrients by the body. Electrochemical detection (ECD) is a useful alternative to conventional UV-VIS detection methods for LC analysis of carotenoids

(Ferruzzi et al., 1998). Detection limits for P-carotene by ECD were measured at ten finole representing a 100- to 1000- fold increase over conventional LC-UV-VIS techniques.

Problems of accurately analyzing carotenoids in human milk samples are numerous and can be related to high variability in the sample compounded by the

10 presence of milk lipid concentrations high enough to interfere with HPLC analysis.

Many previous studies have measured values as total carotene and were unable to separate and quantify individual carotenes (Chappell et al., 1986; Ostrea et al., 1986).

Patton (1990) reported a method to separate and quantify carotenoids in colostrum.

This method has not been applicable to mature milk because it has a significantly higher level of lipid and lower concentration of carotenoids than does colostrum.

Saponification methods with 50% KOH to release the carotenoids from the lipid matrix found in mature breast milk is essential (Giuliano et al., 1992). The effects of time and temperature were found to profoundly effect the concentration of lycopene measured. A 68% loss of lycopene was observed with saponification in breast milk at

45° as compared with 4°. There was also a 25% reduction in the lycopene measured after 16 hrs of saponification as compared with 0.5 hr at 25°. These authors mention the need to add ethanol to denature milk proteins during saponification. An additional step was also required after extracting with hexane to remove water-soluble impurities

(KOH, polar lipids). Absolute ethanol and H 2O were added to the hexane extract to remove impurities

Carotenoids in food and diet

Carotenoids are widely distributed in fruits and vegetables making them the best dietary sources of these compounds. Quantitative results have been combined and tabulated for many fruits and vegetables conunonly consumed in the U.S. (Mangels et al., 1993). According to these data, the highest concentrations of P-carotene are

11 observed in carrot, sweet potato, apricot, and peach; of a-carotene in carrot and pumpkin; of lycopene in tomato, pink grapefruit, watermelon, guava, and apricot; and of lutein + zeaxanthin in dill, mustard greens, kale, parsley, and spinach. The presence and concentration of various carotenoids in a single finit or vegetable is dependent on cultivar/variety, maturity, and growing conditions (Gross, 1991).

Consumption of the cultivated tomato (Lycopersicon esculentum) is second only to the potato in the United States. A net total of 13.3 million tons of tomatoes were available for consumption and processing in 1995. Nearly 85% of this was consumed as juice, paste, puree, and sauce, with the remainder consumed as fresh tomatoes (USDA, 1995-1996). Tomatoes are modestly high in concentrations of folate (13 ug/lOOg) and vitamin A (743 lU/lOOg) with concentrations of folate found to be 1/10^ of spinach and vitamin A concentrations substantially lower than in carrots. Tomatoes are rich in potassium (279 mg/lOOg) and vitamin C (23 mg/lOOg) as compared with carrots and spinach. Lycopene is the carotenoid of highest concentration in tomato products followed by p-carotene, y-carotene, and phytoene as well as several other minor carotenoids (Tonucci et al., 1995). The vitamin A activity of tomatoes comes from p-carotene and y-carotene.

Yellow and orange fruits and dark green leafy vegetables are often promoted as a means to improve vitamin A status (Tang et al., 1999). Such foods are rich in provitamin A carotenoids, expecially P-carotene which is oxidatively cleaved to all- trans retinal and then reduced to retinol (Ong et al., 1994). Vitamin A (retinol) is essential for normal vision, growth, development, and differentiation of epithelial

12 cells. Several studies have shown an inverse correlation in risk of morbidity and mortality in children and vitamin A status (DeSole et al., 1987; Hussey and Klein.,

1990; Rahmathlluh et al., 1990). This is of particular concern in developing countries where the mother meets the majority of the vitamin A requirements for infants entirely from breastfeeding. Requirements are met by consumption of plant products (i.e., carotenoids) when the dietary supply of preformed vitamin A is limited. Several reports indicate that the amount of vitamin A in human milk decreases with maternal deficiency of the vitamin and increases with excessive intake (Butte and Calloway,

1981; Ajans et al., 1965). Increased intake of fruits and vegetables has been shown to be related to improved vitamin A status in many cross-sectional, case-control and community-based studies, but causality has not been proven (de Pee and West, 1996).

The provitamin A properties of some carotenoids have been the focus of many studies in human milk. P-carotene can be cleaved to vitamin A, but the relationship between vitamin A status and carotenoid concentration is not clear (Canfield et al.,

1995). Milk retinol concentration of mothers who took vitamin supplements (n=39,60 ug/lOOg) was found not to be significantly different from those who did not take supplements (n=15,49 ug/lOOg) (Kim et al., 1990) although the group size is unbalanced. Mean milk retinol concentration was highest in women with infants at one month of age and decreased at three-four months of age, but did not reach significance due to the small number of samples in each age group. The correlation with milk a-carotene was the greatest of all the carotenoids (r=0.52, p<0.01).

13 Beta- and alpba-carotene, lycopene, beta-cryptoxanthin and lutein are among tbe 36 carotenoids found in buman milk at about one-tentb tbe concentration found in serum (Giuliano et al., 1994). A recent study bas expanded tbe database to 34 carotenoids including 13 geometric isomers and eight metabolites in buman milk and serum (Kbacbik, et al., 1997). Tbe characteristic yellow color of buman colostrum is due to carotenoids (Goodwin, 1984). Adipose tissue of tbe breast is commonly observed at surgery to be yellow. Tbe bright yellow pigmentation of tbe fat globules comprising buman colostrum at parturition is evidence that carotenoids are concentrated and stored in tbe breast. Following a few days of breast-feeding, tbe color of tbe globules is greatly reduced. Tbe mean concentration of carotenoids in colostrum is 218 ^g/dL while tbe concentration in mature milk is 50 pg/dL (Patton et al., 1990). Qualitatively tbe carotenoids in mature milk are tbe same as those in colostrum, with tbe levels decreasing to ~50 pg/dL (Giuliano et al., 1992; Chappell et al., 1985). A steady and significant increase in tbe serum concentration of both p- carotene and vitamin E is observed in breast-fed infants from day two onward, while formula-fed infants experienced a significant rise only in vitamin E levels and not in

P-carotene levels (Ostrea et al., 1986).

Milk is complex and its major components include casein, a-lactalbumin, secretory IgA, lactoferrin, lysozyme, lactose, mono- and divalent ions, and triacylglycerol and other lipids in tbe milk fat globule (Neville, 1991). There are substantial differences between tbe milk of mothers at differing lengths of gestation.

Plasma concentrations of tocopberols, carotenoids, retinol and lipid are substantially

14 lower at birth (Kiely et al., 1999). No significant relationships were found between maternal plasma carotenoids and cord blood plasma carotenoids at birth in this study.

In another study, plasma concentrations of lycopene showed no correlations between maternal and cord plasma unless an adjustment for plasma triglycerides were made, making the correlation the highest of all the carotenoids (r=0.975, p<0.0001) (Yeum et al., 1998). Colostrum is the fluid secreted by the mammary immediately following parturition. The intense yellow color of colostrum is a result of high concentrations of

P-cryptoxanthin, p-carotene, lutein and zeaxanthin, and a-carotene. The carotene content of colostrum is about ten-fold higher than mature milk (0.34 to 7.57 mg/liter compared with 0.1 to 0.3 mg/liter, respectively (Patton et al., 1990)). Transitional milk is milk of the post colostral period (7-21 days post partum). Mature milk is all milk after 21 days. After birth the milk composition changes from a solution rich in sodium and chloride, immunoglobulins, and lactoferrin to a solution rich in lactose with only moderate protein levels (Neville et al., 1983). Additionally, content of human milk differs at the time of day when samples are obtained and especially with the diet of the mother. Lipid is by far the most variable with the total fat content of

24-hour milk samples varying from less than 20 g/L to more than 50 g/L (Milk

Composition, 1991). During the first three to four days of lactation, pre-term milk (the milk secreted by mothers who delivered prematurely) has higher protein, sodium and chloride concentrations and lower lactose concentrations than milk secreted by mothers of full-term infants.

15 Human milk is recommended as the optimal feeding choice for the first six months of an infant’s life (AAP, 1997). From six-twelve months, breastfeeding should be complemented with the introduction of solid foods. Benefits to the infant include fewer ear infections and less frequent incidence of diarrhea, respiratory illness, allergies and urinary tract infections than formula-fed infants (MCH Links, 1997).

Additional benefits from breastfeeding include immunologic, developmental, psychological, social, economic and environmental (AAP, 1997). Docosahexanoic acid is essential for brain and eye development and arachidonic acid is associated with immune function and infant growth and both are increased in human milk (Oski,

1997). Breastfeeding was associated with significantly higher scores of cognitive development than was formula feeding (Anderson et al., 1999).

Studies of feeding practices during the first year of life indicate that 98% of the infants bom in Africa, 96% of those bom in Asia, and 90% of those bom in South

America are breast fed for some part of the first six months of life (CHD, 1991).

Infant mortality rates among industrialized nations have Finland, Japan and Sweden first with only four deaths/1000 live births, and the United States ranks 21* with eight deaths/1000 live births (MCH Links, 1997). Breastfeeding has been shown to play an important role in preventing infant mortality particularly in developing countries.

16 Metabolism

The human body appears to absorb carotenoids by passive diffusion throughout the duodenal mucosal cells similar to the absorption of triglycerides and cholesterol (Parker, 1996). This includes release from the food matrix and incorporation into bulk lipid droplets. The particles are then broken down into small lipid micelles through the action of bile salts and pancreatic lipase. Carotenoids diffuse passively through the duodenum as determined by the concentration gradient.

Once in the intestinal mucosa cells, carotenoids undergo modifications and are incorporated into chylomicrons which enter the blood stream. In the blood stream, tissue uptake occurs giving rise to a chylomicron remnant. The liver absorbs the remnant where the carotenoids can be acted on in a number of ways (Parker, 1996).

They can be metabolized further to retinal, retinol, and apo-carotenals, or they can be repackaged in very low-density lipoprotein (VLDL) and resecreted in the blood stream. In plasma, carotenoids are distributed into LDL (|3- and a-carotene, and lycopene) and HDL (p-cryptoxanthin, lutein) (Vliet, 1996).

The mammary gland is a compound tubuloalveolar organ embedded in a cushion of adipose tissue. Milk secretion is continuous and results in the accumulation of milk in the mammary alveoli and ducts near the cells that secrete it.

Composition of milk is not altered by storage in the alveoli or passage through the ducts and is entirely determined by the mammary epithelial cells. Nearly all components of milk are derived from the blood. Triacylglycerols are synthesized in the mammary alveolar cell from fatty acids and glycerol derived either from the

17 plasma or from endogenous synthesis. Mammary tissue has the capacity for de novo synthesis of cholesterol, although it is not clear what portion of milk cholesterol arises from de novo synthesis and what portion from plasma (Neville et al, 1983). Lipid droplets accumulate in the cytoplasm of the mammary alveolar cell and migrate from the basal to the apical surface of the cell. The exocytotic process by which lipids are secreted by enterocytes and hepatocytes is not the same as the secretion process unique to mammary epithelial cells. When the lipid droplet reaches the membrane, a mammary-specific apical membrane protein, butyrophilin, interacts with the droplet to encapsulate and extrude it from the cell.

Fat is the main source of energy in milk and is among the most variable and difficult nutrients to measure accurately in human milk, both within and between individuals. Mature human milk has a 3.5% to 4.5% fat content. Within one woman, the fat content of milk increases from the beginning to the end of a single nursing, fat concentration being lowest in fore milk (1-2 g/lOOml) and gradually increasing to highest levels in hind milk (4g/100 ml) (Neville et al., 1983). The fat in human milk is different from that in the milk of other animals and is better absorbed by the infant’s gut (Emmett et al., 1997). Type of fat and amount in the maternal diet can effect the composition of the milk (Hall, 1979). The creamatocrit is a simple clinical technique for estimating fat concentration in human milk samples (Lucas et al., 1978).

Absorption efficiency of ingested carotenoids varies from as low as 1-2% to as high as 50% (Stahl and Sies, 1992; Goodman et al., 1966). There are a number of dietary factors that influence the absorption and utilization of carotenoids from foods.

18 The level and origin of dietary fat is important and a low fat intake has been shown to be the primary cause of vitamin A deficiency in some regions (Roeis et ai., 1958).

Vitamin E in megadoses can interfere with the conversion of p-carotene to vitamin A in the mucosal cell (Amrich and Arthur, 1980). Very high levels of dietary fiber, especially pectin, may result in reduced carotenoid utilization (Erdman et al., 1986).

Diets enriched with pectin, guar, alginate, cellulose or wheat bran significantly reduced plasma lycopene concentrations by 40-74% while only the water soluble fibers (pectin, guar and alginate) decreased the plasma P-carotene concentration by 33-

43% (Riedl et al., 1999). Adequate protein and zinc status is also required for absorption and or conversion of carotenoids to vitamin A (Simpson and Chichester,

1981). Ethanol has been reported to depress carotenoid bioavailability (Grummer and

Erdman, 1986). The absorption of carotenoids from uncooked foods can be as low as

1%, and moderate cooking can enhance absorption by increasing the digestibility of the food, carrots for example (Rock et al., 1998). Food processing degrades the plant cell constituents and decreases the particle size, which also enhances uptake.

Dietary fat stimulates bile flow from the gall bladder, which facilitates the

émulsification of fat and fat-soluble vitamins into lipid micelles within the small intestine. Without micelle formation, carotenoids are poorly absorbed. Humans transport carotenoids in blood plasma exclusively via lipoproteins (Clevidence and

Bieri, 1993). Under fasting conditions, up to 75% of plasma hydrocarbon carotenoids are found in LDL Auctions. Polar carotenoids are found more equally distributed between LDL and HDL fractions. The transfer of carotenoids from the liver to other

19 tissues is poorly defined but it's believed that tissues with a larger number of LDL receptors accumulate carotenoids passively with the uptake of large amounts of LDL

(Kaplan et al., 1990).

Potential Role in Human Disease

Epidemiologic studies have been responsible for drawing associations in populations around the world concerning eating habits and disease rates. As countries have developed economically, consumption of meat, meat products and dairy products has risen sharply with a consistent decrease in the consumption of foods of plant origin. These diets are lower in fiber and other bioactive compounds found in food of plant origin and higher in sugar (AICR, 1997). The National Cancer Institute estimates that 40% of all cancers can be attributed to diet and up to 70% can be influenced by diet. As preferences in diet have shifted away from those that are plant based, a corresponding increase in the incidence of many cancers has been observed.

An aggressive advertising campaign initiated by agencies in the U.S. Government has publicized guidelines recommending two-four servings of fruit and three-five servings of vegetables per day for optimal nutrition. It is widely believed that carotenoids are among the compounds responsible for the preventative effects observed with consumption of fruits and vegetables.

There are many observed actions associated with dietary carotenoid intake and in most cases the specific carotenoid function responsible for the observed activities

20 bas not been examined. The measurement of plasma carotenoids are useful indicators of vegetable and fhiit intake (Campbell et al., 1994; McEligot et al., 1999).

Epidemiologic evidence demonstrates that diets rich in fruits and vegetables are associated with a reduced risk of several human malignancies (Block et al., 1992;

AICR, 1997). Consumption of cooked vegetables and olive oil was shown to be inversely and significantly associated with the risk of rheumatoid arthritis (Linos et al.,

1999). Cumulative data from separate epidemiological studies has shown that individuals with the highest intakes of carotenoid-rich fruits and vegetables have the lowest risk for many cancers, including those affecting the lung, oral cavity, stomach and esophagus (Bendich, 1993). Most of the measurable carotenoids of human plasma can be increased by moderate alterations in diet within a short time, although the magnitude of the plasma response may be related to the baseline carotenoid concentrations (Yeum et al., 1996). Preliminary evidence indicates that p-carotene supplementation significantly reduces the progression of cardiovascular disease in physicians with preexisting disease (Gaziano et al., 1993). Studies have also found a significant association between carotenoid rich diets and lowered risk of cataracts

(Knekt et al., 1992). Lutein and zeaxanthin can quench singlet oxygen and may reduce the oxidative stress on lens proteins and may play a role in prevention of age- related macular degeneration (Seddon et al., 1994). Clinically, P-carotene has been used successfully to treat inherited light sensitivity disorders (Matthews-Roth, 1993).

Furthermore, the consumption of tomatoes and processed tomato products has been proposed to lower the risk of prostate and several other cancers (Giovannucci et al.

21 1995; Giovannucci, 1999). It has been hypothesized that lycopene may contribute to many of the health benefits of tomato products (Clinton, 1998). Approximately 85% of dietary lycopene, a non-provitamin A carotenoid, is provided by tomato products.

In the United States lycopene accounts for approximately 40% of the total blood carotenoids compared with 10% in Asians (Yeum et al., 1999). When examining tomato intake in 217 case-control and cohort studies of all types of cancer, 71% of the studies showed an inverse risk with consumption of tomatoes (AICR, 1997). Plasma lycopene concentrations are most correlated with lycopene intake (Mayne et al.,

1999). Lycopene is found in the human prostate and other tissues (Clinton et al.,

1996; Stahl et al., 1992; Kaplan et al., 1990; Schmitz et al., 1991). Tissue concentrations of lycopene are also associated with reduced mortality from cardiovascular disease (Klipstein-Grobusch et al., 2000). Greater blood lycopene concentrations are correlated with a lower risk of prostate cancer (Gann et al., 1999), bladder cancer (Helzlsouer et al., 1989), breast cancer (Dorgan et al., 1998) and heart disease (Klipstein-Grobusch et al., 2000).

Antioxidant mechanism and action

Generally speaking, antioxidant carotenoids prevent cancer and degenerative diseases by preventing oxidative damage of proteins, lipids, and DNA by affecting signaling mechanisms initiated by oxidative stress (Levy et al., 1999). Lycopene was determined to be the most efficient singlet oxygen quencher among the biologically

22 occurring carotenoids in-vitro (Di Mascio et a., 1989; Sundquist et ai., 1994).

Lycopene is at least three-fold more effective than P-carotene in quenching NOO

radicals which are present in tobacco smoke (Bohm et al., 1995). Lycopene and p-

carotene decomposed more rapidly when exposed to the in-vitro pro-oxidants NaOCl,

AIBN, and natural sunlight (Siems et al., 1999). Growth stimulation of mammary and

endometrial cancer cells by IGF-I is markedly reduced by physiologic concentrations

of lycopene (0.4-0.8 pM), demonstrating that inhibition of growth factor effects may

be one of the mechanisms by which lycopene exerts its effect as antioxidant (Sharoni and Levy, 1996). It has been suggested that lycopene may interfere at several points of cell cycle progression (Levy et al., 1995) and may also act synergistically with the active metabolite of vitamin D and with retinoic acid. Lycopene may act synergistically with other carotenoids to prevent oxidative damage to multilamellar liposomes (Stahl et al., 1998).

It is hypothesized that the disease preventative activity in tomato products is due to the potent antioxidant properties of lycopene (Clinton, 1998). Lycopene has also been shown to act as an in-vitro (Di Mascio et al., 1989) and in-vivo (Agarwal and Rao, 1998; Rao and Agarwal, 1998a, 1998b) antioxidant. Increasing dietary intake of lycopene in the form of spaghetti sauce (39 mg lycopene), tomato juice (50 mg lycopene), or tomato oleoresin (75 mg lycopene) has been shown to significantly decrease serum lipid peroxidation and LDL oxidation (Agarwal and Rao, 1998;

Fuhrman et al., 1999). It’s also increased serum antioxidant capacity, and possibly decreased the oxidation of protein and DNA (Rao and Agarwal, 1998a). Tomato

23 product consumption may reduce the susceptibility of lymphocyte DNA to oxidative damage (Pool-Zobel et al., 1997; Riso et al., 1999). A two week lycopene free diet significantly increased the serum lipid oxidation level by 25% as measured by TEARS with a corresponding 50% decrease in blood lycopene levels (Rao and Agarwal,

1998b). The same study showed a 25% decrease in serum lycopene levels following a meal as compared with fasting, suggesting that a meal induces metabolic stress that will utilize lycopene stores. The authors demonstrated that after smoking three cigarettes, serum lycopene levels decreased by 40% with a corresponding increase in

TEARS (40%). Consumption of a single serving of tomatoes was shown to decrease levels of mutagenic oxidized purine base 8-hydroxyguanine within 24 hours (Rehman et al., 1999). Increased consumption of vegetables and fruits resulted in a decreased level of 8-OhdG in DNA isolated from lymphocytes (Thompson et al., 1999).

Animai studies

Lycopene’s absorption and biological activity in rodent cancer models has been more recently investigated in view of the epidemiological studies that have promoted reduced risk from tomato product consumption. Crystalline lycopene added to the diet at very low levels (0.5ppm) significantly suppressed spontaneous mammary tumor development in mice (Nagasawa et al., 1995). Another study demonstrated no effect on mammary tumor incidence, latency, multiplicity, volume, or total tumors per group when rats were fed either pure lycopene or a mixed carotenoid oleoresin (Cohen et al., 1999). Lycopene has also been shown to inhibit colonic aberrant crypt

24 formation in rats (Narisawa et al., 1996). A study to better define the tissue uptake

and blood levels of lycopene and related carotenoids in male and female rats used

Betatene, a concentrated carotenoid mixture derived from tomatoes (Zhao et al.,

1998). Approximately 55% of ingested lycopene was recovered in the feces. The

major part of absorbed lycopene was stored in the liver with a distinct dose-related

uptake suggesting selective uptake by the liver. Lycopene was present in lung tissue,

mammary gland, and prostate tissue in levels that closely reflected dietary intake.

Serum concentrations did not closely reflect dietary intake. There was an increase in

hepatic vitamin E and glutathione levels in rats fed high levels of lycopene, suggesting

a possible decrease in overall oxidative stress in these animals. In vivo work with

lymph-cannulated ferrets has shown that c/5-isomers of lycopene are more

bioavailable than /rawj-lycopene (Boileau et al., 1999). The same authors use in vitro

work to suggest the reason for the increased bioavailability of m-isomers is their

increased solubility in bile acid micelles and their preferential incorporation into

chylomicrons. The dietary and metabolic variables influencing lycopene isomer

formation, clearance, and biological function remain to be elucidated.

Human intervention

The presence of lycopene in blood and various tissues has been demonstrated

(Stahl et al., 1992) for liver, adipose tissue, ovaries, kidney, adrenals and testes. The

25 concentration of lycopene in tissues was greater than that in serum for the liver, kidney, adrenals and testes.

Collection of buccal mucosal cells (BMC) is an easy and noninvasive method to measure tissue deposition of carotenoids. The intra-individual variability for BMC carotenoid concentration is small but the interindividual variability is large (Peng et al., 1994). Buccal mucosal cell lycopene levels increased approximately 25% after daily tomato juice consumption (Pateau et al., 1999) over 4 weeks but were not statistically significant. There have been conflicting reports correlating blood lycopene and BMC lycopene levels with some suggesting no correlation (Cooney et al., 1991,

Pateau et al., 1999) or good correlation (Peng et al., 1995). These studies based their correlations on samples of the general population without dietary intervention or estimated intakes. With the intervention study making its comparison at one time point at the end of four weeks of feeding. Several factors influence the lycopene amounts sampled in BMCs. The layer of buccal mucosa removed in the first sampling may contain a much older population of cells than that obtained in successive samplings. These older cells may have lost carotenoids through diffusion and/or chemical breakdown (Cooney et al., 1991).

Very little information is available on lycopene clearance rates from blood in individuals consuming a lycopene free diet. One publication reports that the plasma half-life of lycopene is approximately two - three days (Stahl and Sies, 1996). A two- week washout has been reported to produce a 50% reduction in blood lycopene levels over baseline (Bohm and Bitsch, 1999; Agarwal and Rao, 1998) while another study

26 found no decrease in blood lycopene concentrations after washout of two weeks

(Muller et al., 1999).

Little research has thus far focused on lycopene absorption, distribution, and biological activity on defined diets in humans. Studies suggest that lycopene from fresh tomatoes is not as effective for increasing blood lycopene concentrations as lycopene from processed tomatoes (Porrini et al., 1998; Gartner et al., 1997). Adding lipids to tomato juice increased blood lycopene concentrations after a meal (Stahl and

Sies, 1992) suggesting that the type and quantity of lipid consumed may influence uptake into blood and tissues. Supplementation (Smg lycopene/day) with tomato oleoresin or tomato juice significantly increased plasma lycopene while it remained unchanged with intake of raw tomatoes (Bohm and Bitsch, 1999). Daily consumption of 40 mg lycopene as tomato juice increased the blood concentrations 2.4 fold over two weeks (Muller et al., 1999). Blood lycopene was significantly increased after four weeks of consumption of 70-75 mg lycopene/day as tomato juice, tomato oleoresin or lycopene beadlets (Pateau et al., 1998), however significant increases in buccal mucosal cell lycopene were only observed after consuming oleoresin or beadlets (Pateau et al., 1999).

Lycopene is primarily transported via low-density lipoproteins that also carry the bulk of cholesterol in the plasma. It becomes necessary to investigate the relationship between plasma lycopene levels and plasma cholesterol levels. Many epidemiologic studies (Mayne et al., 1999; Ascherio et al., 1992; Vogel et al., 1997;

Brady et al., 1996) have found significant correlations between plasma lycopene levels

27 and plasma cholesterol levels. These studies contained large subject pools (n=l 11 to

600) with a median age of 6Sy. The mean total cholesterol reported in the epidemiologic studies ranged from 5.2 - 6.1 mmol/L. Studies that have correlated each lipoprotein fraction with serum or plasma lycopene values have found that plasma lycopene values correlate highly with LDL and HDL cholesterol (Perugini et al., 2000). Consistent with the studies that correlate plasma lycopene and cholesterol levels is the negative correlation of age to plasma lycopene in a much older population

(>50y).

Synergistic effects of the various carotenoids have been examined. Large doses of P-carotene has been shown to affect the absorption and clearance of canthaxanthin (White et al., 1994) and lutein (Kostic et al., 1995). Lutein and p- carotene were cleared from plasma at approximately the same rate but P-carotene significantly reduced the serum lutein to 54-61% of control (Kostic et al., 1995).

Another study showed that in the presence of high amounts of P-carotene, there is a preferential uptake of lutein and zeaxanthin in chylomicrons (Gartner et al., 1996). A recent study has shown that ingestion of a combined dose of P-carotene and lycopene has little effect on the absorption of P-carotene but improves that of lycopene in men

(Johnson et al., 1997).

Since very few supplementation studies have been performed with carotenoids in lactating women, there are many aspects of the effectiveness of this approach to be investigated. The kinetics of the response of milk and serum to P-carotene supplementation has been investigated (Canfield et al., 1998). Short-term

28 supplementation of healthy, lactating mothers with purified P-carotene substantially

increased milk and serum P-carotene concentrations but did not effect the concentration of other caroteniods or retinol. Kinetics of milk uptake and decay of P- carotene paralleled those in serum.

Supplementation studies with either 60 or 210 mg P-carotene showed that average serum P-carotene concentrations increased 4.1 and 4.0 fold respectively, and average milk P-carotene concentrations increased 4.1 and 3.0 fold respectively

(Canfield et al., 1997). The same study showed that a single 60 mg supplement of P- carotene did not effect concentrations of other major carotenoids, retinol or a- tocopherol. In another study, either 64 mg of all-tran^ P-carotene or 69 mg of 9-cis P- carotene were fed to lactating women and levels of each geometric isomer was measured in milk, blood serum, and buccal mucosa cells. Bothall-trans P-carotene and 9-cis P-carotene levels in serum, milk and buccal cells increased significantly over baseline by the end of the supplementation period of eight days (Johnson et al., 1997).

The study seems to provide evidence that there is no difference in tissue uptake of all- trans P-carotene and 9-cis P-carotene.

Carotenoids present in the highest concentrations in human milk are lycopene and P-carotene at 31.2 and 45.9 nM respectively (Giuliano et al., 1994). In the same study, several observations were made about the inter/intra-individual variability in major carotenoids of mature human milk, a-carotene and P-carotene concentrations were shown to vary as much as 20-fold between individuals and studies that intend to

29 measure concentrations of these carotenes should have large sample sizes. Lycopene and P-cryptoxanthin varied the most within individuals and are strongly correlated with lipid content. It’s recommended that repeated measures over time are necessary to accurately determine breast milk lycopene and p-cryptoxanthin.

Objective

Numerous studies have recently addressed the ability of dietary intake to positively affect lycopene concentrations in plasma and tissues. The current studies have been most commonly in the form of pilot studies, or small studies of less than 10 subjects. Most frequently the interventions are for a short period of time and the amounts of lycopene consumed are far in excess of what can be considered reasonable dietary intake. Conflicting reports on the effectiveness of equivalent dietary intakes may be resolved if an adequate washout precedes intervention. Many facets of lycopene cis-trans isomerization in plasma and tissues remain to be explored and defined. The research that follows is an attempt to address some of these issues and further the understanding of lycopene absorption and transport in the human body. The studies will focus on utilizing commercially available tomatoes and tomato products to mirror reasonable dietary lycopene intake levels. The objective is to determine the effects of consuming reasonable dietary levels of tomato products on lycopene concentrations in blood, buccal mucosal cells (BMC), and human milk.

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43 CHAPTER 2

Diets Rich in cis-Lycopene Increase Circulating c/s-Lycopene Isomers in Humans.

Charlotte M. Alien', Mario G. Ferruzzi', Minhthy L. Nguyen', Ni Luh Fuspitasari-Nienaber', David Francis^, Steven K. Clinton^, Steven J. Schwartz'*.

'Department of Food Science and Technology The Ohio State University 140 Hewlett Hall, 2001 Fyffe Ct, Columbus, Ohio 43210-1096

^ Department of Horticulture and Crop Science The Ohio State University, Columbus, Ohio

^Comprehensive Cancer Center Arthur 0. James Cancer Hospital and Solove Research Institute The Ohio State University, Columbus, Ohio

44 ABSTRACT

Background - Although tomatoes and processed tomato products contain lycopene almost exclusively in the trans configuration, blood and tissue samples contain mostly ci5 -isomers of lycopene. The mechanisms accounting for the transformation from all- trans to c/5 -lycopene in the body and the resulting biological significance remain unknown.

Objective - This study examines blood and buccal mucosal cell (BMC) lycopene concentrations in subjects who have consumed either a high cû-lycopene variety tomato

(Tangella and Tangerine Golden Jubilee, LA 3002) or the more typical d\\-trans lycopene variety tomato {Roma, Ohio 8245).

Methods - Treatment groups consumed either tomato sauce prepared from Tangerine tomatoes (2.9 mg tetra-m lycopene/serving) or tomato sauce prepared from Roma (8.2 mg all-trans lycopene/serving). Two servings per day (70 g/serving) were fed to 10 healthy volunteers for 4 days in a crossover design with 14 days washout (lycopene free diets) prior to the study and 14 days between different dietary regimens. Plasma and

BMC lycopene isomer concentrations were determined at the beginning and end of each

4-day feeding period.

Results - Total blood lycopene concentrations rose (pmol/L, mean ± SEM) from 0.639

± 0.038 to 0.653 ± 0.046 (2.1%) and from 0.556 ± 0.046 to 0.733 ± 0.065 (24.1%, p <

0.001) for the groups consuming Roma and Tangerine tomato sauce, respectively.

When expressed as a ratio of cisUrans isomers, a significant increase was observed in the % cis composition with consumption of Tangerine sauce only (from 55% cis to 65%

45 cis, p < 0.05). No significant changes were observed during this short feeding study in the BMC lycopene pattern.

Conclusion - This study shows that the consumption of dietary cû-isomers of lycopene from Tangerine tomatoes rapidly alters circulating patterns of c/ 5 -isomers, demonstrating that the tetra-cû form of lycopene is absorbed from the diet.

Key words: lycopene, prolycopene, isomer, Tangerine tomato; 7,9, 7’, 9’ tetra-cw lycopene

46 INTRODUCTION

Lycopene has recently emerged as a potentially beneficial dietary phytochemical in light of accumulating research findings showing an inverse correlation between consumption of food products high in lycopene and the risk of developing certain types of cancer (1). The consumption of tomato products at the rate of one serving per day is related to a lower risk of prostate and other cancers ( 2 ,3) and greater blood lycopene concentrations have been correlated with a lower risk of prostate cancer (4). Clinton et al. (S) observed that the ratio of lycopene cis-trans geometric isomers in biological fluids such as plasma and in tissues such as prostate differ from those isomer ratios in fresh tomatoes. It has previously been hypothesized that the higher percentage of lycopene cis- isomer in human biological samples is due in part to consumption of heat treated tomato products containing cis- isomers of lycopene or production of cis isomers in vivo by unknown mechanisms. Our laboratory has established, however, that in tomato products of various moisture content, fat content, and container type, lycopene, unlike |3-carotene, is remarkably stable to isomerization reactions under typical industrial thermal processing conditions ( 6 , 7).

The mechanisms for the transformation of rra/i 5 -lycopene to cis in the body and the resulting biological significance remain unknown. In vivo work with lymph- cannulated ferrets has shown that c/ 5 -isomers of lycopene are more bioavailable from the diet than rra/i 5 -lycopene ( 8 ). The same authors use in vitro studies to suggest that the increased bioavailability of cisr-isomers results from increased solubility in bile acid micelles and their preferential incorporation into chylomicrons. Daily consumption of

47 40 mg lycopene for two weeks in the form of tomato juice, primarily dX\-trans lycopene, substantially increased the cû-lycopene content in plasma (9). In work from this laboratory ( 1 0 ), we observed a significant increase in cû-lycopene isomers after two weeks on a lycopene free diet (p < 0 .0 0 1 ) that corresponds to a significant decrease in al\-trans lycopene (p < 0.001). After four weeks of feeding daily servings of processed tomato products, the isomer ratios had returned to those observed pre-study.

The vast majority of commercially available tomatoes are > 95% all-trans lycopene. Tangerine tomatoes {Tangella and Tangerine Golden Jubilee tomatoes) are uniquely suited to further study lycopene isomer absorption and distribution in humans due to their biosynthesis and accumulation of a tetra-cû-lycopene isomer and the absence of the aW-trans lycopene isomer. Prolycopene, tetra-cw lycopene, with four (7,

9 ,7’, 9’) of its 11 double bonds in the cis configuration, is a geometric isomer of all- trans lycopene (11) and has a visible absorption spectra 35 nm shorter than the all-trans isomer. Also, it is the primary lycopene component in the Tangerine tomato having undergone a recessive mutation at the / locus giving it a distinctive orange color ( 1 2 ).

Phytoene has been determined through stereochemical assignments to be the metabolic branch point for tetra as-lycopene formation in the Tangerine tomato fruit (13). The stereochemical assignment given to tetra ds-lycopene was confirmed through total synthesis of the pigment (14) and ‘H and '^C NMR data (15, 16).

The objective of this research was to compare lycopene geometric isomer concentrations in blood and buccal mucosal cell of subjects who have consumed sauce

48 made from tomatoes high in dXX-trans lycopene, with blood and BMC from subjects who have consumed sauce made from high tetra m-lycopene tomatoes.

SUBJECTS AND METHODS

Ten (5 male and 5 female) healthy, non-smoking subjects aged 20 - 40 y completed the study. The Institutional Review Board of The Ohio State University approved all procedures and all subjects gave written consent. A crossover design was used where two weeks o f‘washout’ or a lycopene free diet proceeded the first intervention period. Half the subjects were randomly assigned the Roma tomato sauce group with the other half assigned to the Tangerine tomato sauce group. Subjects consumed 2 servings/day (70 g sauce/serving) for 4 days and a total of 65.5 mg all-trans lycopene and 23.9 mg tetra-c« lycopene from Roma and Tangerine, respectively.

Serving sizes were determined by the limited amount of Tangerine tomatoes available for processing. The Hirzel Canning Corporation, Toledo, OH, donated the Roma tomato sauce. Tangerine tomatoes were supplied through the Department of

Horticulture and Crop Science and processed into sauce at the Department of Food

Science and Technology pilot plant at The Ohio State University. Amount of fat was standardized at 10% for both sauces in the ratio of 2:1 olive oil: vegetable oil. Subjects went through a second two-week washout period prior to crossing over to the opposite treatment group. Subjects were instructed to consume no other sources of lycopene for the course of the study.

49 Amounts of lycopene were determined using the extraction procedure of

Nguyen and Schwartz ( 6 ). Briefly, the sauce was weighed and extracted 3x ( 1:1

hexaneracetone), the organic layers were combined and saved, and the extinction

coefficients at 471 nm (1870) and 438 nm (1030) were used to determine the

concentration of ali-trans and tetra-cis lycopene, respectively. An external standard

(Sigma, St. Louis, MO) was used for the aW-trans lycopene. For use as a calibration

standard the tetra-cw lycopene was isolated from raw tangerine tomato by a modiHed

open column method of Rodriguez-Amaya (17). A magnesium oxide/celite column of

12 cm X 22 mm was packed under reduced pressure. The column was topped with 1 cm

of anhydrous sodium sulfate and 2 cm of sea sand. Approximately 3 - 5 ml of

Tangerine tomato extract was loaded on the column and developed with hexane under

vacuum. The tetra-c« lycopene isomer was identified by UV-Vis spectrophotometry.

Purity was determined by HPLC and found to be > 92%.

Blood and BMC sample collection

Blood and BMC samples were drawn late morning by trained phlebotomists at

the General Clinical Research Center (GCRC) of the Ohio State University, Columbus,

OH before the intervention and on day S. BMC samples were then obtained via the

method of Peng and Peng (18). Subjects rinsed their mouths with tap water and discarded the rinse. They then brushed both sides of their cheek with a soft toothbrush

20 times on one side and rinsed with 20 ml of rinse solution (0.4 g table salt and SO ml tap water). The rinse solution was deposited in a 50 ml collection tube coated with 50

50 of 1% butylated hydroxytoiuene (BHT) in methanol. The rinsing was repeated once and the toothbrush was swirled in the remaining rinse solution that was added to the collection tube. Samples were immediately placed on ice and shielded from light.

Sample processing

All samples were processed under dim light within 2 hours of collection. Blood samples were centrifuged (1400 x g, 10 min.) into cells and plasma, and 500 pi aliquots were stored in liquid nitrogen until analysis. BMC samples were centrifuged (1400 x g,

10 min.) and the supernatant discarded. The remaining cell pellet was then washed with

15 ml cold phosphate buffered saline (PBS) (Sigma, St Louis, MO) and vortexed. The sample was centrifuged again (1400 x g for 5 min.), the supernatant discarded, 1.2 ml of cold PBS added, and vortexed. One ml was removed and placed in a 2.0 ml BHT coated microcentrifuge tube and 2 -100 pi aliquots were placed in 15 ml conical tubes for protein determination (Bradford method, Bio-Rad Laboratories, Hercules, CA). The microcentrifuge tube was centrifuged (1 0 , 0 0 0 x g, 1 ,0 min.) after which the supernatant was discarded and the cell pellet flushed with nitrogen gas and stored at -80°C until analysis.

Sample extraction

Carotenoids were extracted from BMCs (18) by adding 200 pi of protease solution (100 mg protease/10 ml PBS) to the thawed cell pellet. The sample was vortexed and incubated at 37*^C (30 min.) in a water bath. Following digestion, 500 pi

51 of SDS-ethanol solution (1.0 ml 20% SDS-water solution added to 19.0 ml ethanol) was added and the cell pellet dispersed (Pellet Pestel, Fisher, Pittsburgh, PA). The sample was extracted with 500 pi of hexaneracetone (2:1) containing 0.2% BHT, vortexed, centrifuged (10,000 x g, 1 min) and the hexane layer removed and saved. The hexaneracetone extraction was repeated once. The combined hexane layers were evaporated to dryness under a stream of nitrogen gas.

Five hundred pi of plasma was deproteinated by addition of 500 pi of ETOH-

0.1% BHT (w/v). Carotenoids were extracted with two 1 ml portions of hexaneracetone

(2rl) containing 0.2% BHT, vortexed, centrifuged (1000 x g, 1 min) and the upper phase removed and saved. The combined extracts were evaporated to dryness under a stream of nitrogen gas.

HPLC analysis

HPLC analysis of plasma was carried out using a Waters 2690 system (Milford,

MA) equipped with a 996 photodiode array detector. A YMC (Wilmington, NC) C 30 reversed phase column (4.6 x 250 mm, 3pm polymeric), a guard column packed with

C18 stationary phase (Vydac, Hesperia, CA) and a pre-column filter (0.5 pm) were used for separation. Both sample (25°C) and column (28°C) were thermostated during analysis. Separations were achieved using a gradient elution method with different concentrations of MTBErl.5% of 1 mol/L ammonium acetate (AA) in ME0HrH20.

The following gradient was used: 0 to 25 min 6 8 % MEOH-AA, 1% H 20,31% MTBE;

52 25 to 38 min a linear gradient to 47% MEOH-AA, 1% H 2 0 ,52% MTBE; hold 17 min then re-equilibrate to beginning solvent.

BMC analysis was performed by the method of Ferruzzi et al. (19) with modifications. Chromatography was carried out using a Hewlett Packard 1050 solvent delivery system (Santa Clarita, CA), autosampler, and solvent prep station. A 4 channel

ESA Coularray™ detector (Chelmsford, MA) was used with potential settings of 220 -

520 mV in 100 mV increments from channel 1 - 4. A YMC (Wilmington, NC) C30 reversed phase column (4.6 x 250 mm. Sum polymeric) and a guard column packed with Ci8 stationary phase (Waters, Milford, MA) were used for separation. An isocratic solvent system was used with 42% solvent A (95 MEOH: 3 MTBE: 2 AA) and 58% solvent B (20 MEOH: 78 MTBE: 2 AA).

Standard curves were constructed for a-carotene, P-carotene, tetra-cw lycopene, and al\-trans lycopene using external standards. The remaining cû-lycopene isomers were estimated using the extinction coefficient for 2A\-trans lycopene.

Statistical analysis

Data were analyzed using StatView 5.0 (SAS Institute, Cary, NC) software. All

the data were combined for each group so that n = 10 for each group. Data were normally distributed based on skewness and kurtosis and were not log transformed.

Descriptive statistics were used to compute means and SEMs. Pre and post intervention concentrations were compared with paired t-tests. Differences between groups were

53 determined with an ANOVA and Tukey’s/Kramer post - hoc test, A p < 0.05 was considered significant

RESULTS

Chromatographic separation of the Tangerine and Roma tomato sauces are illustrated in Figure 1. The most profound differences are the minute amount of the all- trans lycopene isomer and the predominance of the tetra-m isomer in the Tangerine variety. Representative structural diagrams of lycopene geometric isomers are shown in

Figure 2. The Tangerine tomato contains many more carotenoid substances than the

Roma (Table 1). Differences include the larger amounts of P-carotene, phytoene, and phytofluene present in the Tangerine variety. Lycopene and its isomers typically elute with a retention time of > 30 min, so the Tangerine tomato has approximately 82.5% of

Its lycopene in the tetra-cw form, only 0.8% d[\-trans, and 16.7% as other c«-lycopene isomers. The process of making sauce changed the relative isomer distribution to

56.8% tetra-cM, 4.0% sW-trans, and 39.2% other cû-isomers (Figure 1 and 2 in

Appendix B, p. 136-137). Open column separation of the Tangerine tomato extract isolated the relatively pure tetra-cw lycopene component (~ 92% purity). Under iodine isomerization (15 min) the tetra-c» component completely isomerized to other lycopene isomers, including the all-trans isomer (Figure 3). The peak maxima of the resulting lycopene isomers range from 440 to 471 nm and corresponding retention time shift of >

30 min.

54 Both washout periods (Figure 4) resulted in similar baseline lycopene concentrations. Total blood lycopene concentrations increased (Figure S, mean ± SEM) from 0.639 ± 0.038 to 0.653 ± 0.046 (2.1%) and from 0.556 ± 0.046 to 0.733 ± 0.065

(24.1%, p < 0.001) pmol/L for Roma and Tangerine treatment groups, respectively.

Plasma concentrations of total cw-isomers rose from 0.303 ± 0.039 to 0.396 ± 0.046

(23.4%, p < 0.005) and decreased from 0.354 ± 0.028 to 0.336 ± 0.032 (5.1%) pmol/L after consumption of Tangerine and Roma sauce respectively (Table 1 in Appendix B, p. 135). The al\-trans isomer rose from 0.284 ±0.015 to 0.317 ± 0.022 (10.4%, p <

0.05) pmol/L after consumption of Roma sauce and remained essentially unchanged at

0.256 ±0.010 pmol/L in the Tangerine group. Representative chromatograms of plasma extracts from the same subject at baseline and after feeding Roma and Tangerine sauce are shown in Figure 6. The all-trans lycopene increase is evident after consumption of

Roma (B) as well as its subsequent decrease after consumption ofTangerine (C). After consumption of Tangerine (C), the presence of the tetra-cir isomer is demonstrated as well as the increase in other cw-lycopene isomers from 35 - 40 min. When plasma amounts of phytoene and phytofluene were examined as areas under the curve (AUC), mean AUC of phytofluene did not change significantly for either treatment group. Only

3 subjects had detectable plasma phytoene before treatment. After treatment with

Tangerine sauce, phytoene was detected in all subjects with a mean ± std o f40036 ±

17778 AUC. Consumption o f Roma sauce did not change phytoene levels.

Before intervention, all subjects had approximately 55% cis and 45% all-trans lycopene isomers circulating in blood. The intervention significantly (p < 0.05) changed

55 the isomer ratio in the Tangerine group with an increase to 65% cis and a decrease to

35% dAl-trans lycopene but did not significantly alter the isomer ratio in the Roma sauce treatment group. No changes were observed over baseline in the BMC’s.

DISCUSSION

This study demonstrates that consumption of dietary c/5-isomers of lycopene from Tangerine tomatoes rapidly alters circulating patterns of cis isomers. These findings suggest that m>isomers are absorbed and are further isomerized to other cis forms in vivo. The increase in total blood lycopene was significantly higher when the high c/5-lycopene sauce was consumed, suggesting preferential absorption of these geometric isomers, agreeing with previously reported findings (8).

The isolated tetra-cû fraction of the Tangerine tomato remained stable in hexane for up to 1 hour at room temperature shielded from light; thus the hexane extraction from plasma did not contribute to further isomerization of the tetra-cw isomer since the procedure is quick (< 10 min). Previous studies have shown that after heating at 120°C for 10 min, the conversion of tetra-cfj lycopene to other lycopene isomers was 25% and after 2 hours exposure to the sun it rose to 100% (12). In our study, when the Tangerine tomato was processed in to sauce, we observed approximately 25% conversion of tetra- cis to other c»-isomers. When iodine was added to the purified tetra-cis extract and the sample exposed to light, the tetra-cis lycopene was isomerized (100%) to other cis- lycopene isomers as well as the all-franj isomer (Figure 3). Zechmeister (11) utilized open column methods to separate and quantitate the components of the Tangerine

56 tomato. Their research showed a similar peak spectrum for the tetra cw isomer as well as the increased peak maxima of the isomers after exposure to iodine, an unusual observation since isomerization usually results in compounds with decreased peak maxima values. The C30 separation of the iodine treated isomers has enabled us to provide additional information on the individual isomer spectra and retention time which closely match the lycopene isomers detected in blood.

Although lycopene clearance rates fi'om blood have not been clearly defined (20,

21,9), one publication reports that the plasma half-life of lycopene is approximately 2 -

3 days (22). We have demonstrated a 50% reduction of lycopene in plasma after 2 wks

(10). In the present study, the washout period of 14 days was effective at decreasing the total lycopene concentrations to within the initial baseline range. The washout period measured in this study is the one between treatments. It is also important to note that the tetra-cM isomer in blood of subjects who consumed Tangerine sauce as the initial treatment was completely cleared before the second dietary treatment was administered.

The dietary intervention was effective at demonstrating for the first time a significant increase in circulating cû-lycopene after 4 days of consuming Tangerine tomato sauce containing primarily tetra-cis lycopene. Previous studies have shown little increase in total blood lycopene when 5 mg/d for 7 days was consumed in the form of tomato juice (20). Since the feeding period was short (4 days) and the amount of lycopene fed was small (16 mg/d), it was expected that blood lycopene concentrations would not be significantly increased on consumption of Roma sauce. When mainly the tetra-cis isomer was consumed {Tangerine sauce), a significant increase (24%) was

57 observed in total lycopene concentrations. This is important since the treatment groups consumed approximately half as much tetra-crs in the Tangerine sauce as they did all- trans in the Roma sauce. The tetra-cû isomer was detected in plasma after consumption of Tangerine sauce, and this is the first study to demonstrate the presence of that isomer in plasma.

Research has shown that the ratio of lycopene cis-trans geometric isomers in biological fluids such as plasma and in tissues such as prostate differ drastically from those isomer ratios in fresh tomatoes (5). Figure 6 shows the plasma lycopene isomer pattern observed after consumption of Tangerine sauce was very similar to the pattern observed after iodine isomerization of the purified tetra-cû compound (Figure 3). The intervention significantly (p < 0.05) changed the isomer ratios in the Tangerine group with an increase to 65% cis and a decrease to 35% 2X\-trans lycopene but did not significantly alter the isomer ratios in the Roma group. The mechanism for the transformation from al\-trans to cis lycopene in the body and the resulting biological significance remains unknown. In vivo work with lymph-cannulated ferrets has shown that m-isomers of lycopene are more bioavailable than rra/ts-lycopene (8). The same authors use in vitro work to suggest the reason for the increased bioavailability of cis- isomers is their increased solubility in bile acid micelles and their preferential incorporation into chylomicrons. Prostate tissue may contain as much as 85% cis- lycopene as compared with 60% cû-lycopene commonly observed in plasma or serum

(5) and the increase in cû-lycopene may contribute to the protective effect lycopene is believed to have on reducing the risk of prostate cancer.

58 It has been suggested that concurrent consumption of lipid will increase lycopene bioavailability from a meal (23). The amount of fat in each of the sauces in this study was standardized at 10%. Subjects were free living with the exception of the

‘no lycopene’ requirement for the course of the study and dietary composition was not monitored. With the amount of fat in the sauce held constant, we can assume that minor variations in lipid intake among volunteers did not influence the results observed.

Plasma lycopene response is often measured as an indicator of bioavailability, but it does not necessarily reflect absorption and deposition into tissues. Collection of buccal mucosal cells (BMC) is an easy and noninvasive method to measure tissue deposition of carotenoids. BMC lycopene concentrations increased (-25%) after tomato juice consumption (24) with 4 weeks of daily consumption but were not significant. Another study (10) showed an increase in BMC lycopene concentrations after consumption of 21 mg/day lycopene as tomato sauce. Our study did not show any change in the BMC lycopene concentrations after 4 days consumption.

In conclusion, this study has demonstrated that cis isomers of lycopene in the diet can subsequently be detected circulating in plasma. Consumption of dietary cis- isomers of lycopene from Tangerine tomatoes rapidly alters circulating patterns of cis- isomers. The increase in total lycopene blood concentrations was significant when the high c/5-lycopene sauce was consumed, suggesting that the absorption of cis isomers is more efficient. Future studies will have to address the formation, interconversion, and biofunction of lycopene before the reason for the different isomer ratios in biological samples are understood.

59 REFERENCES

1. Block G, Patterson B, Subar A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 1992;18:1-29.

2. Giovannucci EL, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. Intake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer Inst 1995:87:1767.

3. Giovannucci E. Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst I999;9I(4): 317.

4. Gann PH, Ma J, Giovannucci E, Willett W, Sacks FM, Hennekens CH, Stampfer MJ. Lower prostate cancer risk in men with elevated plasma lycopene levels: results of a prospective analysis. Cancer Res I999;59(6): 1225.

5. Clinton SK, Emenhiser C, Schwartz SJ, Bostwick DG, Williams AW, Moore BJ, Erdman JW. Cis-trans lycopene isomers, carotenoids and retinol in the human prostate. Cancer Epidemiol Biomark Prev 1996;5:823.

6. Nguyen ML and Schwartz SJ. Lycopene stability during food processing. PSEBM 1998;218:101-105.

7. Nguyen ML and Schwartz SJ. Lycopene: chemical and biological properties. Food Technol I999;53(2): 38-45.

8. Boileau AC, Merchen NR, Wasson K et al. Cis-lycopene is more bioavailable than trans lycopene in vitro and in vivo in lymph-cannulated ferrets. J Nutr 1999;129:1176-1181.

9. Mueller H, Bub A, Watzi B, Rechkemmer G. Plasma concentrations of carotenoids in healthy volunteers after intervention with carotenoid-rich foods. Eur J Nutr 1999;38:35-44.

10. Moxley C, Schwartz S, Craft N, DeGroff V, Giovannucci E, Clinton S. Blood lycopene concentrations increase in healthy adults consuming standard servings of processed tomato products daily. Presented at American Institute for Cancer Research Meeting. Sept 2-3,1999. (abstr). 60 11. Zechmeister L eds. Cis-trans isomeric carotenoids vitamins A and arylpolyenes. 1962.

12. Ulrich JM and MacKinney G. Photoconversion of prolycopene in the tangerine tomato. Photochem Photobiol 1968;7:315-318.

13. Clough JM and Pattenden G. Stereochemical assignment of prolycopene and other poly-z-isomeric carotenoids in fruits of the tangerine tomato Lycopersicon esculentum var. Tangella. J Chem Soc Perkin Trans 1 1983;12:3011-18.

14. Pattenden G and Robson DC. Total synthesis of prolycopene. Tetrahedron Lett 1987;25(46):5751-4.

15. Mercadante AZ, Britton G, Rodriguez-Amaya DB. Carotenoids of yellow passion fruit(PassiJIora edulis). J Agric Food Chem 1998;46( 10):4102-4106.

16. Englert G. NMR spectroscopy. In Carotenoids Vol IB: Spectroscopy. Britton G, Giaaen-Jensen S, Pfander H eds. Girkhauser:Basel, 1995:147-260.

17. Rodriguez-Amaya DB. Critical review of provitamin A determination in plant foods. J Micronutrient Anal 1989;5:191-225.

18. Peng Y-S and Peng YM. Simultaneous liquid chromatographic determination of carotenoids, retinoids and tocopherols in human buccal mucosal cells. Cancer Epidem Biomark Prev 1992;1:375-382.

19. Ferruzzi MG, Sander LC, Rock CL, Schwartz SJ. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Anal Biochem 1998;256:74-81.

20. Boehm V and Bitsch R. Intestinal absorption of lycopene from different matrices and interactions to other carotenoids, the lipid status, and the antioxidant capacity of human plasma. Eur J Nutr 1999;38:118-125.

21. Agarwal S and Rao AV. Tomato lycopene and low density lipoprotein oxidation: a human dietary intervention study. Lipids 1998;33(10):981-984.

22. Stahl W and Sies H. Lycopene: a biologically important carotenoid for humans? Arch Biochem Biophys 1996;336:1-9.

23. Stahl W and Sies H. Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J Nutr 1992; 122: 2161-2166. 61 24. Pateau I, Rao D, Wiley ER et ai. Carotenoids in human buccal mucosa cells after 4 wk of supplementation with tomato juice or lycopene supplements. Am J Clin Nutr 1999;70:490-494.

62 0.08 Roma sauce

0.04

0

A Tangerine sauce U 0.012

lycopene isomers

0.06

0.0

20 40 60 minutes

Figure 1. C3 0 reversed phase separation of Tangerine and Roma sauce extracts, (tracings at 471 nm).

63 Carotenoid Retention Peak % of total % of total % of total % of total time maxima carotenoids in lycopene in carotenoids lycopene in (min) (nm) Roma sauce Roma sauce in Tangerine Tangerine sauce sauce Lycopene 96.3 64.5 all-bians 52 471 87 0.8 iycopene tetra-cû 18.9 437 0 56.8 lycopene other cis- 30-50 440-470 13 39.2 lycopene Phytoene 11.2 286 3.1 22.3 Phytofluene 12.4 346 nd 9.3 P-carotene 20.5 454 0.6 3.9

Table 1. Peak maxima and retention time of select major carotenoids found in Roma and Tangerine sauces. nd = not detected M-trans lycopene

5-cfs lycopene

a ■ 7,9,9%7lycopene tetra-c/s lycopene JL

Figure 2. Representative structures of lycopene isomers. AU cw-lycopene isomers

5-cis

20 40 60 minutes

Figure 3. Tetra-c/s lycopene before (top) and after (bottom) exposure to iodine and light

66 Tangerine sauce Tangerine sauce n=5 n=5 5 males 5 females Roma sauce Roma sauce n=5 n=5

14 day washout 4 days feeding 14 day washout-2 4 days feeding

Figure 4. Experimental design of the study. 0.25

0.2 a Roma O Tangerine 0.15

o E 3 < 0.05

•0.05 J total tetrads ali-trans other ds lycopene lycopene lycopene lycopenel

Figure 5. Plasma lycopene changes after Intervention. ^ CIS isomer concentrations extimated using extinction coefficient for ali-trans 0 = significant difference from baseline p < 0.05

68 ® P-carotene lycopene isomers

CM

m S-cis

tetra-cii

CO a-carQtene g o>

CM in c*> a> in

10.00 20.00 30.00 40.00 50.00 60.00 70. Minutes

Figure 6. Chromatograms of plasma extracts at baseline (A) and after feeding Roma (B), and Tangerine (C) sauce in subject #693.

69 CHAPTERS

Plasma iycopene concentrations increase in healthy adults consuming standard servings of processed tomato products daily.

Charlotte M. Alien', Steven J. Schwartz', Neal E. Craft^, Valerie De Groff^, Edward L. Giovannucci^, Steven K. Clinton^.

'Department of Food Science and Technology The Ohio State University, Columbus, Ohio

^ Craft Technologies, Inc., Wilson, NC

^Comprehensive Cancer Center Arthur G James Cancer Hospital and Solove Research Institute The Ohio State University, Columbus, Ohio 43210

^Department of Nutrition The Harvard School of Public Health, Charming Laboratory, Department of Medicine Harvard Medical School, Boston MA

70 ABSTRACT

Background: The consumption of tomato products has been associated with a reduced risk of several cancers.

Objective: A study was designed to determine plasma and buccal mucosal cell

(BMC) lycopene concentrations in healthy adults consuming standard daily servings of three processed tomato products: spaghetti sauce, tomato soup, or vegetable juice.

Design: Thirty-six subjects (n = 36,18 male and IS female, ages 18 to 65) consumed a lycopene-free diet for two weeks (washout period) in order to determine lycopene clearance rates. Participants were then assigned to one of three (n = 12,6 male, 6 female) intervention groups consuming daily, single standard servings of sauce (21 mg lycopene/1/2 cup), soup (12 mg lycopene/1 cup), or juice (17 mg lycopene/8 oz) for 4 wk without other sources of lycopene. Fasting blood and BMC samples were obtained at enrollment and weekly thereafter for HPLC analysis of carotenoids.

Results: Total plasma (n = 36) lycopene concentrations (Mean ± SEM) decreased from 1.05 ± 0.07 to 0.54 ± 0.05 pmol/L (p < 0.0001) during the washout period. In all intervention groups, plasma lycopene concentrations increased rapidly for 2 weeks and remained stable from 2-4 weeks. Plasma lycopene concentrations for those consuming sauce, soup, and juice increased over the level at the end of the washout period to 2.08 (192%, p < 0.0001), 0.91 (122%, p < 0.0001), and 0.99 (92%, p <

0.0001) pmol/L, respectively. After 2 weeks of intervention, the effect of treatment on plasma lycopene concentrations was statistically significant for sauce (p < 0.01) when compared with pre-study. Total BMC (n = 24) lycopene concentrations did not

71 change significantly during the washout with a Mean ± SEM at enrollment of 1.03 ±

0.19 and 1.26 ± 0.21 nmol/pg protein at the end of washout (p = 0.16). During the

intervention period, BMC total lycopene levels for those consuming sauce, soup and juice increased to 3.34 (165.0%, p < 0.005), 1.72 (42.1%), and 1.87 (48.4%) nmol/pg

protein, respectively. Plasma isomer values showed an approximate 60:40 ratio of

ds.all-trans at the start of the study. We observed a significant proportional increase

in cw-lycopene isomers after two weeks of washout (p < 0.001) that corresponded to a

significant proportional decrease in dW-tram lycopene (p < 0.001). After 2 weeks of

dietary intervention, cis-trans isomer ratios returned to those observed at the start of

the study in all treatment groups.

Conclusions: This study demonstrates that plasma lycopene decreases approximately

50% after 2 weeks on a lycopene free diet. A single daily serving of processed tomato

products can significantly increase blood and tissue lycopene. Standard servings of

tomato products vary in lycopene content and result in different blood concentrations.

Additional studies of lycopene bioavailability are also necessary in order to understand

epidemiologic relationships between dietary tomato products, lycopene, and disease

outcomes.

KEY WORDS: Carotenoids, lycopene, lycopene isomers, tomato, tomato products,

food processing, blood carotenoids, buccal mucosal cell.

72 INTRODUCTION

Epidemiologic evidence demonstrates that diets rich in fruits and vegetables are associated with a reduced risk of several human malignancies (1,2). Furthermore, the consumption of tomatoes and processed tomato products has been proposed to lower the risk of prostate and several other cancers (3,4). Tissue concentrations of lycopene are also associated with reduced mortality from cardiovascular disease (S).

It has been hypothesized that lycopene may contribute to many of the health benefits of tomato products (6). Approximately 85% of dietary lycopene, a non-provitamin A carotenoid, is provided by tomato products. Lycopene is found in the human prostate and other tissues (7,8,9,10). Greater blood concentrations are correlated with a lower risk of prostate cancer (I I), bladder cancer (12), breast cancer (13) and heart disease

(5).

It is hypothesized that the disease preventative activity in tomato products is due to the potent antioxidant properties of lycopene (6). Lycopene has also been shown to act as an in-vitro (14) and in-vivo (15,16,17) antioxidant. Lycopene was determined to be the most efficient singlet oxygen quencher among the biologically occurring carotenoids in-vitro (14). Increasing dietary intake of lycopene in the form of spaghetti sauce (39 mg lycopene), tomato juice (50 mg lycopene), or tomato oleoresin (75 mg lycopene) has been shown to significantly decrease serum lipid peroxidation and LDL oxidation (15), increase serum antioxidant capacity, and possibly decrease the oxidation of protein and DNA (16). A two week lycopene free

73 diet significantly increased the serum lipid oxidation level by 25% as measured by

TBARS (thiobarbituric acid radical species) with a corresponding 50% decrease in blood lycopene concentration (17). Consumption of a single serving of tomatoes was shown to decrease levels of mutagenic oxidized purine base 8-hydroxyguanine within

24 hours (18). Increased consumption of vegetables and fruits resulted in a decrease of 8-OhdG in DNA isolated from lymphocytes (19).

Many questions remain unanswered regarding carotenoid bioavailability from

various food sources, such as the effects of food processing, the role of other dietary constituents (fat, fiber), and the role of cooking. Diets rich in certain types of dietary

fiber, such as pectin, may result in reduced carotenoid utilization (20). The absorption of carotenoids from raw or uncooked foods can be as low as 1%, and moderate cooking can promote the release of carotenoids from the food matrix, enhance the digestibility of the food, such as carrots and spinach (21). Food processing degrades the plant cell wall and fibrous constituents, decreases the particle size, and enhances uptake. Lycopene from tomato puree contributed to a greater increase in plasma

lycopene than did cooked tomatoes indicating that lycopene from intact cells is less available than that from processed tissue (22).

Little research has thus far focused on lycopene absorption, distribution, and biological activity on defined diets. Studies suggest that lycopene from fresh tomatoes is not as effective for increasing blood lycopene concentrations as lycopene from processed tomatoes (23,24). Adding lipids to tomato juice increased blood lycopene concentrations after a meal (25) suggesting that the type and quantity of lipid

74 consumed may influence uptake into blood and tissues. Supplementation (S mg lycopene/day) with tomato oleoresin or tomato juice significantly increased plasma lycopene while it remained unchanged with intake of raw tomatoes (26). Blood lycopene was significantly increased after 4 weeks of consumption of 70 - 75 mg lycopene/day as tomato juice, tomato oleoresin or lycopene beadlets (27); however, significant increases in buccal mucosal cell lycopene were only observed after consuming oleoresin or beadlets (28).

Improvements in analytical technology in recent years led to the discovery that lycopene in human blood and tissue is distributed among approximately 1 0 - 2 0 different isomers. Interestingly, the ratio of lycopene cis-trans geometric isomers in biological fluids such as plasma and in tissues such as prostate differ from those isomer ratios in fresh tomatoes (7). The typical ‘red’ tomato contains > 95% all-trans lycopene, blood samples are approximately 60% c/s-lycopene and 40% all-trans lycopene, while tissues such as prostate are as high as 80% czs-isomers. It has previously been assumed that the higher percentage of lycopene cû-isomer in human biological samples is due in part to consumption of heat-treated tomato products containing cw-isomers of lycopene. An evaluation of tomato products of various moisture content, fat content, and container type, however, suggests that lycopene is remarkably stable to isomerization reactions under typical industrial thermal processing conditions (29). The dietary and metabolic variables influencing lycopene isomer formation, clearance, and biological function remain to be elucidated.

75 Human epidemiologic studies typically quantify the consumption of standard servings of tomato products, although these products vary in lycopene content and processing conditions. The present study was designed to determine the ability of standard, single servings (based on FDA reference amounts) of three different tomato products consumed daily to influence blood and buccal mucosal cell lycopene concentrations and geometric isomer ratios. In addition, we designed the study to provide new information concerning lycopene clearance rates and lycopene isomer patterns in blood and tissue.

SUBJECTS AND METHODS

Thirty-six (18 male and 18 female) healthy subjects completed the study.

Subjects were non-smoking, non-pregnant individuals between 18-65 years of age.

Subjects were excluded if they suffered from gastrointestinal diseases that may alter food digestibility such as pancreatic insufficiency, hepatic failure, diabetes, and metabolic enzyme deficiencies. The Institutional Review Board of The Ohio State

University Human Subjects Committee approved the protocol and all subjects gave informed written consent. All 36 subjects consumed a lycopene-free diet for 6 weeks, with the exception of the V 8 ® Vegetable Juice, Prego® Spaghetti Sauce, or

Campbell’s® Tomato Soup provided by the investigators during the four-week intervention period. A list of lycopene containing foods to avoid during the study was provided to each subject with instructions to carefully examine ingredient labels for tomato containing products. All 36 subjects consumed a lycopene-free diet for the

76 first two weeks (washout period) in order to determine lycopene clearance rates from blood and BMC. After 2 weeks on a lycopene free diet, participants were assigned to one of three (n = 12) intervention groups consuming single standard servings of sauce

(21 mg lycopene/1/2 cup), soup (12 mg lycopene/1 cup), or juice (17 mg lycopene /8 oz) daily for 4 wk without any other sources of lycopene. Blood and BMC samples were obtained at enrollment and weekly thereafter for HPLC analysis of carotenoids.

Total blood cholesterol (mmol/L) was determined (Sigma diagnostics, method #352,

Sigma, St. Louis, MO) for each time point. The Campbell Soup Company (Camden,

NJ) supplied the tomato products. No restrictions were placed on the timing of intake or the composition of meals consumed with the tomato products.

Compliance to the diet was monitored throughout the study by a written diary of any daily deviations from the prescribed diet as well as recording the daily amount and timing of food consumption. At three points during the study (pre-study, second week of washout, and third week of feeding), dietary patterns were monitored with a 3 day food record. Subjects recorded everything they consumed on three assigned, non- consecutive days. Nutrient intakes were calculated using USDA food composition databases and compared with reference values.

Sample coUection

All blood and BMC samples were collected at the General Clinical Research

Center (GCRC) of The Ohio State University (Columbus, OH). Trained

77 phlebotomists at the GCRC obtained fasting, early morning venous blood samples into heparinized tubes. Samples were immediately shielded from light and placed on ice.

In order to collect BMC samples, subjects rinsed their mouths with tap water and discarded the rinse. They then brushed the inside of their cheek with a soft toothbrush

20 times on one side and rinsed with 20 ml of rinse solution (0.4 g table salt and 50 ml tap water). The rinse solution was deposited in a 50 ml collection tube coated with 50 pi of 1% butylated hydroxytoluene (BHT) in methanol. The rinsing was repeated once and the toothbrush was swirled in the remaining rinse solution that was added to the collection tube. Samples were immediately placed on ice and shielded from light.

All samples were processed under dim light within 2 hours of collection.

Blood samples were centrifuged (1400 x g, 10 min.) into cells and plasma, and 500 pi aliquots were stored at -80°C for carotenoid determination. BMC samples were centrifuged (1400 x g, 10 min.) and the supernatant discarded. The remaining cell pellet was washed with 15 ml cold phosphate buffered saline (PBS) (Sigma, St Louis,

MO) and vortexed. The sample was centrifuged again (1400 x g for 5 min.), the supernatant discarded, 1.2 ml of cold PBS added, and vortexed. One ml was removed and placed in a 2.0 ml BHT coated microcentrifuge tube for carotenoid determination and two-100 pi aliquots were placed in 15 ml conical tubes for protein determination

(Bio-Rad Laboratories, Hercules, CA). The microcentrifuge tube was centrifuged

(10,000 X g, 1.0 min.), the supernatant discarded, the tube flushed with nitrogen gas and the cell pellet stored at -80°C until analysis.

78 Sample extraction

Carotenoids were extracted from BMCs by adding 200 jal of protease solution

(100 mg protease/10 ml PBS) to the thawed cell pellet. The sample was vortexed and incubated at 37°C (30 min.) in a water bath. Following digestion, 500 |il of SDS- ethanol solution (1.0 ml 20% SDS-water solution added to 19.0 ml ethanol) was added and the pellet dispersed (Pellet Pestel, Fisher, Pittsburgh, PA). The sample was extracted with 500 nl of hexaneracetone (2:1) containing 0.2% BHT, vortexed, centrifuged (10,000 x g, 1 min) and the hexane layer removed and saved. The hexaneiacetone extraction was repeated once. The combined hexane layers were evaporated to dryness under a stream of nitrogen gas.

Individual carotenoids, retinol, retinyl palmitate, and tocopherols were measured by HPLC following the method described in Craft (30) and Nomura et al.,

(31). Lycopene levels in BMC as well as plasma lycopene isomer patterns were measured by HPLC-electrochemical detection with an adaptation to the method described in Ferruzzi et al. (32). Buccal cell lycopene isomers were separated using a

YMC C30 reversed-phase column (4.6 x 250 mm, 3 pm polymeric) and measured using liquid chromatography (Hewlett Packard 1050, Wilmington DE) with an ESA

(Chelmsford, MA) model 5600 Coularray™ electrochemical detector equipped with four channels in series. Potentials were applied from 220 to 520 mV in 100 mV increments. Column temperature was held constant (28°C) dining separation. An isocratic gradient was used with 42% of solvent A (95 methanol: 3 methyl-tert-butyl ether (MTBE): 2 ammonium acetate (1 mol/L)) and 58% of solvent B (20 methanol:

79 78 MTBE: 2 ammonium acetate-1 (mol/L)). The individual plasma isomers of lycopene were separated by HPLC and the quantities present were estimated using an al\-trans standard to calibrate the detector response.

Statistical analysis

Thirty-six subjects completed the study with only two missing data points due to illness or travel. Four subjects dropped out of the study due to personal reasons and their values were eliminated. These subjects were replaced with additional volunteers.

Data were analyzed using StatView 5.0 (SAS Institute, Cary, NC) software.

Descriptive statistics were used to compute means and SEMs. Significant differences from baseline were measured by paired t-tests and repeated-measures analysis of variance. Significant differences between treatments were measured by repeated- measures analysis of variance. To assess the plasma-BMC relation of lycopene concentration, Spearman correlation coefficients were computed at each sampling point. A p-value < 0.05 was considered statistically significant for all tests.

RESULTS

The mean age and BMI of the subject population were 35.1 y (range 20-61) and 25.5 kg/m^ (range 18.7 - 29.8) for the males and 38.3 y (range 18 - 60) and 24.5 kg/m^ (range 18.3 - 39.0) for the females. The carotenoid concentrations of the food

80 products used are presented in Table 1. Lycopene is the predominant carotenoid in each food product accounting for 93% of total carotenoids in sauce, 94% in soup, and

87% in juice. As expected, V8® contains a more diverse array of carotenoids since the product is prepared from a mixture of eight vegetable juices including tomatoes, carrots, and spinach. A representative chromatogram demonstrating plasma separation is shown in Figure 1. This separation allows the simultaneous resolution of the carotenoids and tocopherols in one chromatographic run. Based on average nutrient intakes calculated from USDA survey data, the subjects consumed at least an average amount of each nutrient and were in many cases above the average consumption level.

No significant differences in dietary fat, cholesterol or other key dietary components were detected between treatment groups (Appendix C, Table 1, p. 163). During the washout period, lycopene consumption decreased from a daily average of 6.4 mg to

0.31 mg; a 95% reduction.

Plasma was analyzed from each participant at seven time points (Table 2).

There were no significant changes in concentrations of retinol, retinyl palmitate, or the tocopherols for the six weeks of the study. During the washout period, total plasma lycopene concentrations decreased from 1.05 ± 0.07 to 0.54 ± 0.05 pmol/L (p <

0.0001,48.5%). Dietary lycopene intake and plasma lycopene concentrations were significantly correlated (r = 0.578, p < 0.0005) at the start of the study but not at the end of the two week washout (r = 0.072). Spearman correlations did not demonstrate any relationship between blood lycopene and age during the washout (r = 0.12).

81 Gender, also, did not correlate with blood lycopene during washout. Concentrations of other carotenoids measured did not significantly change.

In all intervention groups, plasma lycopene concentrations increased and plateaued between 2 -4 wks. Plasma lycopene concentrations for those consuming sauce, soup, and juice increased over those measured at the end of the washout period to 2.08 (192%, p < 0.0001), 0.91 (122%, p < 0.0001), and 0.99 (92%, p < 0.0001) pmol/L, respectively (Figure 2). Mean increases in total p-carotene of 32.7% (p <

0.01), 35.8% (p < 0.01), and 73.7% (p < 0.01) were observed for sauce, soup and juice, respectively. Also, the V8® group had a significant increase in blood a- carotene and lutein by 79.3% (p < 0.01) and by 14.9% (p < 0.05), respectively, with no significant increase with consumption of sauce or soup. Dietary lycopene intake correlated well (r = 0.499, p < 0.005) with plasma lycopene concentrations after 3 weeks of intervention. Spearman correlations did not demonstrate any relationship between blood lycopene and age in either of the three intervention groups. Gender, also, did not correlate with blood lycopene in either of the three intervention groups.

The 2 major lycopene isomers in blood are all-trans and 5-cis. Plasma isomers were analyzed at baseline and at weeks 2,4 and 6. At baseline, plasma lycopene was an average of 39% all-trans and 61% m (5-cis and other cis isomers). After the 2- week washout, the ratios had significantly changed to 31% all-trans and 69% cis (p <

0.001) for all subjects. After 2 weeks of dietary intervention, the all-trans:cis isomer ratios in plasma had returned to the pre-study ratios in all treatment groups.

82 The buccal mucosal cells (BMC) were analyzed for lycopene content due to limited sample quantity. Sample size was decreased (n = 8 per group) due to the elimination of samples contaminated with small amounts of blood from vigorous brushing. During the washout period, total BMC lycopene concentrations did not significantly decrease at 1.26 ± 1.01 as compared with a baseline of 1.03 ±0.19 nmol/pg protein (p = 0.16). In all intervention groups, BMC total lycopene concentrations significantly increased and plateaued between 2 -4 wks. BMC lycopene for those consuming sauce, soup, and juice increased to 3.34 (164.8%, p <

0.005), 1.72 (42.1%), and 1.82 (48.4%) nmol/pg protein, respectively (Figure 3).

Spearman’s correlations were performed on the blood and buccal lycopene concentrations over the course of the study and those results are presented in Table 3.

Blood and buccal lycopene concentrations correlated significantly at the end of washout when all subjects are considered together (0.409, p < 0.05), and after 3 weeks of feeding soup (0.736, p < 0.05) and juice (0.787, p < 0.05). While correlations were not significant at individual time points in the sauce group, there was a strong and significant (0.697, p = 0.05) correlation for increases observed from weeks 2-4.

BMC isomers were analyzed at baseline and at weeks 2 and 4. Relative cis.trans isomer ratios remained unchanged during the washout and intervention periods.

83 DISCUSSION

This study demonstrates that standard-serving sizes of tomato products effectively increase blood lycopene, however standard servings of different tomato products are not equivalent in their contribution to blood and tissue lycopene concentration. A lycopene free diet will lower blood lycopene by approximately 50% in 14 days. The increase in blood concentration seems to plateau after two weeks of consumption following a lycopene free diet. Based on standard serving sizes, tomato sauce provides a greater concentration of dietary lycopene and has a greater effect on blood concentrations than standard servings of soup or juice. BMC lycopene increases during intervention paralleled changes observed in blood providing evidence that standard daily servings of the tomato products used in the study are sufficient to increase the lycopene deposited in tissues.

Very little information is available on lycopene clearance rates from blood in individuals consuming a lycopene free diet. One publication reports that the plasma half-life of lycopene is approximately 2 - 3 days (33). A two-week washout has been reported to produce a 50% reduction in blood lycopene over baseline (26,15) while another study found no decrease in blood lycopene concentrations after washout of 2 weeks (34). Our study observed a 40% and 48% reduction in blood concentrations after one and two weeks of washout, respectively. Lycopene intervention studies should incorporate a washout period into their study design to control for timing of most recent lycopene intake. At baseline, a portion of the subjects may have consumed a high lycopene meal within the past 24 hours and will demonstrate a

84 greater magnitude of decrease in plasma lycopene over time. The observed decrease

in plasma lycopene should be much less for a subject who has not consumed a high

lycopene meal in >3 days. One week of washout yielded a mean decrease in plasma

lycopene of 40% and the additional week of washout yielded only an additional 8%

reduction, suggesting that perhaps a one-week washout is most effective.

We chose to apply standard serving sizes of processed tomato products for our study rather than control the precise lycopene content of the diet to equivalent amounts. A major goal was to determine the efficiency of the food products tested at increasing plasma lycopene. This should assist us in better interpreting the epidemiologic studies that are also based on estimated intake of standard servings of processed tomato products. We feel that it is important to define the quantitative relationships between commonly consumed servings of tomato products, blood and tissue concentrations, and biological outcomes. This study used standard serving sizes

(as defined by the FDA for nutritional labeling), of three processed tomato products commercially available. We chose a single daily serving amount consistent with findings from epidemiologic studies on lycopene intakes typically associated with a reduced disease risk. The amount of lycopene in each of the food products differed.

The relationship between dietary lycopene consumed during intervention and measured plasma lycopene is significantly correlated (r = 0.499, p < 0.005). The plasma response for the sauce group was slightly higher than expected based on amount consumed and the juice group response slightly lower, suggesting differences in bioavailability from the food matrix and differences in absorption specific to the

85 content of the meals. Some groups have suggested that concurrent consumption of lipid will increase lycopene bioavailability (25). Fat intake was not controlled in our study and subjects were allowed to consume products at their discretion. Estimated total dietary fat intake decreased for the participants consuming the sauce and juice by

19% (84 to 68 g, p < 0.05) and 25% (93 to 70 g), respectively, from the end of washout to the third week of feeding, while the soup group remained essentially constant (61 to 65 g). This finding could perhaps account for a lower than expected plasma response in the juice group based on amount consumed. While total dietary fat intake decreased in the sauce group, it was the only treatment group where there was a consistent amount of fat found in the product. This finding supports the hypothesis that concurrent consumption of fat enhances lycopene bioavailability. While the juice group responded somewhat less than expected based on amount consumed, the soup group responded somewhat better than expected based on amount consumed. Subjects were directed to dilute one can of soup as directed with either water or milk and then consume one cup and save the second cup for the next day. Two factors that may increase the soup’s response are the level of processing and the consumption with whole or 2% milk. We did not collect data on whether the soup was consumed consistently with water or milk. Previous studies have indicated that lycopene was more bioavailable from processed tomato products than from fresh tomatoes or non­ processed juice (23,24,25). A recent epidemiologic study (35) examined the influence of different food sources of lycopene on plasma lycopene levels. Their findings

86 suggested that consumption of lycopene from processed tomato products led to greater

blood concentration of lycopene than from unprocessed products (p = 0.06).

Our study showed an increase in blood lycopene over a two-week period of

intervention and then an apparent plateauing effect from wks 2-4. Two studies by

Pateau et al. (27,28) compared consumption of lycopene rich tomato juice with

lycopene beadlets and tomato oleoresin all containing 70 - 75 mg lycopene. Blood

lycopene concentrations were shown to approximately double over baseline values

after 4 weeks of consumption of all three treatments (27). Another study by Agarwal

and Rao (15) showed an increase in blood lycopene after a one-week consumption of

tomato juice although they employed levels of intake providing 50.4 mg of lycopene.

Both these studies support our results, although the intake levels were 3- and 4 - times

greater than were consumed in our study. A plateauing of the blood concentrations

after 1 - 2 weeks of constant stable consumption was similar to the plateau observed in

our study.

Collection of BMC’s is an easy and noninvasive method to measure tissue

deposition of carotenoids. Buccal mucosal cell lycopene concentrations increased

approximately 25% after daily tomato juice consumption (28) over 4 weeks but were

not statistically significant. This finding was also supported by our study, which

showed an increase (48.4%) in buccal mucosal cell lycopene levels but the change did

not reach statistical significance for consumption of juice. Our study showed a

significant increase in BMC lycopene levels after consumption of sauce containing 21 mg lycopene/serving, a level of intake that is only 1/3"* that consumed in the other

87 study (28). With weekly sampling of BMC we observed a delay in response of cellular lycopene levels compared to blood levels following dietary intervention. The delayed response in BMC coincides with the maximal changes in the blood lycopene concentrations observed after 2 weeks, supporting the hypothesis that circulating levels may change more rapidly and BMC content will respond, but at a more gradual pace. There have been conflicting reports correlating blood lycopene and BMC lycopene levels with some suggesting no correlation (36,28) or good correlation (37).

These studies based their correlations on samples of the general population without dietary intervention or estimated intakes. With the intervention study making its comparison at one time point at the end of 4 weeks of feeding. Several factors influence the lycopene amounts sampled in BMCs. The layer of buccal mucosa removed in the first sampling may contain a much older population of cells than that obtained in successive samplings. These older cells may have lost carotenoids through diffusion and/or chemical breakdown (36). Our study was able to correlate blood and

BMC values at each time point over the course of the study and the correlations varied

(Table 4). Initially our subjects showed a relatively poor correlation at baseline, perhaps reflective of the variation in when the subjects last consumed a lycopene meal. At the end of washout, the correlation was stronger (0.409, p < 0.05), presumably because the blood and BMC’s had equilibrated to the decrease in dietary lycopene intake, making plasma more reflective of body stores. Figures 2 and 3 indicate a brief lag time for an increase after intervention begins in BMCs as compared with plasma. The lag time also impacts upon the correlations subsequently

88 evaluated. A relatively poor correlation is observed in Week 3, and a much stronger relationship is observed in Weeks 4,5, and 6 for those consuming soup and juice.

These two groups demonstrate less of an increase per serving in plasma lycopene than does the sauce group and no increase in BMC lycopene resulting in a better correlation of plasma with body stores. When correlating the increase in blood lycopene with the increase in BMC lycopene (Week 4 - Week 2) we found a strong and significant correlation for the sauce group only (0.697, p < 0.05). Over time, the greater increase in plasma lycopene observed with consumption of sauce will eventually be reflected in

BMC body stores. When correlating the blood and BMC levels it was necessary to decrease the sample size (n = 24) to make a comparison based upon individual subjects. This resulted in a decrease in power. A larger, follow-up study might determine if the amount of lycopene fed would influence the strength of correlation between blood and BMCs.

Lycopene is primarily transported via low-density lipoproteins that also carry the bulk of cholesterol in the plasma. It becomes necessary to investigate the relationship between plasma lycopene and plasma cholesterol. While many epidemiologic studies (35,38,39,40) have found significant correlations between plasma lycopene concentrations and plasma cholesterol concentrations, we did not observe this relationship in our population. In contrast to the other studies which contained much larger subject pools (n = 111 to 600) with a median age of 65 y, our study (n = 36) had a mean age of 37 y. The mean total cholesterol reported in the epidemiologic studies ranged from 5.2 - 6.1 mmol/L as compared with 4.6 - 4.9

89 mmol/L in this study; approximately 10 - 20% lower. Studies that have correlated each lipoprotein fraction with serum or plasma lycopene values have found that plasma lycopene values correlate most closely with non-HDL cholesterol. Consistent with the studies that correlate plasma lycopene and cholesterol levels is the negative correlation of age to plasma lycopene in a much older population (> 50 y). This correlation is perhaps not as easily detected with a younger population (< 50 y) having much lower blood cholesterol concentrations (< 5.0 mmol/L or 200 mg/dL).

Little is known about the uptake and appearance of cû-lycopene in human plasma. It has been established that the majority of lycopene in tomato products is present as the dW-trans form and is not substantially changed by food processing (29,

41). Plasma isomer values in our study showed an approximate 60:40 ratio of mrall- trans isomers of lycopene that agrees with previously published values (7). During the washout period, we observed a significant change in the cis. trans lycopene isomeric ratios from 61% cis: 39% trans before the study to 69% as: 31% trans after two weeks on a lycopene free diet. This may be a result of mobilization of lycopene stores from tissues where lycopene is predominantly found in the cû-form. After 2 weeks of dietary intervention, cis-trans isomer ratios returned to those observed at the start of the study in all treatment groups. The only other study with information on the change of cif-lycopene in plasma (34) during a lycopene free diet did not find a significant difference in cis:trans ratios after a washout period. In vivo work with lymph-cannulated ferrets has shown that m-isomers of lycopene are more bioavailable than fraw-lycopene (42). The authors support their hypothesis with in

90 vitro data suggesting the reason for the increased bioavailability of cû-isomers is their increased solubility in bile acid micelles and their preferential incorporation into chylomicrons. Prostate tissue may contain as much as 85% cis lycopene as compared with 60% cis lycopene commonly observed in plasma or serum (7). Recent findings have demonstrated that the proportion of lycopene in the ail-trans form was not significantly different in plasma, chylomicrons, HDL, VLDL or LDL (22). These and other findings point to a difference in metabolism for cis and trans isomers of lycopene and a possible beneficial effect of high cw-isomer concentrations. Future studies will have to address the formation, interconversion, and biofimction of the lycopene isomers.

In conclusion, standard daily servings, as defined by the FDA reference amounts, of the tomato products used in this study, are effective at substantially increasing plasma lycopene concentrations in healthy volunteers. The blood lycopene amounts produced, provide concentrations in the range associated with a lower risk of several diseases in human epidemiologic studies (11,3,4). Blood and buccal mucosal cell lycopene concentrations correlate well at certain time points (in an intervention trial). This correlation however was not consistent over time to exclude the measurement of either, if information on circulating levels as well as tissue deposition is desired. The cis-trans lycopene isomer balance is a dynamic one that can change upon decreased or increased consumption. Future studies will have to address the formation, interconversion, and biofimction of the lycopene isomers

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95 Soup Juice Sauce mg/IOOg per I cup mg/IOOg per 8oz. mg/IOOg per ‘A cup serving serving serving Lutein 0.06 0.13 0.13 0.31 0.32 0.42 Zeaxanthin 0.02 0.04 0.02 0.04 0.04 0.05 a-CryptoxanthIn nd^ nd nd nd nd nd P-Cryptoxanthln nd nd nd nd nd nd M-trans 4.9 11.4 6.6 16.3 14.6 19.6 Lycopene cw-Lycopene 0.23 0.53 0.29 0.70 1.07 1.43 a-Carotene nd nd 0.15 0.36 nd nd P-Carotene 0.08 0.18 0.47 1.14 0.27 0.36 cb-p-Carotene 0.04 0.09 0.11 0.26 0.16 0.21 Other 0.14 0.32 0.17 0.41 0.45 0.60

Table 1. Carotenoid content of food products as consumed in the study' ' vaules represent mean of 3 extracts. ^ nd = not detected Time (minutes)

Figure 1. Representative Cjg separation of plasma extract. Baseline Weekl Week 2 Week Week Week Week 3 4 4 6 retinol 1.93 1.90 1.96 sauce 1.98 2.14 2.01 1.87 soup 1.85 1.91 2.02 1.92 juice 1.44 2.12 2.02 2.00 retlnyl palmltate 0.03 0.01 0.02 sauce 0.01 0.01 0.02 0.01 soup 0.02 0.04 0.02 0.02 juice 0.04 0.02 0.08 0.05 a-tocopherol 28.47 30.53 32.02 sauce 34.61 35.50 34.09 29.09 soup 29.49 23.80 25.73 26.01 juice 23.77 35.55 34.17 33.51 5-tocopherol 0.20 0.17 0.24 sauce 0.21 0.14 0.12 0.14 soup 0.22 0.20 0.24 0.16 juice 0.13 0.17 0.17 0.10 y-tocopherol 3.65 3.82 4.06 sauce 3.89 3.03 2.97 2.97 soup 3.70 3.71 4.03 3.88 juice 2.24 2.99 3.09 3.00 lutein 0.22 0.22 0.23 sauce 0.23 0.24 0.22 0.20 soup 0.24 0.24 0.26 0.25 juice 0.20 0.26 0.24 0.25 zeaxanthin 0.06 0.06 0.09 sauce 0.11 0.13 0.12 0.12 soup 0.09 0.11 0.12 0.11 juice 0.08 0.12 0.11 0.11 a-cryptoxanthIn 0.06 0.05 0.06 sauce 0.07 0.07 0.06 0.06 soup 0.05 0.06 0.06 0.05 juice 0.04 0.06 0.06 0.05 P-cryptoxanthIn 0.37 0.33 0.36 sauce 0.39 0.49 0.39 0.47 soup 0.29 0.39 0.40 0.35 juice 0.24 0.35 0.33 0.30 a\\-trans lycopene 0.54 0.30 0.24 sauce 0.69 0.99 0.92 0.77 soup 0.37 0.46 0.54 0.54 juice 0.28 0.50 0.50 0.50 c/s-lycopenes 0.52 0.34 0.32 sauce 0.71 1.09 1.02 0.91 soup 0.37 0.45 0.52 0.50 juice 0.27 0.49 0.50 0.48 a-carotene 0.12 0.11 0.13 sauce 0.12 0.13 0.13 0.12 soup 0.09 0.11 0.10 0.11 juice 0.12 0.22 0.22 0.22 P-carotene 0.45 0.41 0.44 sauce 0.40 0.56 0.50 0.53 soup 0.32 0.40 0.42 0.43 juice 0.52 0.88 0.85 0.85 c»-p-carotene 0.07 0.04 0.07 sauce 0.05 0.10 0.06 0.06 soup 0.05 0.08 0.06 0.06 iuice 0.05 0.10 0.07 0.08

Table 2. Mean plasma carotenoid concentrations throughout the study (pmol/L). 98 2.3

1.9

1.5

0 1 1.1

0.7

0.3 ✓ / / •sauce soup V8

Figure 2. Total plasma lycopene concentrations during the course of the studyV 'error bars represent SEM. 2 is significantly different from 3 for all groups. 2 is significantly different from 5 for soup and V8. 4 is significantly different from 2 and 3 for sauce.

99 4

3.4 I 2.8 I 2.2 1.6

1

0.4 N <9

*sauce soup •VS

Figure 3. Total buccal mucosal cell lycopene concentrations during the course of the study\ ^error bars represent SEM. 2 is significantly different from 3.

1 0 0 Group' Baseline Weekl Week 2 Week 3 Week 4 W eeks Week 6 Increase sauce -0.201 0.448" 0.409^ 0.082 0.530 0.275 0.034 0.697* soup -0.201 0.448" 0.409^ 0.455 0.737" 0.736* 0.655 0.309 juice -0.201 0.448" 0.409^ 0.111 0.494 0.787* 0.599 0.639

Table 3. Spearman rank order correlations of lycopene concentrations in plasma and buccal mucosal cells for all treatment groups during the study.

'n=8/group

^Significant for the increase (week 4-week2), p=0.05 CHAPTER 4

Blood and Milk Lycopene Isomer Concentrations Increase in Lactating Women Consuming DaUy Servings of Processed or Fresh Tomato Products.

Charlotte M. Allen', Anne M. Smith^, Steven K. Clinton^, Steven J. Schwartz'*.

'Department of Food Science and Technology The Ohio State University, Columbus, Ohio 43210-1096

^ Department of Human Nutrition and Food Management The Ohio State University, Columbus, Ohio 43210-1096

^Comprehensive Cancer Center Arthur G James Cancer Hospital and Solove Research Institute The Ohio State University, Columbus, Ohio 43210-1096

102 ABSTRACT

Background - The nutritional significance of lycopene in the neonatal, newborn or infant period has not been established. There have been no published studies monitoring uptake of dietary lycopene from food products during lactation.

Objective - The present study was designed to quantitate plasma and milk lycopene concentrations in lactating women (n = 24,4 - 12 wks postpartum) consuming equivalent amounts of lycopene as either fresh tomatoes or processed tomato sauce.

Methods - All 24 subjects initially consumed a lycopene-free diet for 7 days (washout period). Participants were assigned to the control (lycopene free diet, n = 8), fresh tomatoes (n = 8), or processed tomato sauce (n=8) for 3 days without any other sources of lycopene. Each of the tomato groups consumed five servings (~ SO mg total lycopene) over 3 days. Plasma, buccal mucosal cells (BMC) and breast milk samples were obtained for HPLC analysis of lycopene and its geometric isomers (all-trans, 5- cis, all other cis, and total) before and after dietary intervention.

Results - Total plasma lycopene concentrations (Mean ± SEM, pmol/L) increased from

0.780 ± 0.04 to 0.890 ± 0.04 (12.3%, p < 0.05) and from 0.637 ± 0.06 to 0.823 ± 0.06

(22.6%, p < 0.005) in the fresh and processed groups, respectively. No significant change was noted in the control group. BMC lycopene concentrations did not change significantly during this brief study. Milk total lycopene concentrations increased for those consuming processed sauce from 133 ± 10 to 178 ± 17 (25.2%, p < 0.01) nmol/L, but did not change when fresh tomatoes were consumed.

103 Conclusions- In a group of lactating mothers, a daily serving of fresh or processed tomato products will significantly increase plasma lycopene in a short period of time.

Significantly increased breast milk lycopene concentrations are noted in women consuming processed sauce in a study of short duration. Based on equivalent lycopene amounts, processed tomato sauce seems to be more effective in rapidly increasing plasma lycopene concentrations and enhancing the transfer of lycopene to milk compared to fresh tomatoes.

Key words: carotenoids, lycopene, lycopene isomer, human milk, plasma, lactation

104 INTRODUCTION

Human breast milk contains a nutrient pattern ideally suited to meet the physiological requirements of the human infant. Provitamin A carotenoids provide an important source of vitamin A in breast milk, and other carotenoids found in milk have been hypothesized to contribute to protecting a nursing infant from respiratory and gastrointestinal infections (1,2). P-carotene has been extensively studied in breast milk because of it’s provitamin A activity, and supplementation studies have been performed during lactation with P-carotene beadlets or other supplements (3,4). Although 34 carotenoids, including lycopene and it’s isomers, have been identified in milk (5), there have been no published studies evaluating lycopene transfer to milk in lactating women.

Nearly all components of milk are derived from the blood. Fat is the main source of energy in milk and is among the most variable and difficult nutrients to measure accurately in human milk, both within and between individuals. It is hypothesized that carotenoids are contained in the milk fat globules. Beta- and alpha- carotene, lycopene, beta-cryptoxanthin and lutein are among the 36 carotenoids found in human milk at about one-tenth the concentration found in serum (6 ). The mean concentration of carotenoids in colostrum is 218 pg/dL while the concentration in mature milk is 50 pg/dL (7). Plasma concentrations of tocopherols, carotenoids, retinol and lipid are substantially lower in infants at birth ( 8 ). Plasma concentrations of lycopene showed no correlations between maternal and cord plasma unless an adjustment for plasma triglycerides were made, making the correlation the highest of all the carotenoids (r=0.975, p<0.0001) (9).

105 Since very few supplementation studies have been performed with carotenoids in lactating women, there are many aspects of the effectiveness of this approach yet to be investigated. The kinetics of the response of milk and serum to p-carotene supplementation has been investigated (10). Short-term supplementation of healthy, lactating mothers with purified P-carotene substantially increased milk and serum P- carotene concentrations but did not effect the concentration of other caroteniods or retinol. Kinetics of milk uptake and decay of P-carotene paralleled those in serum.

Either 64 mg of all-trans P-carotene or 69 mg of 9-cis P-carotene were fed to lactating women and levels of each geometric isomer was measured in milk, blood serum, and buccal mucosa cells. Both all-trans P-carotene and 9-cis P-carotene levels in serum, milk and buccal cells increased significantly over baseline by the end of the supplementation period of eight days (4). The study seems to provide evidence that there is no difference in tissue uptake ofall-trans p-carotene and 9-cis P-carotene.

Lycopene has recently emerged as a potentially beneficial dietary phytochemical.

Accumulating research findings show an inverse correlation between consumption of food products high in lycopene, particularly tomatoes, and the risk of developing certain types of cancer (11). The nutritional significance of lycopene in the neonatal, newborn or infant period, however, has not been established. Non provitamin A carotenoids such as lycopene possess strong antioxidant capability (12). Consumption of foods rich in carotenoids will alter circulating maternal carotenoid concentrations, and thereby modifying the carotenoid status of the newborn.

106 The objective of this study was to compare plasma, BMC, and milk lycopene concentrations and the changes in lycopene isomer patterns in lactating women before and after a 4 day dietary intervention with either fresh or processed tomato products.

SUBJECTS AND METHODS

Twenty-four healthy, lactating women completed this study. Subjects were non- smokers and between 4 and 12 wks post partum. The Institutional Review Board of

The Ohio State University Human Subjects Committee approved the study and all subjects gave their written consent for participation. Women were asked to limit their intake of lycopene containing foods for 7 days prior to the intervention phase of the study. Participants were assigned to one of three (n = 8 ) intervention groups: control

(lycopene free diet), fresh tomato, or processed tomato sauce. Subjects in treatment groups consumed two daily servings ( 1 0 mg lycopene/serving) of either fresh tomatoes or sauce for the next 3 days (50 ± 2 mg lycopene over 3 d). The control group consumed two daily servings of 10 grapes. Human milk samples were expressed and blood and buccal mucosal cell samples (BMC) obtained to demonstrate baseline levels of lycopene at the end of washout. Another set of samples was obtained on day 4 after intervention. Fresh grapes and fresh Roma tomatoes were purchased locally throughout the year. Tomatoes were purchased for the subjects who consumed fresh tomatoes the day before that subject’s appointment, and the lycopene concentrations obtained. We

107 asked subjects to consume2 tomatoes at each of the five designated dietary intervention

points over 3 days; one intervention on the first day and two interventions each for the

next two days. The tomato sauce was developed in collaboration with Hirzel Canning

Corporation (Toledo, OH) with a specified amount of lycopene. The amount of fresh

tomatoes needed to give participants equivalent amounts of lycopene was then

established. Subjects who consumed sauce were asked to consume one-1/3 cup serving

at each designated time point. Fat was standardized in the sauce at 9 g/125 g sauce.

Sample collection

Breast milk, blood, and BMC samples were collected at the Pediatric Clinical

Studies Center (PCSC) of Children’s Hospital in Columbus, Ohio in the afternoon to

minimize diurnal variation in milk lycopene concentration. To minimize variability

during milk expression and to insure the highest amount of fat and therefore, the richest

carotenoid content (13), only hind milk was used. Milk samples were obtained within

one hour of nursing from each breast. Subjects were provided with an electric breast

pump (White River Concepts, San Clemente, CA) express 2 -4 ounces of milk into a

transparent polycarbonate jar (Fisher, Pittsburgh, PA) to protect the sample from light.

Samples were frozen immediately (-20®C) and then stored at -70°C until analysis.

Blood samples were drawn by trained phlebotomists at the PCSC before intervention and on day 4. Blood samples were centrifuged (1400 x g, 10 nun.) into red cells and plasma, and 500 pi aliquots of plasma were stored in liquid nitrogen until carotenoid determination was made.

108 BMC samples were collected using the method adapted by Peng and Peng (14).

Subjects rinsed their mouths with tap water and discarded the rinse. They then brushed the inside of their cheek with a soft toothbrush 2 0 times on one side and rinsed their mouth with 20 ml of rinse solution (0.4 g table salt and 50 ml tap water). The rinse solution was deposited in a 50 ml collection tube coated with 50 pi of 1% butylated hydroxytoluene (BHT) in methanol. The rinsing was repeated once and the toothbrush was swirled in the remaining rinse solution that was added to the collection tube.

Samples were immediately placed on ice and shielded from light.

BMC samples were centrifuged (1400 x g, 10 min.) and the supernatant discarded. The remaining cell pellet was then washed with 15 ml cold phosphate buffered saline (PBS) (Sigma, St Louis, MO) and vortexed. The sample was centrifuged again (1400 x g for 5 min), the supernatant discarded, 1.2 ml of cold PBS added, and vortexed. One ml was removed and placed in a 2.0 ml BHT coated microcentrifuge tube for carotenoid determination and two 1 0 0 pi aliquots were placed in 15 ml conical tubes for protein determination (Bradford Method, Bio-Rad

Laboratories, Hercules, CA). The microcentrifuge tube was centrifuged (10,000 x g,

1 .0 min.), the supernatant discarded, the tube flushed with nitrogen gas and the cell pellet stored at -80°C until analysis.

109 Sample extraction

All organic solvents were HPLC grade (Fisher, Pittsburgh, PA). Concentration of lycopene in the tomatoes and sauce was determined using the extraction procedure of

Nguyen and Schwartz (15). A given amount of the sauce was weighed and extracted 3x

( 1 :1 hexaneracetone), the hexane layers were combined and saved, and the extinction coefficient at 471 nm (3450) was used to determine the concentration of zW-trans lycopene. An external standard (Sigma, St. Louis, MO) was used to establish a standard curve for the d\\-trans lycopene.

Carotenoids were extracted from BMCs by adding 200 pi of protease solution

(100 mg protease/10 ml PBS) to the thawed cell pellet. The sample was vortexed and incubated at 37° (30 min.) in a water bath. Following digestion, 500 pi of SDS-ethanol solution (1.0 ml 20% SDS-water solution added to 19.0 ml ethanol) was added and the cell pellet dispersed (Pellet Pestel, Fisher, Pittsburgh, PA). The sample was extracted with 500 pi of hexaneracetone (2:1) containing 0.2% BHT, vortexed, centrifuged

(10,000 X g, 1 min), the hexane layer removed and saved. The hexane:acetone extraction was repeated once, then the combined hexane layers were evaporated to dryness under a stream of nitrogen gas. Extract was solubilized in 200 pi MTBE then

2 0 0 pi methanol, filtered through a 0 . 2 pm syringe filter and 2 0 0 pi was injected.

Carotenoids were extracted from plasma after bringing the 500 pi sample to room temperature and adding 500 pi of ETOH-BHT (ethanol with 0.1% BHT) to deprotonate the sample. The sample was extracted twice with 1 ml of hexane:acetone

(2:1) containing 0.2% BHT, vortex mixed, centrifuged (1000 x g, 1 min), the hexane

110 layers removed and saved. The combined hexane layers were evaporated to dryness under a stream of nitrogen gas. Extract was solubilized in 200 pi MTBE then 200 pi methanol, filtered through a 0.2 pm syringe filter and 100 pi was injected. Duplicate samples were analyzed.

Milk was thawed and thoroughly homogenized (Tissue Tearor, Biospec

Products, Bartlesville, OK). Three S ml aliquots were placed in glass vials and frozen at

-20°C until analysis. Samples were analyzed in triplicate. Ten ml of 30% potassium hydroxide (KOH) in methanol and 2.5 ml of 3% ascorbic acid in ethanol was added to the thawed milk. The mixture was saponified (400 rpm) for 45 min. at room temperature and protected from light by aluminum foil. The sample was extracted with

5 ml of hexane:acetone (2:1, with 0.01% BHT), vortexed, and centrifuged (2000 rpm, 5 min). The hexane layer was collected and saved. The extraction was repeated for a total of three times combining the hexane layers. The hexane extract was washed with

3 ml of distilled H 2O and centrifuged. The hexane layer was removed and transferred to another centrifuge tube and the washing step repeated. The hexane was carefully removed and evaporated to dryness under nitrogen. Extract was solubilized in 200 pi

MTBE then 200 pi methanol, filtered through a 0.2 pm syringe filter and 100 pi was analyzed by HPLC.

Ill Milk lipids

The lipid content of the milk was estimated as a percentage of total volume by the ‘creamatocrit’ assay (16). Carotenoid concentrations in milk were expressed as nmol/L as well as nmol/g lipid.

HPLC separation

The cis~ and trans- geometric isomers of lycopene, as well as P- and o-carotene, were measured in milk and plasma using liquid chromatography (Waters 2690 with 996 photodiode array detector, Milford MA). A YMC (Wilmington, NC) C 30 reversed- phase column (4.6 x 250 mm, 3pm polymeric), a guard column packed with C|g stationary phase (Vydac, Hesperia, CA) and a pre-column filter (0.5 pm) were used for separation. Sample (25°C) and column temperatures (28°C) were maintained during separation. Separations were achieved using a gradient elution method with different concentrations of MTBE: 1.5% of 1 mol/L ammonium acetate (AA) in methanol:H20.

The following gradient was used: 0 to 25 min 68% methanol-AA, 1% H 2 0 ,31%

MTBE; 25 to 38 min a linear gradient to 47% methanol-AA, 1% H 2 0 ,52% MTBE; hold 17 min then re-equilibration to beginning solvent.

Buccal cell lycopene isomers were separated via the method of Ferruzzi et al.,

(17) using a YMC C 30 reversed-phase column (4.6 x 250 mm, 3pm polymeric) and measured using liquid chromatography (Hewlett Packard 1050, Wilmington DE) with an ESA (Chelmsford, MA) model 5600 Coularray^ electrochemical detector equipped with four channels in series. Potentials were applied from 220 to 520 mV in 100 mV

112 increments. Column temperature was held constant (28°C) during separation. An isocratic gradient was used with 42% solvent A (95 methanol: 3 MTBE: 2 AA - 1 mol/L) and 58% solvent B (20 methanol: 78 MTBE: 2 AA-1 mol/L). Standard curves were constructed for a-carotene, P-carotene, and al\-trans lycopene using external standards.

Statistical analysis

Data were analyzed using StatView 5.0 (SAS Institute, Cary, NC) software and a p < 0.05 was considered significant for all tests. Descriptive statistics were used to compute means and SEMs. Significant differences from baseline were measured by t- tests with an analysis of variance. Analysis of variance with Fisher’s and Tukey’s test were used to determine significant differences between groups. Spearman’s correlation coeffrcients were calculated for blood-milk relationships. Since the sample size was relatively small for each group in this study, all lycopene concentrations were log transformed and examined for any additional relationships or increased significance.

None were found and the data were not presented.

RESULTS

Twenty-four women completed the study with a mean age of 29 y (range 22 -

39 y). All participants were between 4 -1 2 wks postpartum with the mean of 8 wks. A representative HPLC chromatogram illustrating the separation of breast milk

113 carotenoids is shown in Figure 1. The method was developed for human milk samples in order to yield baseline separation of the majority of the lycopene isomers found in milk and plasma samples. At baseline, the total plasma lycopene concentration (Mean ±

SEM, Table 1 - Appendix D, p.217) was 0.780 ± 0.04 pmoI/L for all subjects combined. There were no significant differences in plasma lycopene concentrations between treatment groups at baseline. A significant increase over baseline was observed with both the fresh and processed groups (Figure 2). After intervention total lycopene concentration in the control group (lycopene free diet) decreased from 0.812 ±

0.06 to 0.760 ± 0.06 (6.4%, p = 0.17) pmol/L. In contrast, an increase from 0.780 ±

0.04 to 0.890 ± 0.04 (12.3%, p < 0.05) and from 0.637 ± 0.06 to 0.823 ± 0.06 (22.6%, p

< 0.005) pmol/L was observed in the plasma of those fed fresh tomatoes and processed sauce, respectively. The total lycopene change over baseline is also significantly different from control for both the fresh (p <0.01) and processed groups (p < 0.0005), but there was no significant difference in the increase between fresh and processed.

After intervention, the P-carotene plasma concentration was 0.306 ± 0.031 pmol/L for all groups considered together, which was not a significant increase from the baseline concentration of 0.289 ± 0.022 pmol/L. a-carotene plasma concentrations remained stable at 0.005 ± 0.002 pmol/L during the study. BMC lycopene concentrations were highly variable and did not change from a baseline of 0.71 ± 0.7 nmol/pg protein.

The breast milk lycopene concentrations were expressed as nmol/L as well as nmol/g lipid based upon the creamatocrit method used to estimate the fat content of milk. A mean milk fat concentration of 48.6 g/L (range 16.3 - 75.7, n = 24) was

114 observed at baseline and a mean of 48.7 g/L (range 16.4 - 83.7, n = 24) after the intervention. The mean milk fat concentrations for all subjects at the first and second milk sampling showed a strong and significant correlation (r = 0.451, p < 0.05), and there was no significant effect of dietary treatment.

At baseline, milk total lycopene was 174 ± 30 nmol/L (Table 2, Appendix D, p.219) and 3.9 ± 0.4 nmol/g fat (Table 3, Appendix D, p.220) for all women in the study. Total lycopene in breast milk increased in the women fed processed sauce from

133 ± 10 to 178 ± 17 (25.2%, p < 0.01) nmol/L, but changed very little in the women fed fresh tomatoes (204 ± 30 to 205 ± 19 nmol/L). There were no significant differences at baseline between the milk lycopene concentrations when an ANOVA was performed with baseline concentrations as covariate. Milk lycopene decreased in the control group fi-om 185 ± 20 to 165 ± 19 (11%, p = 0.06) nmol/L (Figure 3). When considering effect of treatment in milk (nmol/L), the total lycopene increase over baseline for processed is significantly different from control (p < 0.005) and from fresh

(p < 0.05). When expressed as nmol/g fat, changes in total lycopene did not reach significance across treatment groups. The two major lycopene isomers found in plasma and milk were diX-trans and 5-cis. When considering the ratios of d\\-trans\cis lycopene isomers before and after intervention, the change in ratios did not reach significance for any group. Concentrations of P- (88.7 ± 12.6 nmol/L) and a-carotene (1.23 ± 0.635 nmol/L) remained essentially unchanged in milk following intervention.

To determine if there was a correlation between milk and blood lycopene concentrations, Spearman rank order correlations were performed on milk and blood

115 lycopene concentrations (Table I). There was a correlation between plasma and milk

dX\-trans lycopene (r = 0.568, p = 0.14) after consumption of fresh tomatoes. There

were significant correlations between plasma and milk m-lycopene (r = 0.822, p <

0.005) after consumption of processed sauce, but the correlation did not reach

significance for total lycopene (r = 0.580, p = 0.13). There were no significant

correlations between milk and plasma lycopene concentrations for the control group.

No significant correlations (r = 0.327 - 0.353, p = 0.42) were observed between milk

lycopene and milk fat content estimated by the creamatocrit method at baseline or after

intervention in any group.

DISCUSSION

This study demonstrates that in a group of lactating mothers a daily serving of

fresh or processed tomato products will significantly increase plasma lycopene in 3 days. Increased plasma lycopene concentrations resulted in significantly increased milk

lycopene concentrations only when processed tomato sauce was consumed. Based on reasonable dietary intake and equivalent lycopene amounts, tomato sauce seems to be more effective at rapidly increasing plasma and milk lycopene concentrations compared to fresh tomatoes.

We compared our baseline milk and plasma lycopene concentrations to those previously reported for a population of lactating women. The total plasma lycopene concentrations observed at baseline (0.743 ± 0.03 pmol/L for all subjects) are

116 comparable to the 0.67 - 0.84 pmol/L reported previously (3, S, 10). In contrast, baseline milk lycopene concentrations in our study (174 ± 30 nmol/L and 3.9 ± 0.4 nmol/g fat) were higher than the previously reported values of 26.7 nmol/L and 0.46 nmol/g lipid (3), 19.9 nmol/L (5) and 47.6 nmol/L and 0.77 nmol/g lipid (10). These previous studies report data from a smaller number of subjects [n = 5 - 6 (3,10), n = 3

(5)], than that in the current study (n = 24). The large differences in milk lycopene concentrations are most probably due to the sampling of carotenoid rich hind milk, but may also be a result of differences in diet, sample size, or method of analysis.

Lycopene concentrations in blood have been shown to increase afrer as little as 7 days of daily consumption of tomato products (18, 19). Pateau et al. (19) observed an increase in blood lycopene concentrations afrer a one-week consumption of tomato juice in a non-lactating group. We were able to demonstrate a significant increase in total plasma lycopene in both treatment groups afrer only 3 days of consumption in a group of lactating women. The levels consumed in the study are well within the range expected from daily consumption of lycopene containing food products.

Previous studies have demonstrated the increased bioavailability of a processed tomato product over fresh tomatoes (20,21). Total plasma lycopene concentrations were significantly higher afrer tomato puree intake than afrer raw tomato intake (20) each containing 16.5 mg lycopene/serving. Lycopene (23 mg) from a single dose of either fresh tomatoes or tomato paste ingested with com oil yielded a 2.5 - fold higher total concentration when paste was consumed (21). In our study, subjects consumed equivalent (50 mg) amounts of lycopene over 3 days, and we observed a 1.8 - fold

117 higher total concentration in plasma lycopene when processed sauce was consumed.

This result is in agreement with Gartner et al. (21), but the difference in treatment groups did not reach significance as the Porrini et al. (20) study did.

Examining the concentration of lycopene and its isomers in milk is a simple, non-invasive method to investigate transfer of lycopene from blood to the breast and secretion in milk. We observed a significant (25%, p < 0.01) increase in milk lycopene concentrations afrer a 3 day intervention for those in the processed group, perhaps due to greater bioavailability. The first carotenoid intervention study in a group of lactating women was with p-carotene (3), A one time 60 mg supplement of p-carotene sustained elevated (4.1 - fold) p-carotene concentrations in milk for > 1 wk in healthy mothers.

Carotenoids are presumed to enter the mammary epithelial cell via lipoproteins, although the exact method of transport has not been identified.

High variation has been observed in carotenoid concentrations in human milk, both within and between individuals (6). These variations pose significant limits on the design and interpretation of studies of milk carotenoids. Milk lycopene has been shown to be more variable within a person than it is between persons (6). To decrease variability within milk samples, all HPLC analyses were performed in triplicate with results indicating a coefficient of variance of < 5%. It would be ideal to analyze a 24 h pool of milk from each lactating subject, but it was impractical since that would interfere with normal infant feeding patterns.

Carotenoids are associated with the lipid fraction of breast milk and therefore enriched in hind milk. It has been suggested that a complete breast emptying is

118 preferred when quantitation of total breast milk carotenoids is required (22). Since this may adversely effect the infant feeding patterns it is rarely practical in clinical studies.

Fat content of milk is highly variable within and among individuals and can change during nursing (23). These factors contribute to interpretation problems when assessing carotenoid content in milk samples. It has been suggested that when quantifying carotenoids in milk samples from partial breast expressions, carotenoid concentrations are most appropriately expressed relative to lipid (6). Other researchers have exclusively used fore milk and collected samples at the same time of day (4). Lipid content of milk can be estimated through the creamatocrit method of Lucas et al. (16).

Our study collected hind milk, sampled in the afremoon, and calculated lycopene concentrations both with and without accounting for milk fat. The significant increases observed when expressing results on a per liter basis were not as profound when expressed on a per gram fat basis. We did not find a significant correlation (r = 0.327 -

0.353, p = 0.42) between milk fat content and milk lycopene concentrations. Our study may not have been powerful enough to identify significant difference between groups and a larger study should be performed to confirm this finding. Until more data are available regarding carotenoid transfer to milk, it will be most helpful to report findings per volume as well as per gram lipid.

Examining the correlations between plasma, milk and BMC lycopene levels gives us information about how diet influences circulating lycopene levels, tissue deposition and tissue secretion levels. Buccal mucosal cell lycopene levels increased approximately 25% afrer daily consumption of tomato juice (24) over 4 weeks but were

119 not statistically significant. Another study showed an increase (48.4%) in buccal mucosal cell lycopene levels but the change did not reach statistical significance for consumption of juice (18). With weekly sampling of BMC this study observed a delay in response of cellular lycopene levels compared to blood levels following dietary intervention. The delayed response in BMC coincides with the maximal changes in the blood lycopene concentrations observed after 2 weeks, supporting the hypothesis that circulating levels may change more rapidly and BMC content will respond, but at a more gradual pace. There have been conflicting reports correlating blood lycopene and

BMC lycopene levels with some suggesting no correlation (24,25) or good correlation

(26). These studies based their correlations on samples of the general population without dietary intervention or estimated intakes. With the intervention study making its comparison at one time point at the end of 4 weeks of feeding. Our lab has found

(18) a brief lag time for an increase in BMCs as compared with plasma. The present study showed no change in BMC lycopene concentrations after 3 days of supplementation, and therefore no correlation with blood or milk lycopene. Based on studies previously mentioned, this result is expected for the relatively short intervention time period. We observed significant correlations in milk and plasma cû-lycopene when processed sauce was consumed, suggesting a mechanism by which human milk content rapidly responds to increased circulating lycopene concentrations.

The nutritional significance of lycopene in the neonatal, newborn or infant period has not been established. Non provitamin A carotenoids such as lycopene possess strong antioxidant capability (12). Consumption of foods rich in carotenoids

120 should alter circulating maternal carotenoid concentrations, and thereby modify the carotenoid status of the nursing infant. Significant differences were observed between formula fed and breast fed infants, with plasma lycopene concentrations not detected in formula fed infants at age 2 weeks and the concentration in breast fed infants significantly higher than at birth (27). Plasma concentrations of tocopherols, carotenoids, retinol and lipid are low at birth, with no relationship found between maternal and cord blood plasma carotenoids (8). Although no correlations were found between maternal and cord plasma lycopene concentrations, a very strong correlation (r

= 0.975, p < 0.0001) was observed when an adjustment for plasma triglycerides was made, making the correlation the highest of all the carotenoids (9).

In conclusion, our data show that supplementation of healthy, well nourished lactating mothers with tomato based products will substantially increase plasma and milk lycopene concentrations without changing concentrations of p- or a-carotene. Milk lycopene levels seem to be most effected when processed sauce is consumed. Based on equivalent lycopene amounts, tomato sauce seems to be more effective at increasing plasma and milk lycopene concentrations than do fresh tomatoes.

121 REFERENCES

1. Krinsky NI. Actions of carotenoids in biological systems. Annu Rev Nutr 1993;13:561-87.

2. Stoltzfus RJ, Hakima M, Miller KW, Rasmussen KM, Dawiesah SI, Habicht JP, Dibley MJ. High dose vitamin A supplementation of breast-feeding Indonesian mothers: Effects on the vitamin A status of mother and infant. J Nutr 1993; 123:666- 675.

3. Canfield LM, Giuliano AR, Neilson EM, Yap HH, Graver EJ, Cui HA, Blashil BM. P-carotene in breast milk and serum is increased after a single P-carotene dose. Am J Clin Nutr 1997;66:52-61.

4. Johnson EJ, Qin J, Krinsky NI, Russell RM. p-carotene isomers in human serum, breast milk and buccal mucosa cells after continuous oral doses of all-trans and 9- cis P-carotene. J Nutr 1997;127:1993-1999.

5. Khachik F, Spangler J, Smith JC. Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal Chem 1997;69:1873-1881.

6. Giuliano AR, Neilson EM, Yap HH, Baier M, Canfield LM. Quantitation of and inter/intra-individual variability in major carotenoids of mature human milk. J Nutr Biochem 1994;5:551-556.

7. Patton, S., Canfield, L.M., Huston, G.E., Ferris, AM. and R.G. Jensen. Carotenoids in Human Colostrum. Lipids 1990;25(13): 159-165.

8. Kiely M, Cogan PF, Kearney PJ, Morrissey PA. Concentrations of tocopherols and carotenoids in maternal and cord blood plasma. Eur J Clin Nutr I999;53(9):711- 715.

9. Yeum KJ, Ferland G, Patry J, Russell R. Relationship of plasma carotenoids, retinol, and tocopherols in mothers and newborn infants. J Am Coll Nutr 1998;17(5):442- 447. 122 10. Canfield LM, Giuliano AR, Neilson EM, Blashil BM, Graver EJ, Yap HH. Kinetics of the response of milk and serum P-carotene to daily P-carotene supplementation in healthy, lactating women. Am J Clin Nutr 1998;67:276-83.

11. Block G, Patterson B and Subar A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 1992; 18:1-29.

12. Rao AV and Agarwal S. Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: a review. Nutr Research 1999; 19(2):305-323.

13. Neville MC, Allen JC, Watters C. The mechanisms of milk secretion. In Lactation: physiology, nutrition, and breast feeding. Eds. MC Neville and MR Neifert. Plenum Press, New York. 1983:49-92.

14. Peng Y-S and YM Peng. Simultaneous liquid chromatographic determination of carotenoids, retinoids and tocopherols in human buccal mucosal cells. Cancer Epidemoilogy, Biomarkers & Prevention 1992;1:375-382.

15. Nguyen ML and Schwartz SJ. Lycopene stability during food processing. PSEBM 1998;218:101-105.

16. Lucas A, Gibbs JAH, Lyster RLJ, Baum JD. Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk. Br Med J 1978;1:1018-20.

17. Ferruzzi MG, Sander LC, Rock CL and SJ Schwartz. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Analytical Biochemistry 1998;256:74-81.

18. Moxley C, Schwartz S, Craft N, DeGroff V, Giovannucci E, Clinton S. Blood lycopene concentrations increase in healthy adults consuming standard servings of processed tomato products daily. Presented at American Institute for Cancer Research Meeting. Sept 2-3,1999. (abstr).

19. Pateau I, Khackik F, Brown ED et al. Chronic ingestion of lycopene-rich tomato juice or lycopene supplements significantly increases plasma concentrations of lycopene and related tomato carotenoids in humans. Am J Clin Nutr 1998;68:1187- 1195.

20. Porrini M, Riso P, Testolin G. Absorption of lycopene from single or daily portions of raw and processed tomato. Br J Nutr 1998;80:353-361.

123 21. Gartner C, Stahl W, Sies H. 1997. Lycopene is more bioavaîlable from tomato paste than from fresh tomatoes. Am J Clin Nutr 1997;66:116-22.

22. Kim Y, English C, Reich P, Gerber LE, Simpson KL. Vitamin A and carotenoids in human milk. J Agric Food Chem 1990;38: 1930-1933.

23. Hall B. Uniformity of human milk. Am J Clin Nutr 1979;32:304-312.

24. Pateau I, Rao D, Wiley ER et al. Carotenoids in human buccal mucosa cells afrer 4 wk of supplementation with tomato juice or lycopene supplements. Am J Clin Nutr 1999;70:490-494.

25. Cooney RV, Bertram JS, Hankin JH et al. Relationship between dietary, serum, and tissue levels of carotenoids. Cancer Lett 1991;61:81-87.

26. Peng YM, Penh YS, Lin Y et al. Concentrations and plamsa-tissue-diet relationships of carotenoids, retinoids, and tocopherols in humans. Nutr Cancer 1995;23(3):233- 246.

27. Sommerburg O, Meissmer K, Nelle M, Lenartz H, Leichsenring M. Carotenoid supply in breast-fed and formula-fed neonates. Eur J Pediatr 2000; 159:86-90.

124 p-carotene

all-lraifs lycopene a-carotene

Cis lycopene Isomers

«

Figure 1. Representative HPLC chromatogram (C30) illustrating the separation of carotenoids in human milk extract. 0.2

0.15

0.1

I 0.05 Tnm -0.05 ' ^significant change from baseiine (p < 0.05)

-0.1 K total 5-cis all-trans other cis □Control BFresh □ Processed

Figure 2. Plasma lycopene changes after 3-day intervention with lycopene free diet (control), fresh tomatoes (fresh) or processed tomato sauce (processed). 50 40 30

20

1 10 e <3 0 unn -10 n -20 = significant change from baseiine (p < 0.05). -30 total 5-cis all-trans other-cis [DControl 5 Fresh □Processed

Figure 3. Milk lycopene changes after 3-day intervention with lycopene free diet (controi), fresh tomatoes (fresh), or processed tomato sauce (processed). Control Fresh Processed

total lycopene ' 0.012 0.508 0.580 all-trans lycopene ^ 0.066 0.568 0.338 5-cis lycopene 0.193 0.682 0.356 total cis-lycopene * -0.044 0.385 0.822

K Table 1. Spearman rank order correlations of lycopene concentrations in plasma and human milk after intervention for all treatment groups during the study.

' trending for Processed, p=0.13 ^ trending for Fresh, p=0.14 ^ significant for Processed p<0.01 DISSERTATION SUMMARY

The research described in the previous document was designed to evaluate the effects of consuming a reasonable dietary intake of various tomato products on the lycopene concentrations circulating in blood, stored in buccal mucosal cells (BMC), and secreted in human milk. A brief summary of each of the clinical studies follows with suggestions for future work in the area.

The first study investigated plasma and BMC concentrations of subjects who consumed either a high cw-lycopene variety tomato {Tangerine) or the more typical all-trans lycopene variety tomato. The body typically contains a higher percentage of cis lycopene when compared to all-trans. The mechanism by which the conversion from dietary all-trans lycopene to predominantly c/5-lycopene in circulation and storage is unknown. This study demonstrated that cis isomers of lycopene in the diet can subsequently be detected circulating in plasma. Consumption of dietary cis- isomers of lycopene from Tangerine tomatoes rapidly alters circulating patterns of cis- isomers. The increase in total lycopene blood concentrations was significant when the high cû-lycopene sauce was consumed, suggesting that the absorption of cis isomers is more efficient. Future studies should address the formation, interconversion, and biofimction of lycopene for a better understanding of the reason for the different isomer ratios in biological samples. The Tangerine variety tomato is uniquely suited 129 to further study in this area because of the large content of cw-lycopene isomers. Ceil culture work will perhaps define the point of further isomerization during absorption.

Analytical work will be needed to definitively identify the geometric isomers of lycopene and extinction coefficients will need to be established so that concentrations in the body can accurately be determined.

The second study examined the effects of consumption of standard daily servings of commercially available tomato product on lycopene concentrations in plasma and BMC. Epidemiology studies determine reduction in risk factors based on standard serving size (on nutritional label) but few intervention studies have focused on dietary consumption based on the standard serving of lycopene containing products. This study demonstrates that plasma lycopene decreases approximately 40% after 1 week on a lycopene free diet and only an additional 8% afrer 2 weeks. This would indicate that an appropriate intervention trial would include a one-week lycopene free portion to ensure that all subjects begin at a similar intake level. A single daily serving of processed tomato products (spaghetti sauce, tomato soup, or VS juice) can significantly increase blood and tissue lycopene. Standard servings of tomato products vary in lycopene content and result in different blood concentrations.

A two-week intervention of the food products was sufficient to increase plasma lycopene to what is believed to be protective levels for reduction of risk. Additional studies of lycopene bioavailability are also necessary in order to understand epidemiologic relationships between dietary tomato products, lycopene, and disease outcomes. This study also defines parameters of the washout and intervention that are

130 most appropriate to model future studies. A study focusing on a population over age

40 would be necessary to address the correlations between plasma lycopene and cholesterol observed in epidemiology studies as well as changes in absorption patterns that are commonly believed to occur with aging. Intervention studies with subjects having a specific disease process would focus on the consumption of tomato products to lower risk factors for diseases. There have been few studies addressing the consumption of dietary lipid in conjunction with tomato products and a controlled experiment should address the validity of the hypothesis as well as the amount and type of lipid necessary for absorption.

The third study examined the effects of consumption of either fresh or processed tomato sauce on lycopene concentrations in plasma, breast milk and BMC.

For those infants who are strictly breast fed, all beneficial dietary components would need to come from nursing. Lycopene is an antioxidant whose beneficial qualities have not been investigated in breast milk. If lycopene concentrations can be increased in milk then that increase should be transferred to the nursing infant. In a group of lactating mothers, a daily serving of fresh or processed tomato products will significantly increase plasma lycopene in a short period of time (3 days). Significantly increased breast milk lycopene concentrations are noted in women consuming processed sauce in a study of short duration. Based on equivalent lycopene amounts, processed tomato sauce seems to be more effective in rapidly increasing plasma lycopene concentrations and enhancing the transfer of lycopene to milk compared to fresh tomatoes. Processed tomato products have been shown to be more effective at

131 increasing blood and tissue lycopene concentrations in previous studies. The difficulties of analyzing samples from lactating women are many and ideal conditions are often not practical. Future studies should confirm these findings while trying to maintain as close to ideal experimental conditions as possible. A total breast expression or a 24-hour sample would increase validity of the study. Enrollment for studies with lactating women is very difficult and often the limiting factor for completion of the work. Future work would have to identify a large enough population or have the funding to enroll subjects over multiple years to have the desired power. Benefits that the infant may incur from increasing maternal consumption of tomato products need to be addressed. There are many issues of antioxidant potential in the nursing infant that can be explored. As these are addressed, guidelines for dietary consumption while nursing can be revised and recommendations eventually made to infant formula companies for inclusion of lycopene or other phytochemicals.

An overall conclusion from this work is that consumers are able to consume reasonable and beneficial amounts of lycopene from processed products readily available on the market. The food industry will most likely continue to investigate the possibility of making label claims on their tomato based products as the volume of research in this area continues to grow.

132 APPENDIX A

Extraction Procedures

133 Lycopene Extraction Procedure from Tomato Fruit

1. Weigh out - 5.000g of the sample on an analytical scale. 2. Add ~ 1 .OOg calcium bicarbonate and ~ 4.00g of Celite as filtering aid. 3. Add 50ml of Methanol and allow mixture to stand for approx. 1 minute. 4 . Homogenize the mixture for approximately 1 minute. 5 . Filter the sample using Watman paper #1 and #42 (#42 on the bottom). 6. Collect and save the filtrate. 7. Remove the filtrant quantitatively. 8. Add 50.0ml of 1.1 Acetone/Hexane. 9 . Allow the mixture to stand for approximately 1 minute. 10. Homogenize the mixture for approximately 1 minute. 11. Filter the sample using Watman paper #1 and #42 (#42 on the bottom). 12. Collect and save the filtrate. 13. Repeat steps 7 to 12,2x (total extraction = 3x). 14. Discard filtrant. 15. Wash the filtrate with ~ 10ml of distilled water. 16. Remove the bottom layer (waste). 17. Retain top layer and repeat 2x (total rinsing = 3x). 18. Transfer top layer to 100ml volumetric flask. 19. Top off with hexane. 2 0 . UV readings at 471 nm. 21. Transfer five 3.0ml aliquots into glass vials and dry under nitrogen. 22. Discard the rest of the hexane solution. 2 3 . Re-dissolve lycopene in vial with appropriate volumes of MTBE followed by methanol. 2 4 . Filter with 0.2 um syringe filter before injection.

134 Buccal Cell Procedure

A. Collection of Samplgs Collection tubes - add 50 ul of 1% BHT - methanol solution to each tube, vortex, dry in hood.

Rinse tube - 50 ml tube with 0.4 g table salt and 50 ml drinking water.

Method - volunteers asked to rinse mouth with drinking water and then brush inside of cheek with a soft toothbrush, 20 times on each side (one up down stroke = 1). Rinse by swirling vigorously with 20 ml of the table salt solution. Deposit spit in collection tube coated with BHT. Repeat rinsing once. Wash the toothbrush witii the remaining 10ml of salt solution and deposit in tube.

B , Proçgssine of Samples All procedures carried out under dim light. 1. Centrifuge at 1400 x g for 10 minutes under refrigeration. 2. Discard supernatant, add 15ml of cold PBS solution. Vortex, centrifuge at 1400 x g for 5 min. 3. Remove supernatant, add 1.2 ml of cold PBS, vortex. 4. Pipet 1ml of cell suspension into a BHT-coated microcentrifuge tube, centrifuge at 13000 X g for 1 min. 5. Discard supernatant, flush cell pellet with N% for 20 sec. cap tightly, store at — 80°C 6. From step 4, also take 2 separate 100 ul samples and place them in 15 ml centrifuge tubes for protein analysis.

C. Extraction Procedure 1. Thaw samples at RT. 2. To each cell pellet, add 200ul of protease solution (10ml cold PBS + 100 mg protease), digest at 37®C for 30 min. 3. Add 500ul of SDS-ethanol-BHT solution, pellitize. 4. Add 500ul hexaneracetone (2:1) containing 0.1% BHT, vortex 60s., centrifuge 13000xg Imin. 5 . Remove upper hexane level and transfer to microcentrifiige tube. 6. Repeat extraction once. 7. Combine two hexane layers and dry under nitrogen. 8. Resolubitize in 200ul MTBE then 200ul Methanol. 9 . Filter with 0.2 um syringe filter before injection.

135 Plasma Extraction

1. Remove vial from -80®C freezer.

2. When thawed, remove 100 ul and place in microcentrifuge tube.

3. Add 100 ul ETOH with 0.1% BHT and vortex 10 sec.

4. Add 0.5 ml Hexane/Acetone (2:1) with 0.1% BHT, vortex 30 sec.

5. Centrifuge 10,000 rpm for 1 minute.

6. Remove hexane layer and place in 11 ml vial.

7. Repeat Ix and combine hexane layers.

8. Dry under a stream of nitrogen.

9. Bring up in 200 pi MTBE and 200 pi MEOH

10. Filter with a 2 pm syringe filter.

136 Milk Extraction Procedure

1. Defrost milk.

2. Add to beaker: milk (5ml), 3% Ascorbic acid in ETOH (2.5ml), 30% KOH in

MEOH (10ml).

3. Stir at 400 rpm for 45 min at room temperature.

4. Transfer to 50 ml centrifuge tube.

5. Add 5 ml hexane/acetone (2:1) with BHT, vortex, centrifuge 5 min.

6. Remove hexane layer to 15 ml centrifuge tube.

7. Repeat steps 5 and 6 for a total of 3 extractions. Combining hexane layers.

8. To combined hexane layers add 3 ml dH20, vortex, centrifuge 5 min.

9. Remove hexane to another 15ml centrifuge tube, add 3ml dH20, vortex, centrifuge 5 min.

10. Remove hexane to glass vial.

11. Dry under N;

12. Bring up in 200 ul MTBE and 200 ul MEOH.

137 APPENDIX B

Supplements to Chapter 2 - Tangerine study

138 Carotenoid Tangerine - before Tangerine - after Roma - before Roma - after Intervention intervention intervention intervention

total iycopene 0.56 ± 0.05 0.73 ±0.06' 0.64 ± 0.04 0.65 ± 0.05 cis iycopenes 0.30 ± 0.04 0.39 ± 0.05' 0.35 ± 0.03 0.34 ±0.03 tetra-cis iycopene nd 0.08 ± 0.02' nd nd ail-trans Iycopene 0.25 ± 0.01 0.26 ±0.01 0.28 ± 0.02 0.32 ±0.02^ p-carotene 0.44 ±0.06 0.39 ±0.06^ 0.42 ±0.08 0.38 ± 0.08 a-carotene 0.02 ±0.01 0.02 ± 0.01 0.02 ±0.01 0.01 ±0.01

VO

Table 1. Plasma carotenoid levels before and after intervention.'

pmoles/L (mean ± sem) ^ p<0.001 ^p<0.005 ^|X0.05 0.03

tetra-ci5 0.15 Iycopene isomers

0.0 AU 0.06 phytofluene

phytoene 0.03

0.0

20 40 60 minutes

Figure 1. Tangerine tomato (LA 3002) extract at 471 nm (top) and a max plot of 250 - 550 nm (bottom).

140 0.012

Iycopene isomers 0.006

0.0 AU

phytofluene unknown product phytoene

0.025

0.0

20 40 60 minutes

Figure 2. Tangerine tomato sauce (LA 3002) extract at 471 nm (top) and a max plot of 250-550 nm (bottom).

141 General Clinical Research Center

Protocol Title;

The effect of tomato intake on Ivcopene levels in serum and buccal cells. A pilot study

Submitted by: Steven J. Schwartz, Ph.D. Charlotte Moxley

142 BASIC INFORMATION

PrQtQCol Data

1. Protocol Title The effect of tomato intake on Iycopene levels in serum and buccal cells.

2. Start/End Date: 9/1/98-9/1/99

3. Resources requested Nursing, Computer, Biostats, Core Lab

Total IP Days - 0 Total OP Days - 40 Total Patients -1 0

IRB Information

1. IRB Number: 98H0254

2. Date of Approval

3. Revised?

Funding Information for PI and each Co-PI (Use additional sheets if necessary)

1. Title: Steven J. Schwartz, Ph.D. Professor

2. Purpose of Research (two or three lines) The purpose of this study is to determine the effects of consuming different tomato varieties on the Iycopene content of blood and buccal cells.

3. Funding source: HAAS Endowed Chair Account

4. Kind: University Endowed Chair Account

5. Agency: OSU

6. Grant/Contract Number: 522256 REF# HAAS

7. Start/End Dates: Continuous

8. Annual Costs: 143 Direct: $500.00 Total: $500.00 9. Percent Commitment: 100%

10. Suggested reviewers for this protocol.

Name: Anne Smith, Ph D. Office address: 343E Campbell Hall, 1787 Neil Ave Phone: 2-0715 Fax: 2-8880 E-mail: [email protected]

Name: Steven K. Clinton, M.D., Ph D. Office address: B402 Starling-Loving Hall, 320 West 10* Ave. Phone: 3-8396 Fax: 3-4372 E-mail: [email protected]

144 CORE LABORATORY UTILIZATION WORKSHEET

Principal Investigator Steven J. Schwartz. Ph.D.

Protocol Title The effect of tomato intake on Ivcopene levels in serum and buccal cells

Sources of funding for proposed project (please check as appropriate)

X Other: HAAS Endowed Chair Account

Has this proposal been approved by funding agency? Yes 522256 REF# HAAS

Will this study be performed on a GCRC? Yes Study is now being submitted for approval

GCRC Core Laboratory: assays requested.

Name of assay # of analysis

145 GENERAL CLINICAL RESEARCH CENTER BODY OF THE PROTOCOL

A. Name and Title of Investigators

Steven J. Schwartz, Ph D. Professor 140 Howlett Hall 2001 Fyffe Ct. Columbus, OH 43210

Charlotte Moxley Graduate Research Assistant 0598 Howlett Hall 2001 Fyffe Ct. Columbus, OH 43210

B. Abstract

The purpose of this study is to determine the effects of consuming processed tomato products, ie. spaghetti sauce, on the carotene content, specifically Iycopene, of blood and buccal cells. Individuals will be asked to consume a given quantity of spaghetti sauce from either red tomatoes or orange tomatoes after a period of up to two weeks of no tomato consumption. Food will be provided and will be commercially processed. If consent is given for blood sampling, blood samples will be drawn and buccal cell samples obtained before and after the feeding period of 4 days. Following a period of 2 weeks of no tomato intake, subjects will donate blood samples as well as buccal mucosal cell samples to establish low levels of Iycopene. Subjects will then be asked to consume a given amount of spaghetti sauce from orange tomatoes per day for the next 4 days. Blood and buccal mucosal cell samples from the subjects, will be drawn on a

146 consensual basis. The subjects will then limit their intake of tomato products for 2 weeks. Blood and buccal cells samples will be taken to once again establish low levels of Iycopene. Subjects will then be asked to consume a given amount of spaghetti sauce from red tomatoes per day for the next 4 days. Blood and buccal mucosal cell samples from the subjects, will be drawn on a consensual basis. Blood sampling will be done at the OSU Clinical Research Center, by qualified medical personnel.

C. Hypothesis and Specific Aims.

The proposed research has been designed to answer the following questions:

1. What are the differences in plasma profiles after consumption of zW-trans Iycopene from red tomatoes as compared with poly-c/5 Iycopene from orange tomatoes?

2. Do buccal cell Iycopene profiles correlate with plasma profiles?

The study is designed to limit the dietary intake of certain carotene containing foods for up to two weeks before the first sampling is performed. The subjects will then be required to consume a predetermined amount of spaghetti sauce that will be provided for 3 days. A second sampling will hopefully show an increase in the carotenoid levels in blood and buccal cells corresponding to an increase in dietary intake. The differences anticipated in red and orange tomatoes will be seen in the m-isomer content of the tomatoes and, secondly, if after consumption these differences reflect the amount of cM-isomer in the blood and buccal cells.

D. Background and significance

Lycopene has recently emerged as a potentially beneficial dietary phytochemical in light of accumulating research findings which show an inverse correlation between consumption of food products high in lycopene and the risk of developing certain types

147 of cancer (Block et al., 1992; Giovannucci et al., 1995). Other studies involving lycopene reveal it’s superior ability to quench singlet oxygen among dietary carotenoids (Di Mascio et al., 1989), chemopreventative properties in animal models and cell cultures (Levy et al., 1995), as well as interesting bioavailability and tissue deposition patterns in terms of concentration and isomeric distribution (Clinton et al., 1996; Gartner et al., 1997; Schierle et al., 1997). Clinton et al. (1996) observed that the ratio of lycopene cis-trans geometrical isomers in biological fluids such as plasma and in tissues such as prostate differ from those isomer ratios in fresh tomatoes. It has previously been assumed that the higher percentage of lycopene cis- isomer in human biological samples is due in part to consiunption of heat treated tomato products containing cis- isomers of lycopene. Our laboratory has recently established, however, that in tomato products of various moisture content, fat content, and container type, lycopene, unlike P-carotene, is remarkably stable to isomerization reactions under typical industrial thermal processing conditions (Nguyen and Schwartz, 1998).

Two different varieties of tomatoes are used in this study: red tomatoes with typical carotenoid composition and orange tomatoes with high prolycopene levels to monitor the serum and buccal cell lycopene response. Tangerine-type tomatoes (Tangella and Tangerine Golden Jubilee tomatoes) are uniquely suited for this study due to their biosynthesis of ds-isomers of lycopene, especially poly-ds lycopene such as prolycopene. Prolycopene, with four of its eleven double bonds in the cis conflguration is the geometrical isomer and the biochemical, metabolic precursor to 2\\-trans lycopene (Zechmeister, 1962).

E. Experimental Design and Methods

1. Patient selection criterion. Patients have been recruited within the Food Science Department at Ohio State University. Subjects must be non-smokers and not pregnant. Every effort has been made to include a subject profile from a variety of ethnic groups.

148 American Asian or Black, Non- Hispanic White, Non- Total Indian Alaskan Pacific hispanic Hispanic Native Islander Female 0 2 0 0 3 5 Male 0 1 0 0 4 5 Total 0 3 0 0 7 10

Subjects must also be willing to follow the dietary restrictions for the wash out periods. Foods to be avoided include: tomatoes, spaghetti sauce, tomato juice, salsa, ketchup, watermelon, and any product containing either fresh or processed tomato products such as lasagna and pizza.

2. Method of patient evaluation. Patient evaluation will involve an informational sheet and a dietary record for the course of the study. Patients will be asked to sign an Informed Consent. Patients will be contacted by telephone to ensure compliance with sampling dates and dietary requirements. Patients must be willing to consume tomato products. To assist in monitoring compliance, the lunch feeding will be occur in the Food Science Department and feeding will occur as a group. Subjects will be required to return the empty food containers for the dinner feeding.

3. Trial design. In each investigation the control will consist of the baseline samples obtained before the desired amount of food is consumed and after the intake of carotene containing foods has been limited.

To investigate "What are the differences in plasma profiles after consumption of all-trans Ivcopene from red tomatoes as compared with poly cis Ivcopene from orange tomatoes?” Subjects will be asked to limit their intake of tomato products, for up to 2 weeks prior to the start of the study. Blood and buccal cell samples will be obtained to demonstrate lowered levels of lycopene. Subjects will then be asked to consume a given portion of spaghetti sauce from orange tomatoes for the next 4 days. A second sample of blood and buccal cells will be obtained. The same 10 subjects will then be asked to limit their intake of tomato products for 2 weeks. A third sample of blood and buccal cells will be obtained to demonstrate a return to lowered levels. Subjects will then be asked to consume a given portion of spaghetti sauce from red tomatoes for the next 4 days. A fourth sample of blood and buccal cells will be obtained.

149 To investigate "Do buccal ceil Ivcopene profiles correlate with plasma profiles?” Lycopene profiles of buccal cells will be compared with plasma profiles.

4. Treatment schedule. When patients have been contacted and have signed the consent form, they will be asked to limit their intake of some carotene containing foods for up to two weeks. Subjects will be scheduled for their first sampling. Subjects will be instructed to consume a specified portion of spaghetti sauce from orange tomatoes per day. On day four, subjects will be scheduled for their second sampling. Subjects will be asked to again limit their intake of some carotene containing foods for two weeks. Subjects will be scheduled for their third sampling. Now the same subjects will be given spaghetti sauce made from red tomatoes, they will then be given a processed food product and vice versa. Subjects will then be instructed to consume a specified portion of the provided food product per day. Four days later, subjects will be scheduled for their fourth sampling.

F. Biostatistical Design and Analysis

Registration and assignment of patients to groups. Patients will be registered when contact is made and they have signed an informed consent. All patients will consume the orange tomato sauce first and then will consume the red tomato sauce second. The study will be of a crossover design in that all subjects will participate in both treatment groups with a washout period afrer the first treatment is completed.

Sample size choice; Ten subjects will be used for this pilot study. The choice of sample size is governed by availability of orange tomatoes. Limited crop availability will limit the sauce that can be made with the tomatoes. With an effect size of 0.8 and an alpha of 0.05 the power of the study will be 0.53 (ni = 10, U 2 = 10).

Monitoring of trial progress. Progress will be monitored to ensure an equal number of subjects are in each group and subjects are adhering to guidelines. Subject dropout may be a factor and may result in less than 10 subjects participating. Dropout will be considered a failure if the study is not completed. Compliance will be monitored by conducting the lunch feeding as a group and requiring the return of the empty containers for the dinner feeding.

Forms of data handling Informational forms and dietary questionnaires will be kept in a locked file cabinet and will be used to assign subjects to treatment groups. Analysis of the blood serum

ISO and buccal mucosal cells for carotenoid profile will be performed in the above researcher’s laboratory by HPLC and or HPLC/ECD (Electrochemical Detection).

Protocol deviations Protocol deviations will be kept to a minimum. The anticipated deviations are dropout and non-compliance to the dietary restrictions. Frequent reminders and close monitoring will hopefttlly keep this to a minimum.

Plans for statistical analysis Differences in carotenoid content between processed and unprocessed food groups, as well as before and after supplementation will be determined by analysis of variance (ANOVA). Tukey’s HSD test will be used to detect the differences between groups. Correlation coefficient will be obtained by correlation analysis to examine relationship between dietary intake, serum levels, and buccal cell levels of lycopene and prolycopene.

G. Bibliography

Block, G., Patterson, B., and A. Subar. 1992. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer IS: 1-29.

Clinton, S.K., Emenhiser, C., Schwartz, S.J., Bostwick, D.G., Williams, A.W., Moore, B.J., and J.W. Erdman. 1996. Cis-trans lycopene isomers, carotenoids, and retinol in the human prostate. Cancer Epidemiology, Biomarkers, and Prevention. 5: 823-833.

Di Mascio, P., Kaiser, S., and H. Sies. 1989. Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274:532-538.

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

Giovannucci, E.L., Ascherio, A., Rimm, E.B., Stampfer, M.J., Colditz, G.A., and W.C. Willett. 1995. Intake of carotenoids and retinol in relationship to risk of prostate cancer. J. Natl. Cancer Inst. 87: 1767-1776.

Levy, J.J., Bosin, E., Feldman, B., Giat, Y., Munster, A., Danilenko, M., and Y. Sharoni. 1995. Lycopene is a more potent inhibitor of human cancer cell proliferation than either a-carotene or P-carotene. Nutr. Cancer. 24:257-266.

Nguyen, M.L. and S.J. Schwartz. 1998. Lycopene stability during food processing. Pro. Soc. Exp. Bio. Med. 218: 101-105.

151 Schierle, J., Bretzel, W., Buhler, L, Faccin, N., Hess, D., Steiner, K., and W. Schuep. 1997. Content and isomeric ratio of lycopene in food and human blood plasma. J. Agric. Food Chem. 96:459-465.

Zechmeister, L. 1962. cis-trans Isomeric Carotenoids, Vitamins A and Arylpolyenes. Academic Press, New York.

H. Resource Request

Limited resources will be needed from the GCRC. Qualified individuals able to perform venipuncture will be needed to draw blood from the subjects. Buccal mucosal cell sampling will be performed by the above researchers. HPLC analysis will be performed on the blood serum, so blood will need to be separated into serum, lymphocytes, and red blood cells if possible. Samples of serum should be stored at -80°C until pick up by the researcher. Carotenoids are light sensitive and every effort should be made to limit exposure to light sources. In addition to the above laboratory assistance, any biostatistical assistance would be appreciated.

H. Justification for Use of the Facility

The GCRC will provide invaluable resources that are necessary for the successful completion of this research study. All of the above requested resources are not available to the researchers except from the GCRC.

I. Human Subjects

See enclosed

152 Biomedical Sciences

Institutional Review Board

Research Involving Human Subjects

The Ohio State University

153 BIOMEDIC.-SI, SCIENCES X Original Re%i«:w INS Hi m ON A L REVIEW BOARD Continniiig Review RESEARCH INN'OLVLNG rn.f>UN SUBJECTS " Amendment THE OHIO ST ATE L^TVERSITY Reactivation

ACT ION OF THE REVIEW BO.VRD

With regard to the emploj-mcnl of human $tibjccts m the proposed research:

9SH0254 THE EFFECT OF TOMATO INTAKE ON LYCOPENE LEVELS IN SERUM AND BUCCAL CELLS. Steven J. Schwartz. Foinl. .Agricultural, and En\ tronmcril.al

THE BIOMEDIC.-VL SCIENCES R E V IE W BQ.ARD IL\S TAKEN THE FOLLOWING ACTION:

APPROVED DISrSPPRQA'ED

X APPROVED WITH SriPUL.ATION(S)* ____ WAIVER OF WRITTEN CONSENT GRANTED

*Stipi’.latiou( 5t stated by the IRB have been met by the invcstig.ntor. and therefore, the protocol is APPROVED.

It is the respoiisibilitv' of the principal investigator to retain a copy of each signed consent form for at least three (3) years beyond the termination of the subject's participation in the proposed activity. Should the principal investigator leave the University, signed consent forms are to be transferred to the Hnman Subjects Review Board for the required retention period. This application has been approv ed for the period of one year. You arc reminded that yon must promptly report any problems to the Review Board, and that no procedural changes may be made without prior review and approval. You are also rcmiuded^uit the identity of the research pSfticipants must be kept conBdeutial.

Date: September 2 1 .199S Signed: ^ t.'hairpersori

154 1 1ll-: o u u ) SI A11*: iiNivKusi rv orritc lise: UIOMUDK'AU.St II-NCF-S l*rii(

l*rn(itc«>U receivetl iit (he o n ite i>f Rr^tarcU RUks PrnlecliMn after the ffratlline date (first Moiulay nf each imiiilh) Mill lie sclinliitrtl fur (he fnlhiMiiig mimtU's *nee(i»g. Raceptiim: Hie ilradline Is innvni to (he Friday liefiire Mmiday hnlldays If all (line slnls are filled and a {irntticol is rrceisrd on or before the deadline «late, the prolttcnl Mill lie scheduled for the following month's meeliuR Ihe IRH meets on (he third Monday of eaili iittinlh). Only {irotmols that are complete will he schednled for reviewr. Incomplète prntncnls will he rednnecl. riin ri|i;il Ittc csti^aior: Or. Steven .1. Scliwnriz < i S U / (i« iiff, K t /*/ ifiOtd / IpCl/ .V,H Ac.iilcitiic Title Proft's.snr i’hiiiic Nil 2-2') V» r.u

Kiioil, /\(>rirnlliii;il anil Vliivironnirntnl l)cti.ir(mcnt/Nc irU ïïyIfe.j;i.)uci..

{' c i Invcsligatiits' ______/ iV.o'ie S»r»n»nne

t \ fr.l V .m if V«C’'i»Ow»e

I'riiliiciil l iilc; lliij crrncl: nf toiivil ii ini aka on lyctnn’no levels in r.i'i'um anil Imt ra l rcU

*'cp;ictmcut I lcair(s) Fudnisctuenl: Dr. Ken Lee } .V.iMie

Crnpnsctl Ucsenrch Involves

\\S Si) □ B InvcMignlnnul Dtuyls) nt invcstig.Kittii.d use ul nuikcleil diiigs(s| If yes. ptuvide INI) Number Issued ui Clcucnc N.iiuc:

□ S luvcMigaticuuil Dcviccs(s) If yes. imhe.dc NSR . ut SR »f SR. ptttvnlc 11 )T N uiuKt ______. issued lu ______

□ S Kmliunclivc Drugs ur Unimml n%pusutcId r.xlcriul R;ulin(um Apptuval hy the Medic.d R.idwmuc.ltdc Cumimtlcc (Phoiic. 2^2 0122) IS ictiuitcd ptiur Ut aclivatiun. (ttvcsltgatur ts responsible fur ubiaimiiR approval from buili euiutudtccs.

□ a CaitLcr rclaicd Activutcs. Approval by (he fames Cancer Center Cliiiicnt Scientific Review Cumiuinec (Phone; 20 \ 107b) is rctpitrcd prior tu nctivaltutt. fnvcstigaUir is tespcmsddc for obtaining approval from both ctnnnnilecs.

□ □ Pregnant Women. Approval by MaternalT-cial cimmtiHcc (Phoiic; 2*73 S736) is rcr|uircd prior to aciivatnm InveMigaiur is responsible fur obtaining approval from both committees

□ □ Minors (f/iulcr (X ycats of age) □ 0 Cognitively Impaired □ □ l eioses/bi Viim I ciidi/ation □ □ Prisim cis

155 CA rKCOUII-S OK UIÛSKAIU II KLICIIII.K KOIt F.XIMCDITKI) KKVIKW UV HUMAN SUUJKCrS RKVIKW COMMHTKK

UcM-Micll aclivitics involving no mon: lhan iiiiniiiinl risk tjnil in wliidi the only involvement of hnman snhjetts will he m oitc or mote of the following categories (cairieil inn through stariilanl iriethoilO may Ire reviewed throueh the cxperliled review ptocerlritc:

Colleelioii of. hair and nail elip|iiiigs. in a noiithvlignring inaniier. ilev iihions teelh, and periiinncm teeth if paiieiil eaic imlic.iics a need (ür cxtraLtion

I ^ C'lillecKim Ilf cxL'icla ami c k Ic i i u I M xtclimis incliuting vwcat. uncaiiniilacat taliva. pt.ictrnia rcim ivcj al delivery, ami anmiodc llutd at liic time of luptuic of the inciiiluanc prior to or dm mg lalior

[ \ Rccimlmg of data fioiu mhjcctN IK years of age or older using nun invasive procedures routinely employed in clinical pintlice l lns mclmles the use ol physical scnsois that ate applied either to (he stii face of the hody or at a distance and do not involve input of matter or significant aiiiouiils of energy into the suh|cit ur an invasion of the suhject’s piivat y It also includes sm It proceduics as sseighing. testing sensory acuity, ciccirocatdiography, electroencephalography, thcriiiogiaphy. detection ol natiiially occinring radioactivity, diagnostic echography, and cicctioictinograpliy li il*-cs not incluile csposuie to cicclioiiiagiietic radiation outside the visible range, i e . s rays, nm lovvavcs

CnIIevlion ol libmil samples hy xeiiipuiictiiic. in aimuinls not exceeding -tVl millihiets in an eight week period and no more often than issu tunes per >^cck. from subjects IK years «I .igc or older ami who arc m g»un| health and not pregn.rnt

Collection of both supra and subgnigiv.il dental pl.it|ue,aiid calculus, provided the proccdtiic is not more mv.isive than routine ptophvlauic scaling of die teeth and the process is accomplished in accoolance with accepted prujdiylaclic icchmi|ues.

Voice rcciudmgs made for lese.m h puiposcs such as investigations ol speech defects

Moderate csercise by healthy solunteets.

I I I lie study of csisling data, duciiments. tecoids. pathological specimens, or diagnostic specimens

Research on individual or giotip Indiavior ur charnt let istics of individuals, such as studies of perception, cognition, game theory, or test development, where the investigator does not manipulate suh|ccts" Ixdiavior and the research will not involve Stress to subjects.

Rescan h on drugs or devices for which an investigational new ditigcsenipiton or an investigational device cscmptton is not rcipiired.

(For an nxrnfX T f-n R K yni^^ltt^k the appropit.ite box and return with the ciuiijiletc protoçoL]

IIS tuvi)(iir;2)

156 INIRODHCnON

l.ycopctie lias recently emerged as a potentially tiencficial dietary pliytochcinical in light of accumulating rcseaicli Imdings which show an inverse corrélation between consumption of food pioducts high in lycopene and the risk of developing certain types of cancer (Dlock et a l. Oiovamuicci et a l, 1995) Other studies involving lycopene reveal it's superior ability to iliiench singlet oxygen among dietary carotenoids (Di Mascio et a l. 1989), chemopreventative properties in animal models and cell cultures (Levy ct al, 1995). as well as interesting bioavailabilrtv and tissue deposition patterns in terms of concentration and isomeric distribution (( linton et a l. I99f>. Canner et al . |9<)7. Schierle et a l, 1997) Clinton et al (1996) observed that the ratio ol lycopene as-rnins geometrical isomers in biological fluids such as plasma and in tissues such as prostate dit 1er from those isomer ratios in fresh tomatoes It has previously been assumed that the higher percentage o f lycopene err- isom er in Inrmarr biological sam ples is due in part to corrsunrption of heat treated tomato prodrrcts currtairrirrg t/.r- isomers of lycopene Our laboratory Iras recently established, however, that in tomato products of various nruisturc content. Iht content, and container type, lycopene. unlike (l-caiuterrc. is remarkably stable to isomer i/at ion reactions tiinler typical industrial thermal processing conditions (Nguyen and .Schwart/, 1998)

Two dilTerent varieties of tomatoes arc used in this study ted tomatoes with typical carotenoid composition and orange tomatoes with high prolycopene levels to monitor the serum and huccal cell lycopene response Tangerine-type tomatoes (Tangella and Tangerine Golden Jubilee tomatoes) ate unnprely suited for this study due to their biosynthesis of f/.r-isorners of lycopene. especially poly cr.\ lycopene such as prolycopene Prolycopene. with four of its eleven dvnible bonds in the u s configuration is the geometrical isomer and the liroclremical. metabolic precursor to all-tnr/r.s lycopene (Zechmeister. 1962)

RESKARC 11 OB.IECn V ES

T.xpcrimerits have been designed to investigate the quest ions

1 What arc the di (Terences in plasma profiles aller consumption of all-rnrw lycopene from red tomatoes as compared with poly-tv.r lycopene from orange tomatoes'^

2 Do buccal cell lycopene profiles correlate with plasma profiles'

EXPERIMENTAL DESIGN

Subjects Terr non-smoking, non-pregnant individuals over the age of 18 will be required for this pilot study Blood scrum and buccal cells will be analy/.ed from these subjects

157 DMigii To invcsligaic ‘'Whal arc the dinctcticcs_.in plasma profiles ji.fter_cpnsumptio.n_or_.aJIjrans l_yeopenc_ltoijt red. toiiiatocsjs _compared with poly cis Ivcopene ftoin orange toiiiaitics>" Subjects will he asked to limit their intake of tomato products, for up to 2 weeks prior to the start of the study Ulood and buccal cell samples will be obtained to demonstrate lowered levels of lycopene. Subjects will then be asked to consume a given portion of spaghetti sauce from orange tomatoes for the next 4 days A second sample of blood and buccal cells will be obtained The same 10 subjects will then be asked to limit their intake of tomato pioducts for 2 weeks A third sample of blood and buccal cells will he obtained to demonstrate a return to lowered levels Subjects will then he asked to consume a given portion of spaghetti sauce from red tomatoes foi the next 4 days A fourth sample of blood and buccal cells will be obtained

To investigate "Do buccal cell lycopene profiles corrclatc_with plasma profiles'’" l.ycopenc prolilcs of buccal cells will be coinpaicd with plasma piofilcs

lU SI ARC II Mi rilODOl ()C;Y

food Red and mange tomatoes will be commercially processed into spaghetti sauce A specific amount of sauce will he given to the subjects each day

DIogd.sampling filood samples will be collected and centrifuged into serum, lymphocytes, and red cells and stored at -7l)”C immediately

Duccal cdl_sampling Buccal cells will be collected with a tooth brush in a tube that will be centrifuged, the cell pellet collected and stored at -70'’C immediately

Lycopene_lcvcls Levels will be measured in blood and buccal cells using lirpiid chromatography (Wateis 2ü‘)0 with a 9')(i photodiode array detector) with a C30 reverse phase column (NIST) coupled with electrochemical detection (PSA model 5600 Coularray detector with I channels)

DA l A AiSALYSIS

Data will be analyzed using the SAS software program Di (Terences in carotenoid content between red and orange tomato spaghetti sauce will be determined by analysis of variance (ANOVA) Tukey’s USD test will be used to detect the differences between treatment groups C orrclation coelTicients will be obtained by correlation analysis to examine relationships between blood and buccal cells

SIGNIFICANCE

This pilot study will determine what, if any. cITcct tomato variety has on bioavailability of Ivcopene and prolycopene If it can be demonstrated that tomato variety docs have an effect on the bioavailability of lycopene. a larger study would be indicated

158 Block. Ci. Pallcrson. R . aiul A Subar I9‘>2 Fruit, vcgclablcs. and caiicct prcvciilion a review ol the epidemiological evidence S’lilr. I ’utiu r 18 1-29

Clinton. S K . Enicnhiser, C . Schwartz. S J . Bostwick. D G , Williams. A W . Moore. D J . and J W Frdman 1996 Cis-trans lycopene isomers, carotenoids, and retinol in the human prostate ( 'tiiitvr l\[iitk'iiui)li>f^\ liiiinnirkcr.s, tiinl I'n'iviiiinii 5 823-83 J

Di Mascio. P . Kaiser. S . and 11 Sics 19.89 l.ycopenc as the most cfTicicnt biological carotenoid singlet oxygen quencher /I/ih. Himlifm. Umphys 27>I 532-538

(iaitnei. C . Stahl. W and II Sies (1997) l.ycopenc is more bioavailable from tomato paste than Irom fresh tomatoes A m ../. Clin. S’tiir. 60 116-122

Giovannucci. II I ,. Ascherio. A . Rimm. I'. R . Stampfer. M J . Colditz. G A . and W C Willett 1995 Intake of carotenoids and retinol in relationship to risk of prostate cancer ./. Natl. (

Nguyen, M I. and S J Schwartz 1998 l.ycopcne stability during food processing/Vo. .Voc / ry Hi». Me,I 218 101-105

Schierle. J . Qtctzel. W . Rubier. I . Faccin. N . I less. D . Steiner, K . and W Schuep 1997 Content and isomeric ratio of lycopene in food and human blood p la s m a . Aync. Fuad ('hem 90 459-165

Zechmeister. I. 1962 a.s-tran.i l.vmiern: t'arntemndi, l'itamiits A and Aryl/'alyenes Academic Press. New York

159 IIIDMKIHC AI.SC ifMfS SIII\IMARVSIIF.F.T.S ADDRKSS FA{ II 11 KM l.N A ( (IM ri.KTK AN» < ONCISK MANNF.R. (Ilii ii

1. Ahstract (overview) Tlie purpose of tins sludy is to Jet ermine the e fleets of cniisuinmg processed tonuito products, ic spaghetti sauce, oil tJic carotene content, specifically Ivcopeiie. of blood and buccal cells Individuals will be asked to consiiiiie a given qiiaiitilv o f spaghetti sauce from either icd toiualoes or orange tomatoes after a period o f up to two weeks o f no tomato consumption Food will he provided and will bo commercially processed If consent is given for blood sampling, blood samples will be diawn and buccal cell samples obtained before and after the feeding period o f I days DIood sampling will be done at the OS Li riiiiical Research Center, by qualified medical personnel

2. Describe I lie requlrcmeiils for a lubjrrl pnpiilalimi and ciplain the rationale for mnng in this population special groups siirh as prisoners, chiUlrcii, the mentally disabled or groups whose ability to give voluntary informed consent may be in question. Address means of pregnancy screening for females. "Die population of interest is non-piegiiaiit. non-smoking adults over llie age of 18 Individuals will be asked to limit their intake of some carotene containing foods for up to two weeks prior to tlie stait of tiic stiiily Blood samples as well as buccal mucosal cell samples from the subjects, will be drawn on a consensual basis to establish low levels of lycopenc Subjects will then be asked to consume a given amoiiiit of spaghetti sauce from orange tomatoes tier dav for the next I days Blond and buccal mucosal cell samples from the subjects, will be drawn on a consensual basis Tlic subjects will then limit their intake o f tomato products for 2 weeks Blood and buccal cells samples will be taken to once again establish low levels of lycopenc Subjects will tlien be asked to consume a given aiiioniit of spaghetti sauce from red tomatoes per day for tlie next I days Blood and buccal mucosal cell samples from the subjects, will be drawn on a consensual basis

.). Describe and assess any potential risks- pbysical, psyrhulugical, social, legal, financial, or other - and assess the likelihood and seriousness of such risks. If methods of research create potential risks, ilescribe other methods, if any, that were considered and why they will not be used. Potential risks from blood drawing by venipuncture (taking blood fiom a vein) include the possible occurrence of discomfort and/or biiiising at the site of tlie puncture Less commonly, a small blood clot, swelling of tlie vein, or bleeding may occur at the piinctiiic site No risks arc anticipated from buccal mucosal cell saniplmg

•4. Describe consent procediiies to he followed, including how and where informed consent will be obtained. (The use of a finder’s fee for recruiting subjects is not permitted) WLieii an eligible subject has been identified. Iie/slie will be told tlic nature o f tlie study After expressed willingness is obtained, he/she will be requested to sign an Tnfoimed Consent' form (see attached) for tests performed on liimselfftieiself A photocopy will be supplied if desired

5. Describe procedures (including confidcntiality safeguards) for protecting against or ininimir.ing potential risks and an assessment of their likely effectiveness. Potential risks arising from venipuncture will be minimized by having tlie blood drawn only by a certified phlebotomist or physician All records of participants in tins study will be maintained in a confidential fashion With the exception of the General Information sheet, subjects will be identified on all rccoids only by tlieir subject number and initials Tlic General liiforniatioii sheet will be kept iii a locked file Data will be analyzed by using tlie subject iiurnber ratlier tJian name There will be no release or publication o f tliis data tliat would reveal tlic identity o f any subjects without permission Subjects may withdraw tlieinselves at any time witliout prejudice or loss o f aiiv benefits to which tlic subject is otherwise entitled Subjects will be provided with phone numbers for reaching tlic investigators during tlie day and after hours

160 ntciM F.nu'.vi. « iF.Nc Fs SUMMARY SIIF.F.TS ADDRESS F.ACI1 ITF.M IN A COMPI.ETF. AND CONCISE MANNF.R. (Do noi leave any iletii blank wilh “See allacheü") Use continuation pages when necessary

6. Assess the potential benrltts to be gained by the individual subject, as w ell as benefits which may accrue to society in general as a result of the planned work. Wliilc paiticipatms in die study, die subjects will receive die benefit of a personal in-dcpdi carotene profile Tlic overall benefit of the study is the possible iniproveincnt in die recommendations regarding nutrient intake of lycopenc containnig processed foods from different tomato varieties

7. Compare the risks versus the benefits. Tlie value of die knowledge to be gained justifies die potential risks of discomfoit and iiicoiiveiiiencc involved in die study

8. Will the subjects for the study be _t _N o ____ Yes paid for participating in the study?

Will subjects be paid for selected Activities (e g . blood diaxvnii;) or for general paiticipation in die study '

•NOTE All infonnation concerning payments, including die aniount and schedule of payment, must be included in the consent form Is there any other indiiccnient^ If so. please describe \ No Yes please describe

Will ailvertising be used to recruit subjects? s _ No Yes" " I f yes. attach a copy of die proposed advertisement SOURCE OF FUNDING FOR PROPOSED RESEARCH (check A or B) A OSURF Sponsor RF Proposal/Project N o ____ B Odier (Identify) Haas Endowed Chair Account No REFW HAAS In for mat ion about die finiduig/sponsorsliip o f liiiman subjects research activities ______

161 ri iH ()i iio SIA I r. UNivr.Rsi rv pi»i,koi no

CONSI-NT 10 INVCSTlCJAriONAI. 1 RRAIMI-NT OR RROCt-IHIRF.

*•______. Iicrchy .tiilliorizr orilirrri Sccvcii J. Schwartz. rii.D.. ««nrinirs or Msislnnls of lii.Vlicr chousing, lo perform the follutving (real me ni or proccilnrr (ilescrihe in gener.ll (ernis): In lliis siuilv. llic siib|ccts aie supposed lo he nonsniokeis. noi pregnant and over ihc âges of 18 As pan ol the studv. 1 agree to be asked to do the Ibllowing things -I will keep an aeeuiate dietary record during the com sc of the study -I will consume the food piovided for the course of the study (food will be spaghetti sauce) -I will visit the data collection site a total of'I times to have a blood sample diawn from a vein about 20ml ( I teaspoons or 2/5 of an ounce) and to donate a buccal mucosal cell sample upon ______(myself or name of subject) riic cipcrimrnlal (research) portion of the treatment or proceiliirc is: In order to study the elTcct of consuming dilTcient varieties of processed tomato products on the lycopenc content m blood and buccal cells, lycopenc levels of buccal cells and blood w dl be measured aller a measuicd period of intake I here is no experimental treatment involved This is done as part of an investigation entitled: The elTcet of tomato intake on lycopenc levels in serum and buccal cells

1. Purpose of the procedure or treatment: The purpose of the study is to provide information about how lyeopene levels change with di lièrent types of tomatoes consumed

2. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): I may choose not to paiticipate or to witluliaw at any time without loss of any benefits to which I am otherwise entitled

J. Uiscnmforts and risks reasonably to he expected: There could be risks of vcnipunctuie (taking blood from a vein) including discomfoit and'or bruising at the site of the puncture Less commonly, a small clot, swelling of the vein, or bleeding may occur at the puncture

4. Possible benefits for subjects/society: I will receive the benefit of a personal in-depth carotene profile The overall benefit of the study is the possible improvement in the recommendations regarding nutrient intake o f lycopenc containing processed foods from different tomato varieties

5. Anticipated duration of subjects participation (including number of visits): I will be asked to come to the data collection site 4 times at specific intervals (total duration to complete participation would be approximately 6 weeks)

162 I htfïhy xcknow tctlceth»l ______liai p r o v i d e t linfwrmalinn A b o u tthe p r o c e d u r e described above, about niy rights as a subject, and he/slie answered all questions to iiiy satisfaction. I understand that I may contact him/her at phone H AN 292 40*9 should I have additional questions. Ilc/she has etplained the risks described above and I understand them; he/she has offered lo explain all possible risks or complications.

I understand that, where appropriate, the II.S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during iiiy participation in this study that may contain my name or other personal idcntilicrs may hr made available to the sponsor of this study. Beyond this, I understand that my participation will remain confidential.

I understand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing future care. No guarantee has been given to me concerning this treatment or procedure.

I understand in signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and I am not releasing the investigator, the sponsor, the institution, or its agents from any legal liability for damages that they might otherwise have.

In the event of injury resulting from participation in this study, I also understand that immediate medical treatment is available at llniversity lluspitals of The Ohio State University and that the costs of such treatment will he at my expense: financial com|iensation beyond that required by law is not available. Questions about this should be directed to the Office of Research Risks Protection at 292-59M.

I have read and fully understand the consent form. I sign it freely and voluntarily. A copy has been given to me.

Date:______Time______Signed ______(subject) Witness (es| If (person authorized to consent for Required ______subject if required)

I certify that I have personally completed all blanks in this form and explained them to the subject or his/her representative before requesting the subject or his/her representative to sign it.

Date:______Signed: (Signature of project director or his/her authorized representative)

163 Abstract for Experimental Biology Meeting 1998, Washington, DC

Diets Rich in cû-Lycopene Increase Circuiating m-Lycopene Isomers in Humans.

Charlotte M. Alien', Mario G. Ferruzzi', Minhthy L. Nguyen', Ni-Luh Puspitasari-Nienaber', David Francis^ Steven K. Clinton^, Steven J. Schwartz'.

The consumption of diets rich in tomato products is associated with a reduced risk of several malignancies and cardiovascular disease. Lyeopene, a carotenoid derived from tomatoes, may contribute to these observations. Although tomatoes and processed tomato products contain lyeopene almost exclusively in the dX\-trans configuration, blood and tissue samples contain mostly cis isomers of lyeopene. The rationale for the transformation from d\\-trans to cis lyeopene in the body and the resulting biological significance remains unknown. This study compares blood lyeopene concentrations of subjects who have consumed either a high cw-lycopene variety tomato (>90% tetra-cw lyeopene, Tangella and Tangerine Golden Jubilee) or a high all-trans lyeopene (>90% aW-trans lyeopene) variety tomato. Treatment groups consumed either tomato sauce prepared from tangerine tomatoes (T, mg tetra-m lycopene/serving) or tomato sauce prepared from ‘red tomatoes’ (R, 8.1 mg a\\-trans lycopene/serving). Two servings per day (70g/serving) were fed to 10 healthy volunteers for 4 days in a crossover design with 14 days washout (lyeopene free diets) prior to the study and 14 days between different dietary regimens. Blood plasma lyeopene isomer concentrations were determined at the beginning and end of each 4-day feeding period. Total blood lyeopene levels rose (mean ± sem) from 0.624 ± 0.038 to 0.652 ± 0.046 (4.2%) and from 0.556 ± 0.046 to 0.819 ± 0.045 (32.1%, p<0.0001) for R and T, respectively.

164 Blood levels of total c/s-isomers rose from 0.303 ± 0.039 to 0.561 ± 0.037 (45.9%, p<0.0001) after consumption of T, while remaining unchanged in R. This study shows that the consumption of dietary cû-isomers of lyeopene from tangerine tomatoes rapidly alters circulating patterns of cis isomers, suggesting that they are absorbed in the cis form from the diet.

165 APPENDIX C

Supplements to Chapter 3 - Campbell’s study

166 Nutrient Reference Baseline W ashout Intervention sauce' intervention soup' Intervention juice' * (mean) (meantôEM) (mean±SEM) (mean±SEM) (mean±SEM) (mean±SEM) Fat(g) 74 78 ± 6 80 ±42 68 ±22 65 ±27 70 ±31 Beta-carotene 248.1 ±34.7 237.7 ± 178.5 269.7 ± 163.8 228.7 ± 102.4 441.0 ±405.7 Lyeopene(ug) 5300 6379.6 ± 1128.8 309.81 ±1109.1 21429.5 ± 1018.5 12679.3 ± 1877.6 15790.7± 7319.2 Energy (Real) 1884.5 2231.6 ± 138.4 2168.1 ±915.5 1933.4 ± 456.1 1956.3 ±729.5 2155.1 ±496.5 Saturated FA (g) 25.7 26.8 ±2.1 26.8 ± 13.4 23.5 ± 8.5 22.8 ± 11.2 24.8 ± 12,8 PUFA(g) 14.8 14.8 ± 1.5 15.4 ±8.7 11.5 ±5.2 12.3 ±5.0 13.0 ±5.1 MUFA(g) 27.6 30.6 ±2.9 31.6 ±19.6 25.4 ±9.5 25.6 ±10.5 26.9 ± 12.8 Cholesterol (mg) 288 235 ±22 241± 134 225 ± 149 216± 100 259±136 Sodium (mg) 3190 3965 ± 263 3614± 1288 3735 ± 835 3715± 1200 4138 ±952 Carbohydrates (g) 224 295 ± 18 278± 119 257 ±70 253 ± 106 296 ±67 ^ Total dietary fiber (g) 14.5 19.6 ± 1.1 17.5 ±8.1 17.0 ±5.8 15.0 ±7.9 18.9 ±5.8 Protein (g) 77.2 81.2 ±4.3 81.2 ±33.5 72.1 ± 18.0 67.7 ± 26.4 81.0 ±32.7 Vitamin A (lU) 6521 10653 ±2456 7189 ±9346 8620 ± 5944 5779 ±3853 10411 ±10299 Vitamin C (mg) 96.5 145.7 ±15.1 148.7 ± 126.7 121.8 ±82.6 128.1 ±135.8 200.5 ± 133.7 Calcium (mg) 737.5 857.4 ±46.5 859.6 ±357.7 856.1 ±392.1 718.8 ±433.0 859.6 ±483.2 Iron (mg) 14.2 17.4 ±1.4 16.3 ± 7.5 15.5 ±4.8 13.7 ±7.2 16.7 ±4.6 Retinol 555.6 ±24.5 568.6 ± 146.1 572.6 ± 104.9 578.4 ± 184.3 587.5 ± 153.0

Table 1. Nutrient intakes of subject population based on diary information.

' Mean value for individuals 20 and over. Reference USDA NFS Report No. 91-2 from 1989-1991 unless otherwise noted. ^n=36. GENDER Subj.# Height WEIGHT AGE FOOD (LBS) M 896 5'9" 190 51 V8 M -DR2 709 5'10" 180 57 M 769 5’5” 140 36 V8 M 559 5’8” 165 28 V8 M 691 5’9” 170 20 V8 M 102 5’10” 200 60 V8 M 831 6 3 " 150 25 V8 F 978 5’10" 160 29 V8 F 795 5’6" 125 33 V8 F 214 5’4" 128 45 V8 F 341 4’11” 115 45 V8 F 850 5’3" 220 49 V8 F -M 4 019 5’2” 160 57 V8 F-D R 3 734 5’5" 160 27 V8 M 008 5’7" 190 32 Soup M 767 5’8" 150 36 Soup M 926 5’7" 170 30 Soup M 668 5'7" 185 31 Soup M 090 5’8" 135 29 Soup M 528 6’2" 160 30 Soup M -D R 3 189 5’9.5” 160 30 Soup F 119 5 6 " 164 53 Soup F 852 5’ 95 29 Soup F-DR2 481 5’4" 120 43 F 969 5’2” 175 58 Soup F 165 5’6" 127 26 Soup F 293 5’5” 110 36 soup F 076 5’2" 120 35 soup M 788 5’10” 165 44 Sauce M 655 6' 190 26 Sauce M 727 5’8” 186 61 sauce M 154 5’9" 172 46 Sauce M 028 5 9 " 180 32 Sauce M 618 6'1" 225 35 Sauce F 240 5’5" 140 26 Sauce F 499 5’6” 160 27 Sauce F 126 5'7" 130 26 Sauce F 363 5’10" 142 18 Sauce F 589 4’11.75” 158 60 sauce F -M 7 610 5'4" 148 38 sauce

Master table of subject demographics.

168 Subject BMI v alues ft inches lbs kg inches meters sq meters BMi M 896 5'9" 190 86.4 69 1.75 3.07 28.12 M 769 5'5" 140 63.6 65 1.65 2.73 23.35 M 559 5'8" 165 75.0 68 1.73 2.98 25.14 M 691 5'9" 170 77.3 69 1.75 3.07 25.16 M 102 5'10" 200 90.9 70 1.78 3.16 28.76 M 831 6'3" 150 68.2 75 1.91 3.63 18.79 978 5'10 " 160 72.7 70 1.78 3.16 23.01 795 5'6" 125 56.8 66 1.68 2.81 20.22 214 5'4" 128 58.2 64 1.63 2.64 22.02 341 4’i r 115 52.3 59 1.50 2.25 23.28 850 5'3" 220 100.0 63 1.60 2.56 39.05 F-MD4 19 5'2" 160 72.7 62 1.57 2.48 29.33 M 8 5'7" 190 86.4 67 1.70 2.90 29.82 M 767 5'8" 150 68.2 68 1.73 2.98 22.86 M 926 5 7 ” 170 77.3 67 1.70 2.90 26.68 M 668 5 7 " 185 84.1 67 1.70 2.90 29.04 M 90 5’8” 135 61.4 68 1.73 2.98 20.57 M 528 6'2” 160 72.7 74 1.88 3.53 20.59 119 5’6" 164 74.5 66 1.68 2.81 26.53 852 5' 95 43.2 60 1.52 2.32 18.59 969 5'2" 175 79.5 62 1.57 2.48 32.07 165 5'6” 127 57.7 66 1.68 2.81 20.54 293 5'5" 110 50.0 65 1.65 2.73 18.34 76 5'2" 120 54.5 62 1.57 2.48 21.99 M 788 5’10" 165 75.0 70 1.78 3.16 23.72 M 655 6' 190 86.4 72 1.83 3.34 25.82 M 727 5’8" 186 84.5 68 1.73 2.98 28.34 M 154 5’9" 172 78.2 69 1.75 3.07 25.45 M 28 5'9” 180 81.8 69 1.75 3.07 26.64 M 618 6’1” 225 102.3 73 1.85 3.44 29.75 240 5’5” 140 63.6 65 1.65 2.73 23.35 499 5’6" 160 72.7 66 1.68 2.81 25.88 126 5 7 " 130 59.1 67 1.70 2.90 20.40 363 5'10" 142 64.5 70 1.78 3.16 20.42 589 411.75" 158 71.8 59 1.50 2.25 31.98 F-MD7 610 5’4" 148 67.3 64 1.63 2.64 25.46

m ean 25.03 std dev 4.49

169 Subject buccal proteins (ug protein). subject# draw 1 draw 2 draw 3 draw 4 draw 5 draw 6 draw 7 896 80.69 71.54 78.88 68.68 88.13 51.32 45.69 102-1 25.37 41.68 50.27 43.11 75.64 75.35 103.30 769 28.23 11.54 82.41 6.87 18.41 33.57 22.70 559 33.00 49.98 43.40 45.31 47.69 50.36 53.51 691 6.77 7.51 18.12 16.12 -6.49 37.20 30.90 831 43.78 23.66 24.90 47.60 30.62 44.16 31.10 978 27.18 24.90 16.31 14.02 35.20 33.48 21.08 795 37.20 42.25 22.51 14.50 5.06 20.41 15.45 214 27.95 9.92 19.36 10.87 19.46 33.00 32.33 341 14.69 3.53 12.50 13.64 6.87 30.71 4.29 850 22.61 20.79 14.12 19.94 43.59 30.62 19 126.10 25.28 31.57 30.71 19.74 31.48 27.47 734 17.65 20.22 21.37 8 15.64 16.22 22.32 25.66 2.77 31.10 31.57 767 91.66 37.01 63.72 71.25 54.27 65.24 97.96 926 29.66 9.35 4.58 65.43 23.94 1.34 11.92 668 26.42 22.61 5.06 21.37 24.04 10.59 21.84 90 60.28 67.15 99.39 31.57 13.74 41.68 44.45 189 52.37 50.84 26.61 528-1 28.52 17.36 50.84 83.56 32.91 19.46 39.11 119 35.39 13.07 41.30 34.81 33.48 27.95 24.23 852 76.12 30.14 63.14 17.07 35.10 45.50 41.68 969 40.16 21.18 17.93 29.28 18.50 26.42 22.42 165 21.65 18.79 -6.49 28.52 17.93 28.42 26.14 293-1 37.10 32.72 45.78 35.39 24.70 29.47 59.42 76 62.19 28.71 29.00 42.06 26.14 20.51 47.69 788 44.16 40.25 39.11 37.77 66.67 50.93 44.54 655 31.86 46.83 31.00 64.00 25.85 51.60 16.79 727 69.25 52.27 72.21 78.12 56.09 62.57 71.73 154 25.18 78.12 58.09 91.85 92.24 60.47 36.82 28 39.68 47.02 29.95 26.42 22.51 25.37 29.47 618 11.92 21.75 22.51 34.62 59.14 35.29 25.94 240 10.97 32.43 14.50 8.78 18.70 12.21 10.97 499 27.57 15.64 14.02 20.41 25.56 24.99 12.97 126 39.30 31.00 29.00 35.10 24.80 35.20 29.76 363 22.80 11.26 13.54 15.45 25.47 26.52 24.23 589 20.70 20.41 5.06 8.58 10.87 11.73 20.60 610 29.00 17.74 26.80 13.93 33.00 20.70

170 Total Cholesterol Results

Values are expressed in mg/dL

8 181.9 187 197.0 206.1 207.7 194.0 183.4 19 269.4 266.4 258.9 287.0 265.0 288.0 28 153.5 181.7 175.2 165.5 182.3 180.0 169.3 76 176.4 169.7 187.1 195.7 190.5 194.0 160.6 90 153.8 137.8 143.4 147.5 153.8 182.0 148.2 102 185.6 156.4 165.9 179.4 191.0 203.4 217.2 119 179.8 171.4 177.8 166.2 213.2 222.0 209.5 126 181.5 198.1 211.6 197.1 198.1 190.0 193.1 154 204.8 199.7 193.7 213.9 263.3 194.0 206.2 165 148.4 152.1 155.6 147.5 158.9 150.0 165.6 214 157.6 161.5 170.5 136.7 157.0 167.0 145.2 240 136.1 136.4 151.0 154.8 160.7 146.0 142.5 293 125.5 140.8 142.4 144.7 141.8 141.0 140.5 341 156.2 155.1 176.5 161.7 183.0 152.0 159.2 363 129.2 120 129.5 151.1 142.8 145.0 115.7 499 98.5 98.2 101.7 88.8 106.1 117.0 91.9 528 159.7 162.7 153.0 167.6 175.4 160.0 163.9 559 183.2 193.3 180.8 197.1 181.0 201.0 196.1 589 197.9 210.6 205.0 228.3 217.3 252.0 200.1 610 164.8 200.1 199.0 195.5 180.6 181.0 618 230.4 248.9 228.5 229.0 232.1 242.0 198.1 655 211.3 234.3 187.4 217.4 199.8 195.0 216.1 668 145.6 161.1 146.4 142.6 155.2 165.0 148.5 691 181.2 128.5 136.4 163.8 172.3 179.0 173.7 727 213.7 191.2 190.4 171.2 190.9 177.0 189.4 767 150.8 161.1 142.4 160.0 153.1 173.0 140.5 769 133.7 106.8 125.5 133.6 123.6 135.0 125.4 788 217.1 206 230.5 225.5 234.1 214.0 238.4 795 148.4 151.7 154.0 163.9 170.3 168.0 168.0 831 154.5 150.7 169.9 172.4 196.0 149.0 164.3 850 258.1 307.5 178.1 245.3 286.3 286.0 277.5 852 190.1 192.7 194.7 200.2 197.7 193.0 171.3 896 236.6 245.8 252.6 226.9 247.9 251.0 220.2 926 186.3 171 201.3 186.2 188.1 180.0 169.6 969 215.4 218.6 238.1 236.3 234.1 239.0 251.1 978 148 133.4 161.6 156.1 165.5 162.0 168.3 m ean 176.8 178.0 178.2 179.3 190.0 187.3 180.5

Expected values (desirable blood ctwlesterol = <200mg/dL, borderline high = 200-239, high-risk - >239

171 Abstract for poster presented at American Institute for Cancer Research Meeting September 2-4,1999

Blood lyeopene concentrations increase in healthy adults consuming standard servings of processed tomato products daily. C Mozley, S Schwartz, N Craft, V DeGrofT, E Giovannucci, S Clinton. The Ohio State University Comprehensive Cancer Center and Dept of Food Science and Technology, Craft Technologies, Inc., and The Harvard Schooi of Public Health

The consumption of tomato products is associated with a reduced risk of several cancers. Overall, human studies suggest that benefits may be achieved with approximately one serving of tomato products per day. Lyeopene, the predominant carotenoid in tomatoes, is hypothesized to be one component contributing to the health benefits of tomato products. High plasma lyeopene concentrations are associated with a reduced risk of prostate cancer in the Physicians’ Health Study (Gann, et.al, 1999.).

The present study was designed to determine plasma lyeopene concentrations in healthy adults (n=36, ages 18 to 65) consuming standard daily servings of three processed tomato products: Prego™ Spaghetti Sauce (SS), Campbell’s Tomato Soup

(TS), or VS™ vegetable juice (J). All 36 subjects consumed a lycopene-free diet for the first two weeks (washout period) in order to determine lyeopene clearance rates from the blood. Participants were assigned to one of three (n=12) intervention groups consuming single standard servings of SS (21 mg lyeopene/1/2 cup), TS (12 mg lycopene/lcup), or J (17 mg lycopene/8 oz) daily for 4 wk without any other sources of lyeopene. Blood samples were obtained at enrollment and weekly thereafter for

HPLC analysis of carotenoids. Total plasma lyeopene concentrations (Mean i: SE) decreased from 1.05 ± 0.07 to 0.54 ± 0.05 umoles/L (p<0.0001) during the washout

172 period. In all intervention groups, plasma lyeopene concentrations increased and plateaued between 2-4 wk. Plasma lyeopene levels for those consuming SS, TS, and J increased to 2.08 (192% p < 0.0001), 0.91 (122%, p<0.0001), and 0.99 (92%, p<0.0001) umoles/L, respectively. This study demonstrates that lyeopene is cleared from the plasma with a T*'^ of approximately 14 days. Lyeopene is readily absorbed from SS, TS, and J although bioavailability differs for each product. The study demonstrates that a single daily serving of processed tomato products can signifîcantly increase blood lyeopene. [Supported by NIH-NCI ROl 72482, NIH-NCIROICA 74666,

NIH-NCIP30 CA16058 to the OSU Comprehensive Cancer Center, OARDC

Competitive Grants Program and The Campbell's Soup Co.]

173 IKSTIWriOHU. «IVT». lOkRD „„„

Protocols for full comiiiittcc review received in (he Office of Research Risks Protection after the deadline dale (first Monday of each month) will he scheduled for the followin;; month's meetinu (third Monday of each month). Only protocols that arc complete will he scheduled for review. Incomplete protocols will he returned.

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rn c.incer-reUte.l Ancivities Approval by Clio J.nmos C.nncor Conter Clinical Soiencitic Re-.-iew Committee (Phono 291 493SI in requirol prior to activation. Inventiq.ntor is renponsilile □ for obC«iiitinq «pprovAl from both comm ittees. r n pr^qn.nnc Wnm«n Approval hy MAtf*rn.i I • Feta I Cmmmitree (293 0716» is roqnir«tl p r io r to □ activation. Invest iqatoi is responsible for obtaining approval from botli rommitte«'s I I |T] Minors (Under lA years of ago)

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174 BIOMEDICAL SCIENCES SUMMARY SHEETS ADDRESS EACH ITEM IN A COMPLETE AND CONCISE MANNER. (Do not leave any item blank with "See attached”) Use continuation pages when necessary.

1. Abstract (overview)

The objective is to determine the ability of tomato soup, tomato sauce and V8 juice consumption to enhance the levels of the carotenoid lyeopene in blood and buccal cells. Lyeopene is the major antioxidant carotenoid in tomato products and very little is known about its digestion and absorption from foods or its distribution in blood and tissue. Healthy volunteers will be asked to avoid tomato containing foods for 2 weeks. Paticipants will then consume 7 servings/week either tomato soup (I cup/8 oz), tomato sauce (lcup/8oz) or V8 juice (8 oz.) for 4 weeks. Commercially available soup, sauce and juice will be provided to participants by the investigators. Participants will provide a blood and buccal cell sample once per week starting at the time of enrolment for a total of 7 samples. Blood and buccal cell sampling will be done at the OSU Clinical Research Center, by qualified medical personnel.

2. Describe the requirements for a subject population and explain the rationale for using in this population special groups such as prisoners, children, the mentally disabled or groups whose ability to give voluntary informed consent may be in question. Address means of pregnancy screening for females.

Participants will be non-pregnant, non-smoking adults (18 male, 18 female) between the ages 18-65. Exclusions include those with gastrointestinal disease that may alter food digestibility including diseases such as: pancreatic insufficiency, hepatic disease, diabetes, and metabolic enzyme deficiencies. Subjects will be questioned about disease status and pregnancy.

Subjects will be asked to modify their diet and avoid lyeopene containing foods (tomato products, watermelon, pink grapefruit) for 7 weeks except for the juice, sauce or soup provided. After two weeks on a lyeopene free diet all of the 18 male and 18 female subjects will be randomized and asked to consume either 7 servings/week either tomato soup (I cup/8 oz), tomato sauce (I cup/8 oz) or V8 juice (8 oz.) for 4 weeks. Subjects will be asked to provide a blood sample and buccal cell sample at the time of enrolment and every 7 days therafter until the completion of the study (7 samples total).

3. Describe and assess any potential risks- physical, psychological, social, legal, financial, or other - and assess the likelihood and seriousness of such risks. If methods of research create potential risks, describe other methods, if any, that were considered and why they will not be used.

Potential risks include those associated with drawing blood by venipuncture (taking blood from an anticubital vein). Mild discomfort is common and bruising at the site o f the puncture may occasionally occur. Less conunonly, a localized blood clot may develop within the vein resulting in some swelling of the vein or arm. Rare bleeding may occur at the puncture site. No risks are anticipated fix>m buccal ceil sampling. There are no known or anticipated risks in

175 healthy (including pregnant) individuals associated with consuming a lycopene-free diet or the consumption of V8 juice, tomato sauce or tomato soup in the amounts provided.

4. Describe consent procedures to be followed, including how and where informed consent will be obtained. (The use of a finder’s fee for recruiting subjects is not permitted)

Subjects will be recruited via word of mouth in the Departments of Food Science and Nutrition and Internal Medicine at The Ohio State University. When an eligible subject has been identified, he/she will be informed of the objectives, requirements, risks/benefits of the study. After expressed willingness is obtained, he/she will be requested to sign an ‘Informed Consent’ form (see attached) for tests performed on himself/herself. A photocopy will be supplied if desired.

5. Describe procedures (including confidentiality safeguards) for protecting against or minimizing potential risks and an assessment of their iikely eifectiveness.

Potential risks arising from venipuncture will be minimized by having the blood drawn only by a certified phlebotomist or physician. All records of participants in this study will be maintained in a confidential fashion. With the exception of the General Information sheet, subjects will be identified on all records only by their subject number and initials. The General Information sheet will be kept in a locked file. Data will be analyzed by using the subject number rather than name. There will be no release or publication of this data that would reveal the identity of any subjects without permission. Subjects may withdraw themselves at any time without prejudice or loss of any benefits to which the subject is otherwise entitled. Subjects will be provided with phone numbers for reaching the investigators during the day and after hours.

6. Assess the potential benefits to be gained by the individual subject, as well as benefits which may accrue to society in general as a result of the planned work.

Lyeopene is one o f the major dietary carotenoids found in human blood and many tissues. Lyeopene exhibits may biological properties suggesting that it may protect against premature aging and many chronic diseases associated with oxidative stress including vascular disease, cancer, autoimmune disease, and specific types of dementia. This study is one of the first to investigate the quantitative relationship between intake of tomato containing foods and distribution in blood and tissue (buccal cells). Additional information on the bioavialabiltiy of lyeopene from food will allow investigators to better define a healthy diet for humans.

7. Compare the risks versus the benefits.

The value of the knowledge obtained from the proposed study is significant while the risks associated with the dietary intervention and blood sampling are minimal.

176 BIOMEDICAL SCIENCES SUMMARY SHEETS ADDRESS EACH ITEM IN A COMPLETE AND CONCISE MANNER. (Do not leave any item blank with **See attached") Use continuation pages when necessary

8. Will the subjects for the study be No _ x _ Y e s paid for participating in the study?

Will subjects be paid for selected activities (e.g., blood drawing) or for general participation in the study? ïes, they will be paid for participation in the study

Subjects will be paid $150 for completion of the study (all 7 blood and buccal cell samples) or $75.00for completion of the washout period (first 3 blood and buccal cell samples)

*NOTE: All information concerning payments, including the amount and schedule of payment, must be included in the consent form.

Is there any other inducement? If so, please describe _x N o ___ Yes—please describe

9. Will advertising be used to recruit subjects? _x __ No Yes** **If yes, attach a copy of the proposed advertisement.

SOURCE OF FUNDING FOR PROPOSED RESEARCH: (check A or B):

A. OSURF: Sponsor ______RF Proposal/Project No. ______B. Other (Identify)

Dr. Clinton’s new faculty start up funds. Dr. Schwartz's discretionary funds.

Information about the funding/sponsorship of human subjects research activities

177 THE OHIO STATE UNIVERSITY Protocol No. 98H0353 CONSENT TO INVESTIGATIONAL TREATMENT OR PROCEDURE

I,______, hereby authorize or direct associates or assistants of his/her choosing, to perform the foilowing treatment or procedure (describe in general terms): In this study, the subjects are to be healthy, nonsmokers, not pregnant and between the ages 18-65 with no history of active gastrointestinal disease. Exclusions include those with gastrointestinal disease that may alter food digestibility including diseases such as: pancreatic insufficiency, hepatic disease, diabetes, and metabolic enzyme deficiencies.

As part of the study, I agree to be asked to do the following things:

-I will complete a dietary history questionnaire at the beginning o f the study.

- 1 will consume a diet free of tomato products, pink grapefruit, watermelon, and guava for 6 wks except for the foods provided.

-I will consume 7 servings/week of either tomato soup (1 cup/8 oz), tomato sauce ( Icup/Soz) or V8 juice (8 oz.) for 4 weeks.

-I will visit the data collection site a total of 7 times to have a blood sample drawn from a vein about 20ml (4 teaspoons or 2/5 of an ounce) and to donate a buccal mucosal cell sample (the surface cells on the inside of my cheek) by brushing the inside o f my cheek with a toothbrush.

upon

(myself or name of subject)

The experimental (research) portion of the treatment or procedure is: To compare the ability of three different tomato products to increase the blood and tissue levels of lyeopene, the main antioxidant carotenoid in tomatoes. There is no treatment of a disease process.

This is done as part of an investigation entitled: The effect of commercially available tomato product consumption on lyeopene levels in tissue and serum.

1. Purpose of the procedure or treatment: The purpose of the study is to provide information about how lyeopene levels change during a period of controlled consumption.

2. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): 178 I may choose not to participate or to withdraw at any time without loss of any benefits to which I am otherwise entitled.

3. Discomforts and risks reasonably to be expected: There could be risks of venipuncture (taking blood from a vein) including discomfort and/or bruising at the site of the puncture. Less commonly, a small clot, swelling of the vein, or bleeding may occur at the puncture. No risk is anticipated from buccal cell sampling. 4. Possible benefits for subjects/society: The overall benefit of participation in the study is the knowledge that my effort contributes to the knowledge regarding nutrition and health, and that these results may lead to improvements in the current recommendations regarding healthy dietary intake of fruits and vegetables. Each participant will be provided $150 for completing the 6 week study ($75 for completion of the initial 2 week component) as compensation for travel expenses incurred and time commitment.

5. Anticipated duration of subjects participation (including number of visits): I will be asked to come to the data collection site 7 times at specific intervals (total duration to complete participation would be approximately 6 weeks).

I hereby acknowledge that ______has provided information about the procedure described above, about my rights as a subject, and he/she answered ail questions to my satisfaction. I understand that I may contact him/her at phone # 614- 292-4069 should I have additional questions. He/she has explained the risks described above and I understand them; he/she has offered to explain ail possible risks or complications.

I understand that, where appropriate, the U.S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during my participation in this study that may contain my name or other personal Identifiers may be made available to the sponsor of this study. Beyond this, I understand that my participation will remain confidential.

I understand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing future care. No guarantee has been given to me concerning this treatment or procedure.

I understand in signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and I am not releasing the Investigator, the sponsor, the institution, or its agents from any legal liability for damages that they might otherwise have.

In the event of injury resulting from participation in this study, I also understand that immediate medical treatment is available at University Hospitals of The Ohio State University and that the costs of such treatment will be at my expense; financial

179 compensation beyond that required by law is not available. Questions about this should be directed to the Office of Research Risks Protection at 292-5958.

I have read and fully understand the consent form. I sign it freely and voluntarily. A copy has been given to me.

Date: ______Time______Signed______(subject) Witness (es)______

If (person authorized to consent for Required______subject if required)

1 certify that I have personally completed all blanks in this form and explained them to the subject or his/ber representative before requesting the subject or his/her representative to sign it.

Date: ______Signed:______(Signature of project director or his/her authorized representative)

180 INTRODUCTION

Lyeopene has recently emerged as a dietary phytochemical having potential health benefits. Epidemiologic studies are beginning to accumulate showing an inverse correlation between consumption of food products rich in lyeopene (primarily tomatoes) and the risk of developing chronic diseases (Block et al., 1992; Clinton, 1998; Giovannucci et al., 1995). Lyeopene is a carotenoid with a superior ability to quench singlet oxygen compared to all major dietary carotenoids (Di Mascio et ai., 1989). Studies with animal models and in vitro investigations suggest beneficial effects (Levy et al., 1995). Overall, lyeopene exhibits extreme variability in absorption from foods and its bioavialability will depend upon many factors such as food processing, cooking methods employed, and the content of fat or fiber in the diet. Furthermore, our laboratories were the first to identify 12-18 different isomers of lyeopene in human blood and tissues (Clinton et al., 1996; Gartner et al., 1997; Schierle et al., 1997; Nguyen and Schwartz, 1998; Ferruzzi, et al. 1998; Emenhiser, et al. 1996).

Very little information is available concerning the uptake and absorption of lyeopene into the bloodstream after consumption of various processed tomato products. Furthermore, little is also not well established how rapidly lyeopene is cleared fi*om the blood while consuming tomato free diets. Additional information concerning lyeopene bioavailability and metabolism will contribute to efforts underway to establish healthy dietary guidelines.

RESEARCH OBJECTIVES

A pilot experiment has been designed to investigate the following questions:

1. How rapidly is lyeopene cleared form the blood and buccal cells while consuming a lyeopene free diet?

2. How rapidly will the consumption of V8 juice, tomato sauce or tomato soup (7 servings per week) increase the concentration of lyeopene in the blood and buccal cells?

3. What is the variability in blood and buccal cell lyeopene between individuals consuming a lyeopene fi*ee diet or while consuming V8, tomato sauce or tomato soup?

181 EXPERIMENTAL DESIGN

Subjects Eighteen male and eighteen female, healthy, non-smoking, non-pregnant individuals between 18-65 with no history of gastrointestinal disease will be required for this pilot study.

Design Thirty-six individuals placed on a lyeopene free diet for six weeks, with the exception of the V8, tomato sauce, or tomato soup provided by the investigators. After two weeks on a lyeopene free diet, the individuals will be assigned to receive V8, tomato sauce or tomato soup. Participants will continue on the study for four additional weeks with no lyeopene other than that provided by the V8, tomato sauce or tomato soup products. Intake of V8, tomato sauce or tomato soup will be 7 servings per week of 8oz. We will obtain blood samples and buccal cells at seven time points (-2, -1,0, +1, +2, +3, +4wks).

EXPERIMENTAL DESIGN

V8. Males, n = 6

V8, Females, n = 6 18 Males Tomato sauce. 6 Males “Wash-Out” Females Tomato sauce, 6 Females

Tomato Soup, 6 Males

Tomato Soup, 6 Females

-2 -1 +1 +2 +3 +4

WEEKS

182 To investigate “How rapidly is Ivcopene cleared form the blood and buccal cells while consuming a lyeopene free diet? We will examine lyeopene profiles for subjects on a lyeopene free diet for 2 weeks.

To investigate “How rapidly will the consumption of V8 juice, tomato sauce or tomato SOUP (1 servings per week) increase the concentration of Ivcopene in the blood and buccal cells?”After 2 weeks on a lyeopene free diet, subjects will consume either V8 juice, tomato sauce, or tomato soup for 4 weeks with a weekly assessment of lyeopene concentrations in blood and buccal cells.

To investigate “What is the variability In blood and buccal cell Ivcopene between individuals consuming a Ivcopene free diet or while consuming V8. tomato sauce or tomato soup?” Prior studies suggest significant heterogeneity in lyeopene bioavailability among humans consunung lyeopene containing products. We will determine the variation in response through our statistical evaluation of the blood and buccal cell lyeopene patterns over the duration of the study. RESEARCH METHODOLOGY

Food: The dietary interventions will be commercially available V8 juice, tomato sauce, or tomato soup.

Blood sampling: Blood samples will be collected using sterile standard techniques by trained personnel and immediately processed stored at -70°C for subsequent analysis.

Buccal cell sampling: Buccal cells will be collected with a tooth brush in a tube that will be centrifuged, the cell pellet collected and stored at -70°C.

Lyeopene concentrations: Carotenoid profiles will be measured in blood using liquid chromatography (Waters 2690 with a 996 photodiode array detector) via the method of Nomura, etal. (1997). Buccal cell levels will be measured using a C 3 0 reverse-phase column (NIST) coupled with electrochemical detection (ESA model 5600 Coularray detector with 4 channels).

DATA ANALYSIS

Data will be analyzed using the SAS or Statview software programs. Differences in carotenoid content between individuals in the same group will be determined by analysis of variance (ANOVA). Increases or decreases of lyeopene in blood of individuals in the same group will be measured and compared via correlation coefficients.

183 SIGNIFICANCE

This pilot study will determine whether the consumption of three major types of tomato products rich in lyeopene will increase the lyeopene content of blood or buccal cells. The study will also provide critical data concerning the rate of clearance of lyeopene from the blood while consuming a lyeopene free diet. Data will be derived to define the inter-individual variability expected in the general population. Results from these studies will allow researchers to adequately design a larger and more comprehensive study to assess lyeopene bioavailability from tomato containing foods and the role of tomato products in disease prevention. REFERENCES

Block, G., Patterson, B., and A. Subar. 1992. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18: 1-29.

Clinton, SK. 1998. Lyeopene: chemistry, biology, and implications for human health and disease. Nutrition Reviews. 56 (2ptl):35-51.

Clinton, S.K., Emenhiser, C., Schwartz, S.J., Bostwick, D.G., Williams, A.W., Moore, B.J., and J.W. Erdman. 1996. Cis-trans lyeopene isomers, carotenoids, and retinol in the human prostate. Cancer Epidemiology, Biomarkers, and Prevention. 5: 823-833.

Di Mascio, P., Kaiser, S., and H. Sies. 1989. Lyeopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274:532-538.

Emenhiser, C., Simunovic, N., Sander, L.C., and S.J. Schwartz. 1996. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. J. Agric. Food Chem. 44:3887-3893.

Ferruzzi, M.G., Sander, L.C., Rock, C.L., and S.J. Schwartz. 1998. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Analytical Biochemistry. 256:74-81.

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

Giovannucci, E.L., Ascherio, A., Rimm, E.B., Stampfer, M.J., Colditz, G.A., and W.C. Willett. 1995. Intake of carotenoids and retinol in relationship to risk of prostate cancer. J. Natl. Cancer Inst. 87: 1767-1776.

184 Levy, J.J., Bosin, E., Feldman, B., Giat, Y., Munster, A., Danilenko, M., and Y. Sharoni. 1995. Lycopene is a more potent inhibitor of human cancer cell proliferation than either a-carotene or P-carotene. Nutr. Cancer. 24:257-266.

Nguyen, M.L. and S.J. Schwartz. 1998. Lycopene stability during food processing. Pro. Soc. Exp. Bio. Med. 2li: 101-105.

Nomura, A.M.Y., Stemmermann, G.N., Lee, J., and N.E. Craft. 1997. Serum micronutrients and prostate cancer in Japanese Americans in Hawaii.. Cancer Epidemiology, Biomarkers, and Prevention. 6: 487-491.

Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and W. Schuep. 1997. Content and isomeric ratio of lycopene in food and human blood plasma. J. Agric. Food Chem. 96:459-465.

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186 THE OHIO STATE UNIVERSITY Protocol No. 98H03S3 CONSENT TO INVESTIGATIONAL TREATMENT OR PROCEDURE

I,______, hereby authorize or direct associates or assistants of his/her choosing, to perform the following treatment or procedure (describe in general terms): In this study, the subjects are to be healthy, nonsmokers, not pregnant and between the ages 18-65 with no history of active gastrointestinal disease. Exclusions include those with gastrointestinal disease that may alter food digestibility including diseases such as: pancreatic insufGciency, hepatic disease, diabetes, and metabolic enzyme deficiencies.

As part of the study, I agree to be asked to do the following things:

-I will complete a dietary history questionnaire at the beginning of the study.

-I will complete a three day dietary intake record at three different times during the study.

- 1 will consume a diet hree of tomato products, pink grapefruit, watermelon, and guava for 6 wks except for the foods provided.

•I will consume 7 servings/week of either tomato soup (I cup/8 oz), tomato sauce (lcup/4oz) or V8 juice (8 oz.) for 4 weeks.

-I will visit the data collection site a total of 7 times to have a blood sample drawn from a vein about 20ml (4 teaspoons or 2/5 of an ounce) and to donate a buccal mucosal cell sample (the surface cells on the inside of my cheek) by brushing the inside of my cheek with a toothbrush.

upon (myself or name of subject)

The experimental (research) portion of the treatment or procedure is: To compare the ability of three different tomato products to increase the blood and tissue levels of lycopene, the main antioxidant carotenoid in tomatoes. There is no treatment of a disease process.

This Is done as part of an Investigation entitled: The effect of commercially available tomato product consumption on lycopene levels in tissue and serum.

3. Purpose of the procedure or treatment: The purpose of the study is to provide information about how lycopene levels change during a period of controlled consumption.

187 4. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): I may choose not to participate or to withdraw at any time without loss of any benefits to which I am otherwise entitled.

3. Discomforts and risks reasonably to be expected: There could be risks of venipuncture (taking blood from a vein) including discomfort and/or bruising at the site of the puncture. Less commonly, a small clot, swelling of the vein, or bleeding may occur at the puncture. No risk is anticipated from buccal cell sampling.

4. Possible benefits for subjects/society: The overall benefit of participation in the study is the knowledge that my effort contributes to the knowledge regarding nutrition and health, and that these results may lead to improvements in the current recommendations regarding healthy dietary intake of fruits and vegetables. Each participant will be provided $ ISO for completing the 6 week study ($75 for completion of the initial 2 week component) as compensation for travel expenses incurred and time commitment.

6. Anticipated duration of subjects participation (including number of visits): 1 will be asked to come to the data collection site 7 times at specific intervals (total duration to complete participation would be approximately 6 weeks).

I hereby acknowledge that ______has provided information about the procedure described above, about my rights as a subject, and he/she answered all questions to my satisfaction. I understand that I may contact him/her at phone # 614-292-4069 should I have additional questions. He/she has explained the risks described above and I understand them; he/she has offered to explain all possible risks or complications.

I understand that, where appropriate, the U.S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during my participation in this study that may contain my name or other personal identifiers may be made available to the sponsor of this study. Beyond this, I understand that my participation will remain confidential.

I understand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing future care. No guarantee has been given to me concerning this treatment or procedure.

I understand In signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and I am not releasing the investigator, the sponsor, the institution, or its agents from any legal liability for damages that they might otherwise have.

In the event of injury resulting from participation in this study, I also understand that immediate medical treatment is available at University Hospitals of The Ohio State University and that the costs of such treatment will be at my expense; financial compensation beyond that required by law is not available. Questions about this should be directed to the OfUce of Research Risks Protection at 292-5958.

188 I have read and fully understand the consent form. I sign it freely and voiuntarfly. A copy has been given to me.

Date:_ Time Signed, (subject) Witness (es)_

If (person authorized to consent for Required subject if required)

I certify that I have personally completed all blanks in this form and explained them to the subject or his/her representative before requesting the subject or his/her representative to sign it. Date:______Signed: ______(Signature of project director or his/her authorized representative)

189 April 27, 1999

Dr. Ray Margorien Office of Research Risks Room 300, Research Foundation Building 1960 Kenny Road Campus

Dr, Margorien,

Please accept this request for addendum to the protocol entitled: The effect of commercially available tomato product consumption on Ivcopene levels in serum. IRB#98H0353

It is believed that lycopene is carried throughout the body with cholesterol. We have found it necessary to measure the total cholesterol levels of the plasma samples that were collected. Several of the subjects may be taking prescription medication that may alter cholesterol levels. We would like to request information firom the subjects concerning any prescription medication they may be taking. Since the majority of the subjects have completed the study, we will be voluntarily requesting the information through a mailing. A sample of the letter is attached.

Thank you for your assistance in coordinating this study.

Sincerely,

Steven K. Clinton M.D., PhD. Principal Investigator

190 (Date)

Dear (Subject Name),

Thank you once again for participating in the tomato product study at Ohio State University. We have been able to collect a significant amount of important information from the study. Before we can come to any conclusions regarding the results we would like to accoimt for any pharmacological interactions with normal digestive patterns. With this in mind, the information requested on the enclosed survey would be helpful in our analysis. We are asking that you complete the survey on a voluntary basis and return it in the enclosed envelope.

Thank you again.

191 General Clinical Research Center

Protocol Title:

The effect of commercially available tomato product consumption on lycopene levels in tissue and serum. A pilot study

Submitted by: Steven K. Clinton, M.D., Ph D. Steven J. Schwartz, Ph D. Charlotte Moxley

192 BASIC INFQRMAHON Protocol Data

1. Protocol Title The effect of commercially available tomato product consumption on lycopene levels in tissue and serum

2. Start/End Date: 1/1/99-8/1/99

3. Resources requested Nursing, Computer, Biostats, Core Lab

Total IP Days - 0 Total OP Days - 252 Total Patients - 36

IRE Information

1. IRE Number: 98H0353

2. Date of Approval 11/16/98

3. Revised?

Funding Information for PI and each Co-PI (Use additional sheets if necessary)

1. Title: Steven K. Clinton M.D., Ph.D.

2. Purpose of Research (two or three lines) The purpose of the study is to provide information about how lycopene levels change during a period of controlled consumption.

3. Funding source: New faculty start up funds and donations

4. Kind:

5. Agency: OSU

6. Grant/Contract Number:

7. Start/End Dates: 1/4/99 - 7/1/99

193 8. Annual Costs: Direct: $6000.00 Total: $6000.00 9. Percent Conunitment: 100%

10. Suggested reviewers for this protocol.

Name: Anne Smith, Ph D. Office address: 343E Campbell Hall, 1787 Neil Ave Phone: 2-0715 Fax: 2-8880 E-mail: [email protected]

Name: Tammy Bray Office address: 347 Campbell Hall, 1787 Neil Ave Phone: 2-4485 Fax: 2-8880 E-mail: [email protected]

194 GENERAL CLINICAL RESEARCH CENTER BODY OF THE PROTOCOL

Name and Title of Investigators

Steven K. Clinton, M.D., PhD. Arthur G. James Cancer Hospital The Ohio State University 3402 Starling-Loving Hall 320 W. lO*" Ave Columbus, OH 43210 614-293-7560 Clinton-1 @medctr.osu.edu

Steven J. Schwartz, Ph D. Professor 140 Howlett Hall 2001 FyffeCt. Columbus, OH 43210 614-292-2934 Schwartz. 177(^osu.edu

Charlotte Moxley Graduate Research Assistant 0598 Howlett Hall 2001 FyffeCt. Columbus, OH 43210 614-292-4069 moxley. 1 [email protected]

Abstract

The objective is to determine the ability of tomato soup, tomato sauce and V8 juice to enhance the levels of the carotenoid lycopene in blood and buccal cells. Lycopene is the major antioxidant carotenoid in tomato products and very little is known about its digestion and absorption fiom foods or its distribution in blood and tissue. Healthy volunteers will be asked to avoid tomato containing foods for 2 weeks. Participants will then consume 7 servings/week either tomato soup (1 cup/8 oz), tomato sauce (I cup/4oz.), or V8 juice (8 oz.) 195 for 4 weeks. Commercially available soup, sauce and juice will be provided to participants by the investigators. Participants will provide a blood and buccal cell sample once per week starting at the time of enrolment for a total of 7 samples. Blood and buccal cell sampling will be done at the OSU Clinical Research Center, by qualified medical personnel.

Hypothesis and Specific Aims.

The proposed research has been designed to answer the following questions;

4. How rapidly will lycopene be cleared from the blood and buccal cells while consuming a lycopene free diet?

5. How rapidly will the consumption of V8 juice, tomato sauce or tomato soup (7 servings per week) increase the concentration of lycopene in the blood and buccal cells?

6. What is the variability in blood and buccal cell lycopene between individuals consuming a lycopene free diet or while consuming V8, tomato sauce or tomato soup?

This pilot study is designed to limit the dietary intake of lycopene containing foods for six weeks. After 2 weeks, the subjects will be required to consume a predetermined amount of either V8 juice, tomato sauce or tomato soup for an additional four weeks. Blood and buccal cell samples will be obtained at one week intervals beginning when the subject has enrolled, continuing for six weeks, for a total of 7 sampling times. The study will define the range and variability of responses expected with V8, tomato sauce and tomato soup in a free-living, diverse group of healthy individuals. The baseline and two samples of no tomato consumption will determine how quickly blood lycopene levels drop over a period of time on a lycopene free diet. The four weeks of feeding will determine the shape of the curve and magnitude of change for blood lycopene over a period of four weeks. The quantitative change expected is necessary for calculating the statistical power of larger, more definitive, future studies of disease prevention. 196 Background and significance

Lycopene has recently emerged as a potentially beneficial dietary phytochemical in light of accumulating research findings which show an inverse correlation between consumption of food products high in lycopene and the risk of developing certain types of cancer (Block et al., 1992; Clinton, 1998; Giovannucci et al., 1995). Other studies involving lycopene reveal it’s superior ability to quench singlet oxygen among dietary carotenoids (Di Mascio et al., 1989), chemopreventative properties in animal models and cell cultures (Levy et al., 1995), as well as interesting bioavailability and tissue deposition patterns in terms of concentration and isomeric distribution (Clinton et al., 1996; Gartner et al., 1997; Schierle et al., 1997). Clinton et al. (1996) observed that the ratio of lycopene cis-trans geometrical isomers in biological fiuids such as plasma and in tissues such as prostate differ from those isomer ratios in fresh tomatoes. It has previously been assumed that the higher percentage of lycopene cis- isomer in human biological samples is due in part to consumption of heat treated tomato products containing cis- isomers of lycopene. Our laboratory has recently established, however, that in tomato products of various moisture content, fat content, and container type, lycopene (unlike P-carotene) is remarkably stable to isomerization reactions under typical industrial thermal processing conditions (Nguyen and Schwartz, 1998). Although lycopene and its’ cis-isomers can be measured in foods, blood and biological tissues independently (Ferruzzi, et al. 1998; Emenhiser, et al. 1996), further information is necessary to monitor the uptake and absorption into the bloodstream after typical tomato product consumption. It is also not well established how rapidly lycopene is cleared from the blood while consuming lycopene free diets. Data is needed to determine the period of consumption and amount of tomato products in the diet necessary to modulate circulating lycopene levels.

197 Experimental Design and Methods

1. Patient selection criterion. Patients will be recruited within the Food Science Department and Internal Medicine Department at Ohio State University. Subjects must be non-smokers, not pregnant, and healthy. Exclusions include those with gastrointestinal disease that may alter food digestibility including diseases such as; pancreatic insufficiency, hepatic disease, diabetes, and metabolic enzyme deficiencies, with no history of gastrointestinal disease. Every effort has been made to include a subject profile firom a variety of ethnic groups. Below is a sample of the projected subject profile bases on previous experience by the investigators. Lycopene patterns normally observed between ethnic groups are very similar.

American Asian or Black, Non- Hispanic White, Non- Total Indian Alaskan Pacific hispanic Hispanic Native Islander Female 0 6 2 0 10 18 Male 0 7 1 0 10 18 Total 0 13 3 0 20 36

Subjects must also be willing to follow the dietary restrictions for the wash out periods. Foods to be avoided include: tomatoes, spaghetti sauce, tomato juice, salsa, ketchup, watermelon, guava, and any product containing either fresh or processed tomato products such as lasagna and pizza.

2. Method of patient evaluation. Patient evaluation will involve an informational sheet and a dietary record for the course of the study. Patients will be asked to sign an Informed Consent. Patients will be contacted by telephone to ensure compliance with sampling dates and dietary requirements. Patients must be willing to consume tomato products. Subjects will be required to return the empty food containers to ensure compliance.

3. Trial design. In each investigation the control will consist of the baseline samples obtained when the subject is enrolled and before the washout period has begun. Eighteen male and eighteen female, non-smoking, healthy, non-pregnant between 18-65 will be required for this pilot study. There will be three groups of twelve subjects each (six men and six women per group); one group will consume V8 juice, one group will consume tomato sauce, and one group will consume tomato soup. Blood serum and buccal cell samples will be analyzed firom these subjects.

198 To investigate “How rapidly is Ivcopene cleared form the blood and buccal cells while consuming a lycopene free diet? We will examine lycopene profiles for subjects on a lycopene free diet for 2 weeks. For our purposes it is not important to show clearance as it would be for a drug. Our main focus is to measure the decrease over the two week period.

To investigate “How rapidlv will the consumption of V8 juice, tomato sauce or tomato soup H servings per week) increase the concentration of Ivcopene in the blood and buccal cells?” After 2 weeks on a lycopene free diet, subjects will consume either V8 juice, tomato sauce or tomato soup for 4 weeks with a weekly assessment of lycopene concentrations in blood and buccal cells.

To investigate “What is the variability in blood and buccal cell Icvopene between individuals consuming a Ivconene free diet or while consuming V8. tomato sauce or tomato soup?” Prior studies suggest significant heterogeneity in lycopene bioavailability among humans consuming lycopene containing products. We will determine the variation in response through our statistical evaluation of the blood and buccal cell lycopene patterns over the duration of the study.

4. Treatment schedule. When patients have been contacted and have signed the consent form, they will have their first baseline sample of blood drawn and buccal cells obtained. Subjects will then be asked to limit their intake of lycopene containing products for one week. The second sampling will be performed. Subjects will then continue their limited lycopene intake for one more week. The third sampling will be performed. Subjects will be instructed to consume a specified portion of either V8 juice, Prego or tomato soup 7 times/ week for four weeks. Samples 4-7 will be obtained at the end of weeks 1-4 of feeding.

Biostatistical Design and Analysis

Registration and assignment of patients to groups. Patients will be registered when contact is made and they have signed an informed consent. Subjects will be randomly assigned to groups with each group containing 6 men and 6 women.

Sample size choice: Thirty-six subjects will be used for this pilot study. The choice of sample size is governed by expense of the study and time constraints. With an effect size of 0.8 and an alpha of 0.05 the power of the study will be 0.53. A much more precise power calculation will be performed for the larger, more definitive study based on the information gained from this pilot.

Monitoring of trial progress. 199 Progress will be monitored to ensure an equal number of subjects are in each group and subjects are adhering to guidelines. Subject dropout may be a factor and may result in less than 36 subjects participating. Data obtained from the washout will constitute partial completion and will result in partial compensation for the study. Full completion will occur if all 7 sampling times are completed and will result in full compensation of the study. Compliance will be monitored by phone calls to the subjects to remind them of appointments and to identify problems. Subjects will be requested to return empty containers to signify consumption of required food.

Forms of data handling Informational forms and dietary questionnaires will be kept in a locked file cabinet and will be used to assign subjects to treatment groups. Analysis of the blood serum and buccal mucosal cells for carotenoid profile will be performed in the above researcher’s laboratory by HPLC and or HPLC/ECD (Electrochemical Detection).

Protocol deviations Protocol deviations will be kept to a minimum. The anticipated deviations are dropout and non-compliance to the dietary restrictions. Frequent reminders and close monitoring will hopefully keep this to a minimum.

Plans for statistical analysis Statistical analysis will be performed by Dr. Edward Giovannucci at Harvard University. The 36 subjects will be characterized according to baseline demographic characteristics (age, sex, height, body weight) and dietary intake for various nutrients (especially fat and carotenoid) based on the food frequency questionnaire. At each time point, blood values for lycopene will be determined for all 36 participants. Descriptive statistics for all subjects combined, and stratified by use of the 3 different tomato products will include mean and median values, and standard deviations. The mean and standard error of the mean for lycopene for each time point will be plotted to define the shape of the curve. The main test for difference in lycopene level by tomato product group will be determined by ANOVA methods. Transformations to achieve normality may be required. If distributions are very skewed, not-parametric procedures (Kruskal-Wallis test) will be used. The main test will be for differences at the end of the entire trial. Because multiple time points will be assessed, differences among groups could arise by chance due to multiple statistical testing; such comparisons will be interpreted with caution. We will also use multiple logistic regression methods to adjust for age and gender. We will used paired t-tests including all subjects to examine if oxidative markers change from time I (beginning of feeding) to time 2 (end of feeding). Again, transformation may be necessary to achieve normality of data or non- parametric tests (Wilcoxon test) may be used with non-normal data. Similar analyses will be conducted for buccal lycopene. In addition, the correlation between the change in blood lycopene (end of trial minus baseline) and the change 200 in buccal lycopene will be examined to determine if individuals respond similarly for these markers. Also, the mean and standard error for buccal lycopene level at each time point will be contrasted with the same blood lycopene to examine for different patterns in the response of buccal cell and blood (for example, if blood lycopene changes more quickly than buccal lycopene).

G. Bibliography

Block, G., Patterson, B., and A. Subar. 1992. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18: 1-29.

Clinton, SK. 1998. Lycopene: chemistry, biology, and implications for human health and disease. Nutrition Reviews. 56 (2ptl):35-51.

Clinton, S.K., Emenhiser, C., Schwartz, S.J., Bostwick, D.G., Williams, A.W., Moore, B.J., and J.W. Erdman. 1996. Cis-trans lycopene isomers, carotenoids, and retinol in the human prostate. Cancer Epidemiology, Biomarkers, and Prevention. 5: 823-833.

Di Mascio, P., Kaiser, S., and H. Sies. 1989. Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274:532-538.

Emenhiser, C., Simunovic, N., Sander, L.C., and S.J. Schwartz. 1996. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. J. Agric. Food Chem. 44:3887-3893.

Ferruzzi, M.G., Sander, L.C., Rock, C.L., and S.J. Schwartz. 1998. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Analytical Biochemistry. 256:74-81.

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

Giovannucci, E.L., Ascherio, A., Rinun, E.B., Stampfer, M.J., Colditz, G.A., and W.C. Willett. 1995. Intake of carotenoids and retinol in relationship to risk of prostate cancer. J. Natl. Cancer Inst. 87: 1767-1776.

Levy, J.J., Bosin, E., Feldman, B., Giat, Y., Munster, A., Danilenko, M., and Y. Sharoni. 1995. Lycopene is a more potent inhibitor of human cancer cell proliferation than either a-carotene or P-carotene. Nutr. Cancer. 24:257-266.

Nguyen, M L. and S.J. Schwartz. 1998. Lycopene stability during food processing. Pro. Soc. Exp. Bio. Med. 21%: 101-105.

201 Nomura, A.M.Y., Stemmermann, G.N., Lee, J., and N.E. Craft. 1997. Serum micronutrients and prostate cancer in Japanese Americans in Hawaii.. Cancer Epidemiology, Biomarkers, and Prevention. 6:487-491.

Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and W. Schuep. 1997. Content and isomeric ratio of lycopene in food and human blood plasma. J. Agric. Food Chem. 96:459-465.

H. Resource Request

Limited resources will be needed from the GCRC. Qualified individuals able to perform venipuncture will be needed to draw blood from the subjects. Buccal mucosal cell sampling will be performed by the above researchers. HPLC analysis will be performed on the blood serum, so blood will need to be separated into serum, lymphocytes, and red blood cells if possible. Samples of serum should be stored at -80°C until pick up by the researcher. Carotenoids are light sensitive and every effort should be made to limit exposure to light sources. In addition to the above laboratory assistance, any biostatistical assistance would be appreciated.

I. Justification for Use of the Facility

The GCRC will provide invaluable resources that are necessary for the successful completion of this research study. All of the above requested resources are not available to the researchers except from the GCRC.

J. Human Subjects

See enclosed

202 Instructions for 3 Day Diet Record Book

This three-day diary will be used to estimate your typical nutrient intake. The dietary information you provide is an important component of the research study.

To estimate your nutrient intake accurately, we need a complete record of what you eat and drink.

Keep a record of everything you eat and drink (including water) for three days. You should choose two non-consecutive weekdays (e.g. Monday and Wednesday) and one weekend day (Saturday fi£ Sunday). Fill in all information requested.

It is best to record what you eat and drink immediately after you consume it. However, if you recall something at a later time, add it to the diet diary. Carry your food record booklet with you throughout the day. Put it in your pocket or handbag. It’s just the right size! ©

Be very specific in describing the foods that you eat. For example

• Types of foods:

Milk - Is it whole, non-fat, 2% or 1% lowfat? Bread - Is it rye, whole wheat, raisin, etc.? Cereal - Brand name? Cookies - Chocolate chip, oatmeal, peanut butter, etc.?

• Food preparation:

Eggs - Fried, poached, scrambled (with what), etc.? Chicken - Roasted, hied, barbecued, etc.? Skin on or off?

• Addition to your food:

Coffee - Cream, milk, sugar, artificial sweeteners, etc.? Toast - Butter, margarine, jelly, etc.? Vegetables - Butter, cheese, margarine, dips, etc.? Sandwich - Mayonnaise, mustard, butter, etc.? Salad - Dressing? What type? Croutons, other toppings?

203 • Amounts consumed:

Eggs - How many? Milk - How much? 1 cup?Vr cup? (I cup = 8 ounces) Meat and Cheese - How many ounces? Check package labels for total amount and estimate how much you are eating. (3 ounces of cooked meat is the size of a deck of cards). Peanuts - Approximately how many? Salted or unsalted? If you ate a package, the weight of the package. Candy bars - How many bars and how many ounces? Cereal - How much? 1 cup? 16 cup? Check package labels. (Don’t forget the milk!)

• Mixed dishes:

You will need to break down certain combination items in your food record using two or more separate lines. For example, a turkey and Swiss cheese sandwich might be:

• Two slices of whole wheat bread • Three slices turkey • Two slices Swiss cheese • One tablespoon mayonnaise

204 Example:

Time Meal Place Eaten Amount Food Description 7AM B Home 1 cup Cheerios 6 ounces 2% milk 2 large Strawberries 5 Vi oz can V8 11AM S At desk 2 inch diam Cherry Danish 12 ounces Coffee 2 packets Sugar 4 packets Half & half 1 PM L School cafeteria 1 Vi cups Chicken noodle soup 20 Goldfish 12 ounces Diet Pepsi 7 PM D Boston Market 1 medium Chicken leg and thigh w/skin, roasted % cup Stuffing cup Glazed carrots 3 inch square Cora bread 12 ounces Rolling Rock beer

205 NAME FOOD CONSUMED

DIETARY SCHEDULE

DAY OF DATE DAY OF TIME OF AMOUNT OTHER STUDY WEEK MEAL CONSUMED 1 2 3 4 5 6 7 EVENING 8 GCRC TIME 9 10 11 12 13 14 EVENING 15 GCRC TIME 16 17 18 19 20 21 EVENING 22 GCRC TIME 23 24 25 26 27 28 EVENING 29 GCRC TIME

206 FOODS TO AVOID DIETARY INTERVENTION PERIOD (WEEKS 3-6)

Please list any deviations (mm the required diet on this table.

DAY DATE DAY OF AMOUNT TOMATO CONTAINING FOOD WEEK I 2 3 4 5 6 7 BLOOD DRAW TOMORROW 8 9 10 11 12 13 14 BLOOD DRAW TOMORROW 15 16 17 18 19 20 21 BLOOD DRAW TOMORROW 22 23 24 25 26 27 28 BLOOD DRAW TOMORROW

207 Foods to avoid include:Please remember that fast food restaurants put ketchup on burgers unless you ask for no ketchup. It’s very important to read labels of meals that contain sauce or seasoning packets. Many of them contain tomato powder or flakes.

TOMATOES, RED PASTA SAUCE, TOMATO PASTE, KETCHUP, VEGETABLE SOUP, BARBEQUE SAUCE, SALSA, PIZZA, VEGETABLE JUICE, WATERMELON, PINK GRAPEFRUIT, BLOOD ORANGES, GUAVA. CHILI.

208 FOODS TO AVOID WASHOUT PERIOD (WEEKS 1-2)

Please list any deviations from the required diet on this table.

DAY DATE DAY OF AMOUNT TOMATO CONTAINING FOOD WEEK 1 2 3 4 5 6 7 BLOOD DRAW TOMORROW 8 9 10 11 12 13 14 BLOOD DRAW TOMORROW

Foods to avoid include:Please remember that fast food restaurants put ketchup on burgers unless you ask for no ketchup. It’s very important to read labels of meals that contain sauce or seasoning packets. Many of them contain tomato powder or flakes.

TOMATOES, RED PASTA SAUCE, TOMATO PASTE, KETCHUP, VEGETABLE SOUP, BARBEQUE SAUCE, SALSA, PIZZA, VEGETABLE JUICE, WATERMELON, PINK GRAPEFRUIT, BLOOD ORANGES, GUAVA. CHILI

209 Dietary Information Sheet

Name Date

Address Phone_

Age

Height_ Weight_ Subject #_

How frequently do you consume tomatoes? (fresh or raw) never daily biweekly ^weekly twice/month _monthly

How many servings/day? (Iserving=l/2 tomato or 'A cup chopped or sliced tomato)_

How frequently do you consume tomato juice (not V8 or vegetable juice)? never daHy biweekly ^weekly twice/month _monthly

How many servings/day? (lserving= 8oz/medium glass) ______

How frequently do you consume V8 juice? never daily biweeWy weekly twice/month monthly

How many servings/day? (Iserving= 8oz/medium glass)_

How frequently do you normally consume tomato soup? never daily biweekly weekly _ twice/month _monthly

How many servings/day? (Iserving= 8oz / I cup)_

How frequently do you consume pizza? never daHy biweekly weekly twice/month monthly

How many servings (slices)/day? (lserving=l/8 o f a 12” pie)_

How frequently do you consume red pasta sauce (spaghetti sauce,)? never daHy biweekly weekly twice/month _monthly

How many seivings/day? (lserving= '/i cup) ______

How frequently do you consume watermelon? never daily biweekly ^weekly twice/month _monthly

How many servings/day? (lserving= 1 cup diced or medium wedge).

How frequently do you consume pink grapefruit or pink grapefruit juice? (Separate juice and fruit?) never daily biweekly weeWy twice/month monthly

How many seryings/day? (lserying= 1 cup juice/medium glass or 'A grapefruit) ______

210 How frequently do you consume blood oranges? never daHy biweekly weekly twice/month monthly

How many servings/day? (lserving= 1 medium) ______

How frequently do you consume salsa? neyer daHy biweekly weekly twice/month monthly

How many seryings/day? (lserving= 2 tablespoons) ______

How frequently do you consume ketchup or barbecue sauce? never daily biweekly weekly twice/month monthly

How many seryings/day? (lserving= I tbs.) ______

211 Recipe Suggestions PREGO® Each of the following recipes includes cup Prego in one serving of the recipe.

IVfeatbail Sandwich (1 sandwich)

Heat thoroughly V^ cup Prego Traditional Pasta Sauce and frozen frilly cooked meatballs (no tomato containing ingredients). Serve on hard roll. Sprinkle with mozzarella and parmesan cheese.

Itaiian Sausage Sandwich (3 servings)

1 pound Italian pork sausage, casing removed 1 Vi cups Prego Traditional Pasta Sauce 3 long hard rolls, split

Cook sausage until browned, stirring to separate meat. Spoon off fat. Add spaghetti sauce. Reduce heat to low. Heat through stirring occasionally. Serve on rolls.

Italian Style Sloppy Joes (3 servings)

1 pound ground beef 1 medium onion, chopped 1 tablespoon Worcestershire 1 Vi cups Prego Traditional Pasta Sauce

Cook ground beef and onion until browned, stirring to separate meat. Spoon off fat. Add spaghetti sauce and Worcestershire. Reduce heat to low. Heat through stirring occasionally. Serve open face on rolls.

Chicken Mozzarella Sandwich (1 sandwich)

Vi cup Prego Traditional Pasta Sauce 1 refrigerated frilly cooked breaded chicken cutlet Mozzarella Cheese Roll

Heat spaghetti sauce and chicken cutlet. Top cutlet with cheese, continue heating until cheese begins to melt. Serve on roll.

Chicken Pizza Mufilns

1 English muffrn, split and toasted Vi cup Prego Tradtional Pasta Sauce 1 can (3 ounces) Chunk Chicken, drained '/« cup shredded mozzarella cheese Crushed red pepper, dried oregano, garlic powder (optional)

212 Arrange muffin halves on baking sheet Spread each muffin half with 4 tablespoons spaghetti sauce. Divide chicken and cheese on muffin halves. Sprinkle with red pepper, etc. Bake at 400°F. 10 minutes or until cheese is melted.

Cheesey Vegetable Macaroni (6-1 cup portions)

1! tablespoon olive or vegetable oil 2 medium zucchini sliced (about 3 cups) 3 cups Prego Traditional Pasta Sauce 4 cups cooked corkscrew macaroni (about 3 cups dry) 1 cup shredded mozzarella cheese (4 ounces)

In 10-inch skilled over medium heat in hot oil, cook zucchini until tender crisp, stirring often. Add spaghetti sauce and macaroni. Heat through, stirring occasionally. Stir in cheese.

PREGO® INDIVIDUAL QUICK CHICKEN PARMIGIANA Prep Time: S minutes Cook Time: IS minutes

Frozen fully cooked breaded chicken pattie or refrigerated fully cooked breaded chicken cutlet '/] cup Prego® Traditional Pasta Sauce Grated Parmesan cheese and shredded mozzarella cheese Hot cooked spaghetti

1. Place chicken pattie In shallow baking dish. Top with 1/2 cup pasta sauce. Sprinkle with Parmesan cheese and mozzarella cheese. 2. Bake at 400° P. for 15 minutes or until chicken is hot and cheese is melted. PREGO® MIRACLE LASAGNA (Serves 6) Prep Time: S minutes Cook Time; 1 hour Stand Time: 5 minutes

3 cups Prego® Traditional Pasta Sauce 6 uncooked lasagna noodles 1 container (IS ounces) ricotta cheese 8 ounces shredded mozzarella cheese (2 cups) 1/4 cup grated Parmesan cheese

1. In 2-quart shallow baking dish (11-by 7-inch) spread / cup pasta sauce. Top with 3 uncooked lasagna noodles, ricotta cheese, I cup mozzarella cheese, Parmesan cheese and I cup pasta sauce. Top with remaining 3 uncooked lasagna noodles and remaining pasta sauce. Cover. 2. Bake at 37S°F. for 1 hour. Uncover and top with remaining mozzarella cheese. Let stand S minutes.

213 Campbell’s Tomato Soup Menu and recipe ideas. Portions given will provide K can of Campbell’s tomato soup.

Tomato Beef Stew (Makes 2 servings, about 1 % cups each)

'/ z pound ground beef I can (10 % ounces) Campbell’s condensed tomato soup '/z soup can water I cup frozen cut green beans '/ z cup frozen sliced carrots I teaspoon Worcestershire sauce

1. In I '/z quart sauce over medium heat, cook beef until browned and no longer pink, stirring to separate meat. Spoon off fat. 2. Stir in soup and water. Add beans, carrots and Worcestershire sauce. Heat to simmering. Cook 10 minutes or until vegetables are tender, stirring occasionally.

Tomato-Vegetable Noodle Soup (Makes 2 servings about 1 % cups each)

I can (10 % ounces) Campbell’s condensed tomato soup I soup can water I cup cooked mixed vegetables I cup cooked bow tie noodles (1 cup uncooked)

In I '/z quart saucepan, compine soup and water. Ad vegetables and noodles. Over medium heat, heat through, stirring occasionally.

CAMPBELL’S® BEEF & BROCCOLI (Two servings) Prep Time: 10 minutes Cook Time: 20 minutes

'/z pound boneless beef sirloin or top round steak, 3/4 inch thick I tablespoon vegetable oil I can (10 3/4 ounces) Campbell’s® Condensed Tomato Soup 3 tablespoons soy sauce I tablespoon vinegar 1 teaspoon garlic powder 1/4 teaspoon crushed red pepper (optional) 3 cups fresh or thawed* frozen broccoli flowerets 2 cups hot cooked rice

1. Slice beef into very thin strips. 2. In medium skillet over medium-high heat, heat oil. Add beef and stir-fry until browned and juices evaporate. 3. Add soup, soy, vinegar, garlic and pepper. Heat to a boil. Reduce heat to medium. Add broccoli and cook until tender-crisp, stirring occasionally. 4. Divide mixture in half, serve over cooked rice.

214 V8(D Recipes and Menu Ideas Menu and recipe ideas. Portions given will provide 1 cup V8.

LIME SPLASH i\ splash vtwMn 1 cupofVS) Prep Time; 5 minutes 3 cups V8® 100% Vegetable Juice 1 i/3 cups ginger ale 1 tablespoon lime juice Lime slices for garnish

Mix vegetable Juice, ginger aie and lime Juice. Serve over ice. Garnish with iime siices. Makes about 4 cups.

V8® CITRUS COOLER (2 cups of Citrus Cooler will provide 1 cup of V8) Prep Time: 5 minutes 3 cups V8® 100% Vegetable Juice 1 1/2 cups orange juice 1 1/2 cups seltzer water or club soda 1/4 cup lime juice Orange and lime slices for garnish

Mix vegetable juice, orange juice, seltzer and lime juice. Serve over ice. Garnish with orange and lime slices. Makes about 6 cups.

V8® BLOODY MARY MOCKTAIL (I BLOODY MARY MOCKTAtL will provide 1 cup of VS) Prep Time: 5 minutes 3 cups V8® 100% Vegetable Juice I teaspoon prepared horseradish 1 teaspoon Worcestershire sauce 1/2 teaspoon hot pepper sauce Lemon slices for garnish 1. Mix vegetable juice, horseradish, Worcestershire and hot pepper sauce. 2. Serve over ice. Garnish with lemon slices. Makes 3 cups.

215 TOMATO SAUCE INSTRUCTIONS

WASHOUT and DIETARY INTERVENTION

The first two weeks of the study are called the '"washout period” and the following 4 weeks will be terms the “dietary intervention period”.

FOODS TO AVOID

For the entire six weeks of this investigation, you will be asked to avoid certain foods. Some of these foods are listed on attached “Foods to Avoid” sheet. It is very important that you examine food labels and avoid all foods containing any tomato derived items. If you do consume a food that you suspect or know contains any of the products on the “foods to avoid” list, please record the information (food item and amount) on page 2 of the “Diet Record” sheet and the amount consumed. If you are unsure if the food is a violation, please record it anyway.

DIET Your dietary intervention will begin after two weeks and continue for an additional 4 weeks. You will be consuming one serving of tomato sauce every day for 28 days.

One serving = 4 ounces = 1/2 cup. Use the measuring cup to accurately measure your portion. Please open a new jar every 3 days. Refiigerate unused portion after opening.

If you use on of the recipes, the amount of prepared food you need to consume is listed on the recipe.

You may add any other food to that meal as long as it does not appear on the list of potential violations AND does not have an ingredient label listing ‘tomato’.

The tomato sauce can be consumed at any time of day, except on the day before the GCRC visit when it should be consumed with the evening meal (after 5pm).

DIET RECORD The Diet Record sheet should be used daily to record time of day and amount of sauce consumed. Page 2 of the Diet Record is to be used to record any food violations.

BLOOD AND CHEEK CELL SAMPLE Your schedule for providing blood and cheek cells is also included on the diet record sheet. If for any reason you cannot keep you appointment, please notify a study investigator at your earliest convenience to arrange another visit.

216 QUESTIONS

If you have any questions about foods to avoid or the consumption of tomato sauce, please call one of the study investigators at your earliest convenience.

TOMATO SOUP INSTRUCTIONS

WASHOUT and DIETARY INTERVENTION

The first two weeks of the study are called the "washout period” and the following 4 weeks will be terms the “dietary intervention period”.

FOODS TO AVOID

For the entire six weeks of this investigation, you will be asked to avoid certain foods. Some of these foods are listed on attached “Foods to Avoid” sheet. It is very important that you examine food labels and avoid all foods containing any tomato derived items. If you do consume a food that you suspect or know contains any of the products on the “foods to avoid” list, please record the information (food item and amount) on page 2 of the “Diet Record” sheet and the amount consumed. If you are unsure if the food is a violation, please record it anyway.

DIET Your dietary intervention will begin after two weeks and continue for an additional 4 weeks. You will be consuming one serving of tomato soup every day for 28 days.

Prepare the entire can of soup as directed on the label with either milk or water. Use the measuring cup to measure out a one cup portion to consume that day. Measure out a one cup portion to consume the next day and refiigerate that portion.

If you use on of the recipes, the amount of prepared food you need to consume is listed on the recipe.

You may add any other food to that meal as long as it does not appear on the list of potential violations AND does not have an ingredient label listing ‘tomato’.

The tomato soup can be consumed at any time of day, except on the day before the GCRC visit when it should be consumed with the evening meal (after Spm).

217 DIET RECORD The Diet Record sheet should be used daily to record time of day and amount of soup consumed. Page 2 of the Diet Record is to be used to record any food violations.

BLOOD AND CHEEK CELL SAMPLE Your schedule for providing blood and cheek cells is also included on the diet record sheet. If for any reason you cannot keep you appointment, please notify a study investigator at your earliest convenience to arrange another visit.

OUESTIONS

If you have any questions about foods to avoid or the consumption of the tomato soup, please call one of the study investigators at your earliest convenience.

V8 JUICE INSTRUCTIONS

WASHOUT and DIETARY INTERVENTION

The Grst two weeks of the study are called the “washout period” and the following 4 weeks will be terms the “dietary intervention period”.

FOODS TO AVOID

For the entire six weeks of this investigation, you will be asked to avoid certain foods. Some of these foods are listed on attached “Foods to Avoid” sheet. It is very important that you examine food labels and avoid all foods containing any tomato derived items. If you do consume a food that you suspect or know contains any of the products on the “foods to avoid” list, please record the information (food item and amount) on page 2 of the “Diet Record” sheet and the amount consumed. If you are unsure if the food is a violation, please record it anyway.

DIET Your dietary intervention will begin after two weeks and continue for an additional 4 weeks. You will be consuming one serving of V8 juice every day for 28 days.

You are to consume a total of 10 cans (5.5 oz.) each week. Please alternate one can one day and then two cans the next. For example: one can Monday, then two cans Tuesday, and so on....

If you use on of the recipes, the amount of prepared food you need to consume is listed on the recipe.

218 You may add any other food to that meal as long as it does not appear on the list of potential violations AND does not have an ingredient label listing ‘tomato’.

The V8 juice can be consumed at any time of day, except on the day before the GCRC visit when it should be consumed with the evening meal (after Spm).

DIET RECORD The Diet Record sheet should be used daily to record time of day and amount of juice consumed. Page 2 of the Diet Record is to be used to record any food violations.

BLOOD AND CHEEK CELL SAMPLE Your schedule for providing blood and cheek cells is also included on the diet record sheet. If for any reason you cannot keep you appointment, please notify a study investigator at your earliest convenience to arrange another visit.

OUESTIONS

If you have any questions about foods to avoid or the consumption of the V8 juice, please call one of the study investigators at your earliest convenience.

219 APPENDIX D

Supplements to Chapter 4: Milk Study

220 Control Fresh Processed Baseline After treatment Baseline After treatment Baseline After treatment total lycopene^ 0.812 ±0.034 0.760 ±0.067 0.780 ±0.039 0.890 ±0.044 0.637 ± 0.062 0.823 ± 0.06 all-lraits lycopeme^^ 0.316 ±0.019 0.303 ±0.019 0.292 ±0.011 0.366 ±0.022 0.271 ±0.012 0.319 ±0.02 S-cis lycopene* 0.278 ±0.013 0.274 ±0.012 0.259 ±0.011 0.263 ±0.015 0.246 ±0.009 0.279 ±0.01 other cls-lycopenes^ 0.218 ±0.033 0.184 ±0.041 0.216 ±0.031 0.260 ±0.012 0.120 ±0.046 0.225 ± 0.03 «p-carotene 0.321 ±0.055 0.358 ± 0.088 0.275 ±0.018 0.298 ±0.018 0.271 ±0.033 0.262 ± 0.028 a-carotene 0.007 ± 0.004 0.007 ±0.004 0.004 ±0.002 0.004 ±0.002 0.004 ±0.001 0.004 ±0.001

Table 1. Plasma concentrations of major carotenoids in each treatment group.

' concentration in pmoles/L. ^ significant increase from baseline Fresh (p<0.01) ^ significant increase from baseline Processed (p<0.005) ^ significant increase from baseline Fresh (p<0.005) ^ significant increase from baseline Processed (p<0.05) ^ significant increase from baseline Processed (p<0.01 ) ^ significant increase from baseline Processed (p<0.05) Estimated fat levels by creamatocrit sam ple m ean fat sam ple m ean fat 847-1 45.7 169-1 38.8 847-2 48.1 169-2 25.3 188-1 55.8 595-1 19.4 188-2 70.0 595-2 34.9 791-1 36.7 478-1 61.9 791-2 32.5 478-2 31.4 576-1 29.1 533-1 65.1 576-2 37.6 533-2 45.4 313-1 58.4 646-1 49.9 313-2 75.4 646-2 58.6 969-1 45.0 969-2 67.8 855-1 35.0 855-2 43.3 132-1 66.8 132-2 29.0 896-1 71.6 896-2 62.1 615-1 25.6 615-2 55.5 123-1 75.7 123-2 83.7 441-1 48.6 441-2 79.4 232-1 58.2 232-2 44.3 451-1 48.6 451-2 43.7 369-1 39.1 369-2 45.9 937-1 64.9 937-2 57.5 214-1 16.3 214-2 16.4 557-1 67.1 557-2 41.9 788-1 43.1 788-2 41.0

222 Baseline After treatment

nmol/L nmol/L total lycopene Control * 185 ±20.3 165 ± 19.0 Fresh 204 ± 29.7 205± 19.4 Processed * 133 ± 10.3 178.4 ±17.7 all-traits lycopene Control ‘ 66.1 ±7.49 58.7 ±5.4 Fresh 74.0 ± 10.6 81.7 ±8.93 Processed * 51.8 ± 1.87 67.0 ± 5.8 S-cis lycopene Control 59.1 ±5.52 55.4 ±4.55 Fresh 63.9 ± 8.53 57.6 ±3.37 Processed * 49.1 ± 1.82 58.4 ±3.9 other cfs-lycopenes Control 60.2 ± 7.68 51.4 ±9.74 Fresh 66.1 ± 10.7 65.7 ± 5.86 Processed * 34.2 ± 7.58 53.0 ±8.7 P-carotene Control 102 ±34.1 90.2 ±24.6 Fresh 101 ± 13.1 99.9 ± 17.0 Processed 61 ±8.8 77.1 ± 13.6 a-carotene Control 2.34 ± 1.9 1.78 ± 1.35 Fresh 0.911 ±0.22 0.896 ± 0.29 Processed 0.435 ±0.14 0.823 ±0.33

Table 2. Milk concentrations of major carotenoids in each treatment group expressed as nmol/L.

‘ After treatment < baseline, p baseline, p<0.05

223 Baseline After treatment

nmol/g lipid nmol/g lipid total lycopene Control 4.5 ±0.8 4.5 ±0.9 Fresh 4.17 ±0.5 4.4 ±0.6 Processed 3.4 ±0.7 3.7 ±0.6 M -trans lycopene Control 1.6 ±0.3 1.6 ±0.3 Fresh 1.5 ±0.2 1.7 ±0.2 Processed 1.3 ±0.3 1.4 ±0.2 S-cis lycopene Control 1.4 ±0.3 1.5 ±0.3 Fresh 1.3 ±0.2 1.3 ±0.2 Processed 1.2 ±0.2 1.3 ±0.2 other c/5-lycopenes Control 1.5 ±0.3 1.4 ±0.4 Fresh 1.3 ±0.2 1.4 ±0.2 Processed 0.9 ±0.3 1.1 ±0.2 P-carotene Control 2.4 ±0.9 2.7 ± 1.1 Fresh 2.0 ±0.5 2.0 ±0.2 Processed 1.4 ±0.2 1.6 ±0.3 a-carotene Control 0.06 ±0.05 0.06 ±0.05 Fresh 0.02 ±0.01 0.02 ±0.01 Processed 0.01 ±0.01 0.02 ±0.01

Table 3. Milk concentrations of major carotenoids in each treatment group expressed as nmol/g lipid.

224 Poster presentation at Experimental Biology, 2000 in San Diego, CA.

Blood and milk lycopene Isomer concentrations Increase In lactating women consuming dally servings of processed or fresh tomato products.

CL Moiley', AM Smltb\ SK.CIintan\ SJ Schwartz'.

The Ohio State University Dept of Food Science and Technology', Dept of Human Nutrition^ and College of Medicine and Public Health^ 43210

The present study was designed to quantitate plasma and milk lycopene concentrations in nursing mothers (n=24, 4-12 weeks postpartum) consuming equivalent amounts of lycopene as either fresh tomatoes or processed tomato sauce. All 24 subjects consumed a lycopene-free diet for 7 days (washout period). Participants were assigned to the control (C) group (n=8, grapes) or one of two (n=8) intervention groups instructed to consume five servings of either fresh tomatoes (F) or processed tomato sauce (?) over 3 days (- SOmg total lycopene) without any other sources of lycopene. Blood and milk samples were obtained before and after intervention for HPLC analysis of the geometric isomers (all-trans, 5-cis, all other cis, and total) of lycopene. Total plasma lycopene concentrations (Mean ± SE, pmoles/L) increased from 0.780 ± 0.04 to 0.890 ± 0.04

(12.3%, p<0.05) and from 0.637 ± 0.06 to 0.823 ± 0.06 (22.6%, p<0.005) in the F and P groups, respectively. The only plasma geometric isomer to increase significantly in the

F group was the all-trans lycopene isomer (20.2%, p

225 were seen in the P group which increased from 0,133 ± 0.01 to 0.178 ± 0.01 (25.2%,

p<0.01) pmoles/L. The geometric lycopene isomers increased in the P milk group by

23.8% (p<0.05), 15.5% (p<0.05), and 30.1% (p<0.05) for all-trans, 5-cis, and all other

cis, respectively. This study demonstrates that lycopene from processed tomato products

may be more bioavailable than raw tomatoes and may contribute to a greater transfer of

lycopene isomers into the milk of lactating women in a short-term study. {Supported by

OSU Comprehensive Cancer Center, Hirzel Canning Corporation, and The Ohio

Agricultural Research and Development Center.)

226 Proposal No: ______OARDC Matching Grant Competition New Submission Bioavailability of lycopene from fresh and processed tomatoes.

Steven J. Schwartz, Professor (Contact) Department of Food Science and Technology 140 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210 Phone: 614-292-4069 Fax: 614-292-4233 E-Mail: schwartz.l77(g)osu.edu

Beginning November 1, 1998 for 12 months Amount Requested from OARDC: $6,000 Matching funds from The Hirzel Canning Company: $6,000

227 Project Summary:

Lycopene is the main carotenoid pigment in tomatoes that imparts the characteristic red color. Accumulating epidemiological evidence shows a possible link between the consumption of tomato products with reduced risk of developing certain types of cancer. The bioavailability, as measured by absorption and depositon in the body, of lycopene in tomatoes is believed to be influenced by the amount of cis-isomers present in the foods and thermal processing is partially responsible for the presence of cis- isomers. This pilot project aims to determine the effect food processing has on lycopene isomerization reactions. The researchers will then investigate the bioavailability of lycopene from consumption of fresh and processed tomatoes through analyses of samples of blood serum, buccal cells and human milk.

228 Table of Contents

Objectives 1

Rationale and Significance I

Research Methods 2

Duration of Proposed Studies 3

Budget Narrative 4

Collaborative Arrangements and Matching Funding Plan 4

References 4

List of Potential Internal and External Reviewers 6

Budget 7

Curriculum Vitae 8

Current and Pending Support 10

Abstract 12

Attachment 13

229 Project Description Objectives The following objectives will be investigated in the proposed research. / Determine the effect food processing has on lycopene isomerization reactions by comparing fresh tomatoes and processed tomato sauce. / Determine the bioavailablity of lycopene from fresh tomatoes and processed spaghetti sauce from Hirzel Canning Corporation.

Rationale and Significance

Lycopene has recently emerged as a potentially beneficial dietary phytochemical

in light of accumulating epidemiological findings which show an inverse correlation

between consumption of food products high in carotenoids such as lycopene and the

risk of developing certain types of cancer (Block et al., 1992; Giovannucci et al., 1995).

Limited data exists suggesting that carotenoid compounds within processed food

products are more readily absorbed by humans and are therefore more bioavailable than

non-processed products (Rock et al., 1998 ; Gartner et al., 1997; Stahl and Sies, 1992).

There seems to be indications that consuming processed tomato products

simultaneously with fat will enhance the bioavailability of carotenoids (Gartner et al.,

1997; Stahl and Sies, 1992). Blood and body tissues have been shown to have more

than half of their total amount of lycopene comprised of cw-isomers instead of the all-

trans form (Clinton et al., 1996). It has previously been assumed that the higher percentage of lycopene cis- isomer in human biological samples is due in part to consumption of heat treated tomato products containing cis- isomers of lycopene.

230 Questions, however, remain regarding the presence of cis- lycopene isomers formed by

thermal processing of tomatoes and their physiological significance.

Although lycopene and its’ cis-isomers can be measured in foods, blood and

biological tissues independently (Ferruzzi, et al. 1998; Emenhiser, et al. 1996),

biological sampling presents the greatest challenges. Invasive procedures and difficult

sample composition present obstacles that can in part be solved with appropriate

analytical techniques and convenience of sampling. Determining bioavailability

through uptake in blood as well as body tissues is essential, since there are indications

that isomer content is very different in blood and body tissues. An easily accessible

body tissue is the buccal cell, or cells on the inside of the cheek. These cell samples can

be obtained with a toothbrush. Human milk is a natural secretion that is sampled with relative ease. Both buccal cells (Peng and Peng, 1992) and human milk (Canfield et al.

1998) have not only been shown to contain carotenoids, but to be valuable physiologic sources to monitor bioavailablility and uptake.

The tomato industry has devoted recent attention in how the bioavailability of

lycopene in tomato products is affected by the composition and processing of foods

initiated the formation of the Tomato Research Council. The Council’s purpose is to educate the public about the health benefits of processed tomatoes and encourage further study of the potential nutritional benefits of processed tomato products.

Recently, The Hirzel Canning Corporation agreed to assist us in conducting a pilot research project on how lycopene bioavailability is influenced by food composition and processing. If funded by OARDC, this study will provide a

231 mechanism to create a new industry-university cooperative research effort. The pilot study will provide necessary data on the nutritional benefits of tomato product consumption and how processing affects isomerization reactions, as well as the bioavailability of those products. Indications that processed tomato products increase uptake by the body of valuable nutrients such as lycopene could result in additional marketing opportunities for the tomato industry. When a sufficient volume of research has been generated to support the early claims that have been made, the possibility exists for the addition of health claims to the nutritional label of tomato products. The data generated will be useful to apply for future funding through the USDA Human

Nutrition Competitive Grant Program as well as other federal funding agency programs.

Research Methods Design Three groups of 15 lactating women will be needed for the study. The control group will receive no tomato products but will receive grapes to eat. One treatment group will be required to consume given amounts of fresh tomatoes, and the other treatment group will be required to consume given amounts of spaghetti sauce. There will be a period of time when no tomatoes will be consumed before feeding begins so the first set of samples (milk, blood serum, and buccal cells) will demonstrate lowered levels of lycopene. After 4 days of feeding, a second set of samples will be drawn to show increased levels. All blood sampling will be done at Children’s Hospital Clinical

Studies Center.

232 Food

Fresh Roma tomatoes and grapes will be commercially purchased. Spaghetti sauce will be provided free of charge from Hirzel Canning Corporation. Every subject in the treatment groups will be consuming equivalent amounts of lycopene. The tomatoes selected for this study will be carefully monitored for lycopene content and the processing of the sauce will be carried out according to National Canner’s

Assoication (1976) time and temperature requirements. Sauce will be standardized to lOg fat/serving.

Analysis

Food and biological samples will be analyzed for amounts of all-tra/u lycopene and lycopene cis- isomers using HPLC with either electrochemical detection or UV- visible detection (Ferruzzi et al., 1998; Emenhiser, et al. 1996). Presumably, there will be a higher content of lycopene cû-isomers in the samples from the spaghetti sauce subject group than in the fresh tomato subject group. Comparison of the lycopene amounts in the three different sample systems (blood, buccal cells, human milk) could yield valuable information on the bioavailability of lycopene in blood and body tissues.

Limitations

This protocol has been awarded Ohio State University and Children’s Hospital

IRB approval and advertisements have been posted in local pediatricians offices to recruit interested women for this study. Duration of the study will mainly be determined by the length of time involved in enrolling subjects.

233 Duration of proposed studies

All of the required human subjects protocols have been approved, advertisements are in place and enrollment is ready to begin. It is anticipated that enrollment will be complete within 6 months, along with sample collection. An additional 4 months will be required for sample analysis, compiling data and determining statistical correlations. The remaining 2 months of the 1 year project will be dedicated to preparing manuscripts to be published and presenting results at scientific meetings.

Budget Narrative

The majority of the funding requested is for materials and supplies ($2000).

Charges fi-om Children’s Hospital ($1,350) are necessary to obtain the biological samples. Partial support for a graduate student ($1500) is needed to conduct the analyses and coordinate the study. The sum of $200 is requested for travel to Hirzel

Canning Corporation to coordinate the manufacture of the sauce and to report on the results of the study. The budgeted sum of $500 will be required to publish research results in refereed journals. The total of $6,000 requested reflects 50% the costs necessary to complete this project. Additional funds from Hirzel Canning Corporation

($6,000) is contingent upon matching funds from OARDC.

234 Collaborative Arrangements and Matching Funding Plan

Discussions with Dr. Bill Hirzel at Hirzel Canning Corporation in Toledo, Ohio bave led to an agreement by which Hirzel Canning Corporation agrees to provide the matching funds necessary to support this pilot research project contingent upon receipt of hmds from OARDC. They have also graciously agreed to provide the tomato sauce at no charge for the feeding portion of the study. A copy of the letter of intent is included in the attachments.

References

Block, G., Patterson, B., and A. Subar. 1992. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18: 1-29.

Canfield, L.M., Giuliano, A.R., Neilson, E.M., Blashil, B.M., Graver, E.J., and H.H. Yap. 1998. Kinetics of the response of milk and serum beta carotene to daily beta carotene supplementation in healthy, lactating women. Am. J. Clin. Nutr. 67:276-83.

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

Emenhiser, C., Simunovic, N., Sander, L.C., and S.J. Schwartz. 1996. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. J. Agric. Food Chem. 44:3887-3893.

Ferruzzi, M.G., Sander, L.C., Rock, C.L., and S.J. Schwartz. 1998. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Analytical Biochemistry. 256:74-81.

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

Giovannucci, E.L., Ascherio, A., Rimm, E.B., Stampfer, M.J., Colditz, G.A., and W.C. Willett. 1995. Intake of carotenoids and retinol in relationship to risk of prostate cancer. J.Natl. Cancer Inst. 87: 1767-1776.

235 National Canner’s Association. Processes for Low Acid Canned Foods in metal containers. Bulletin 26-L, 1 i“* ed.; National Canner’s Association; Washington, DC, 1976.

Peng, Y-S and Y-M Peng. 1992. Simultaneous liquid chromatographic determination of carotenoids, retinoids, and tocopherols in human buccal mucosal cells. Cancer Epidemiology, Biomarkers and Prevention. 1:375-382.

Rock, C.L., Lovalvo, J.L., Emenhiser, C., Ruffin M.T., Flatt, S.W., and S.J. Schwartz. 1998. Bioavailability of beta-carotene is lower in raw than in processed carrots and spinach in women. J. Nutr. 128:913-916.

Stahl, W. and H. Sies. (1992) Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J. Nutr. 122(11): 2161-2166.

236 List of potential internal and external reviewers

John B. Allred, PhD. Professor, Food Science Professor, Allied Medicine 122 Vivian Hall 2121 FyffeRoad 614/292-6217 614/292-0218 e-mail: [email protected]

Gary R. Beecher, Ph.D. Resident Chemist, Food Composition Lab USDA 10300 Baltimore Avenue Guilding 161-EE Beltsville, MD 20705 301/504-8356 301/504-8314 e-mail: beechertS.bhnrc.usda.gov

Robert A. DiSilvestro, Ph D. Associate Professor, Human Nutrition 315F Campbell Hall 1787 Neil Avenue Columbus, OH 43210 614/292-6848 614/292-8880 e-mail: disilvestro. 1 (^osu.edu

Mark L. Failla, PhD. Professor and Senior Scientist, Department of Food Nutrition and Food Management University of North Carolina 310 Stone Building Greensboro, NC 27412-5001 910/334-5313 910/334-4129 fax e-mail: faillanK^iris.uncg.edu

237 Budget for 1 year project Name of contact PI: Steven J. Schwartz, Ph D.

Category OARDC funds Hirzel matching Salaries and wages: graduate student $1,500 $1,500 Fringe benefits 0.8% $80 $80 Subject reimbursement $600 $600 Materials and Supplies $2,000 $2,000 Sample collection charges $1,350 $1,350 Travel $200 $200 Publication costs $270 $270 Indirects (The OARDC, OSU Research Enhancement Grants Program does not pay indirect costs or administrative fees.) Total $6,000 $6,000

Approved:

Chairperson Date OARDC Research Advisory Committee

OARDC Director's Office Date

238 April 16, 1998

Dr. Vincent V. Hamparian Chair, Institutional Review Board Children’s Hospital 700 Children’s Drive Columbus, OH 43205

Dr. Hamparian,

Please accept this request for expedited review by the Human Subjects Research Committee. The protocol entitled “The effect of dietary intake on human breast milk and serum carotenoid levels’’ has been approved by The Ohio State University IRB and assigned protocol #97H046I. The subjects for the study will be recruited through PROBE sites affiliated with Children’s Hospital. The study involves a dietary intervention with breast feeding women and their infants. Samples of breast milk, blood serum, and buccal mucosal cells will be analyzed from the women and their infants. Forty-five women will be recruited during a year’s time. Subjects will be requested to participate for 6-8 weeks total. During that time 4 separate visits to selected PROBE sites will be required. Subjects will be compensated $50.00 for full completion of the study.

The study coordinator will be Ms. Charlotte Moxley. She can be reached at 614-292- 4069. The contact person at Children’s Hospital will be Dr. Daniel Coury.

Thank you for your assistance in helping coordinate this study.

Sincerely,

Steven J. Schwartz, Ph.D. Professor Principal Investigator

239 June 18,1998

Dr. Vincent V. Hamparian Chair, Institutional Review Board Children’s Hospital 700 Children’s Drive Columbus, OH 43205

Dr. Hamparian,

Please accept this request for amendment to protocol no. 98HSE019 which is entitled: The effect of dietarv intake on human breast milk and serum carotenoid levels.

This request is for an additional advertisement that will be posted in examination rooms at identified PROBE sites. The requested advertisement is attached.

Thank you for your assistance in helping coordinate this study.

Sincerely,

Steven J. Schwartz, Ph D. Professor Principal Investigator

240 August 12,1999

Dr. Vincent V. Hamparian Chair, Institutional Review Board Children’s Hospital 700 Children’s Drive Columbus, OH 43205

Dr. Hamparian,

Please accept this request for amendment to protocol no. 98HSE019 which is entitled: The effect of dietarv intake on human breast milk and serum carotenoid levels.

This request is for an additional advertisement that will run in the Columbus Dispatch. The requested advertisement is attached.

Thank you for your assistance in helping coordinate this study.

Sincerely,

Steven J. Schwartz, Ph D. Professor Principal Investigator

241 RESEARCH STUDY Nursing Mothers Wanted If you are a nursing mother between 4-12 weeks after delivery, you are eligible to participate in a research study looking at the effects of tomato product consumption on the content of breast milk. The study requires two separate visits to Children’s Hospital within one week for blood (^w s and milk sampling. Compensation is provided upon completion of the study. If you are interested, please call Charlotte Moxley at 292-4069.

242 BIOMEDICAL SCIENCES SUMMARY SHEETS ADDRESS EACH ITEM IN A COMPLETE AND CONCISE MANNER. (Do not leave any item blank with “See attached”) Use continuation pages when necessary.

1. Abstract (overview) The purpose of this study is to determine the effects of consuming processed or fresh food products on the carotene content of human breast milk. The study will also determine what, if any, correlation there is between blood carotene levels of the mother and the infant, with the carotene levels in breast milk. Specifically, women who are totally breast feeding will be asked to consume a given quantity of either fresh or processed food. Food will be provided. A baseline sample of breast milk will be expressed as well as a sample after 4 days. If consent is given for blood sampling, blood samples will be drawn from mother and infant at the same time as the two milk samples are expressed. Dietary intake questionnaires will be requested of all participants for the 4 days participation in the study. A food frequency questionnaire will also be required at the beginning of the study to determine habitual intake of carotene containing foods. Blood sampling will he done at the OSU Clinical Research Center, by qualified medical personnel.

2. Describe the requirements for a subject population and explain the rationale for using In this population special groups such as prisoners, children, the mentally disabled or groups whose ability to give voluntary Informed consent may be In question. Address means of pregnancy screening for females. The population of interest is healthy, totally breast feeding women and their infants. ‘Totally breast feeding’ means that the infant gets no food other than breast milk. Women will be asked to limit their intake of some carotene containing foods for up to two weeks prior to the start of the study. They will then be asked to consume a given amount of a carotene containing food that will be provided. Samples will be taken of mature milk only (at least two weeks after birth) and should be between 2-4 ounces. Samples should be expressed between 12-4 pm daily via breast pump and should consist of only hind milk (milk remaining in the breast after the infant has fed). These requirements will hopefully limit the high variability that is often experienced with breast milk. Subjects will be required to keep accurate dietary records during the study. Maternal blood samples as well as blood samples and/or buccal mucosal cell samples from the infants, will be drawn on a consensual basis to correlate carotene levels in the breast milk, maternal blood serum, and infant blood serum with dietary maternal carotene intake.

3. Describe and assess any potential risks- physical, psychological, social, legal, financial, or other - and assess the likelihood and seriousness of such risks. If methods of research create potential risks, describe other methods, if any, that were considered and why they will not be used. Potential risks from blood drawing by venipuncture (taking blood from a vein) Include: the possible occurrence of discomfort and/or bruising at the site of the puncture. Less commonly, a small blood clot, swelling of the vein, or bleeding may occur at the puncture site. No risks are anticipated from breast milk sampling or buccal mucosal cell sampling.

243 4. Describe consent procedures to be followed, including how and where informed consent will be obtained. (The use of a finder’s fee for recruiting subjects is not permitted) When an eligible woman has been Identified, she will be told the nature of the study. After expressed willingness is obtained, she will be requested to sign an 'Informed Consent’ form (see attached) for tests performed on herself and her Infant A photocopy will be supplied to the mother if desired.

5. Describe procedures (including confidentiality safeguards) for protecting against or minimizing potential risks and an assessment of their likely effectiveness. Potential risks arising from venipuncture will be minimized by having the blood drawn only by a certified phlebotomist or physician. All records of participants in this study will be maintained in a confidential fashion. With the exception of the General Information sheet, subjects will be identified on all records only by their subject number and initials. The General Information sheet will be kept in a locked file. Data will be analyzed by using the subject number rather than name. There will be no release or publication of this data that would reveal the identity of any subjects without permission. Subjects may withdraw themselves or their infants at any time without prejudice or loss of any benefits to which the subject is otherwise entitled. If at any time infants require more nutrition than can be provided by breast milk alone, subjects will be withdrawn from the study without prejudice or loss of any benefits to which the subject is otherwise entitled. Subjects will be provided with phone numbers for reaching the investigators during the day and after hours.

6. Assess the potential benefits to be gained by the individual subject, as well as benefits which may accrue to society in general as a result of the planned work. While participating in the study, the subjects will receive the benefit of a personal in-depth carotene profile. The overall benefit of the study is the possible improvement in the design of recommendations regarding nutrient intake while breast feeding.

7. Compare the risks versus the benefits. The value of the knowledge to be gained justifies the potential risks of discomfort and inconvenience involved in the study.

8. Will the subjects for the study be No _ x _ Y e s —How much? $50.00 paid for participating in the study?

Will subjects be paid for selected Activities (e.g., blood drawing) or Subjects will be paid for general participation for general participation in the study?

*NOTE: All information concerning payments, including the amount and schedule of payment, must be included in the consent form. Is there any other inducement? If so, please describe _x_N o Yes—please describe

9. Will advertising be used to recruit subjects? ___ No _ x _ Y e s ** **If yes, attach a copy of the proposed advertisement. SOURCE OF FUNDING FOR PROPOSED RESEARCH: (check A or B): A. OSURF: Sponsor ______RF Proposal/Project No. ______B. Other (Identiiy) Haas Endowed Chair Account No. 522254 REF# HAAS Information about the funding/sponsorship of human subjects research activities

244 THE OHIO STATE UNIVERSITY Protocol No. 07HO46I

CONSENT TO INVESTIGATIONAL TREATMENT OR PROCEDURE

I,______, hereby authorize or direct Steven J. Schwartz. Ph.D.. associates or assistants of his/her choosing, to perform the following treatment or procedure (describe in general terms): In this study, the subjects are supposed to be nonsmokers, be totally breast feeding (infant receives no other food), be no more than 6 weeks post delivery, and be in generally good health without chronic diseases. As part of the study, I agree to be asked to do the following things: -I will complete a food frequency questionnaire -I will keep an accurate dietary record during the course of the study. -I will consume the food provided for the course of the study (food will include tomato products, i.e. fresh tomatoes and spaghetti sauce) •I will visit the data collection site 4 times to express a sample of my breast milk and have a blood sample drawn from a vein about 20ml (4 teaspoons or 2/5 of an ounce) upon

(myself or name of subject) The experimental (research) portion of the treatment or procedure Is: In order to study the effect of consuming fresh versus processed food products on the carotene content of breast milk and blood, carotene levels of breast milk and blood will be measured at different stages of lactation in totally breast feeding women. The study will be of a crossover design where Group 1 subjects will consume fresh food first, and then four weeks later consume processed food. Group 2 subjects will consume processed food first, and then four weeks later consume fresh food. There is no experimental treatment involved. This is done as part of an investigation entitled: Human breast milk carotenoid levels.

1. Purpose of the procedure or treatment: The purpose of the study is to provide information about how carotene levels in lactating women change with different types of food consumed.

2. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): I may chooK not to participate or to withdraw at any time without loss of any beaedts to which I am otherwise entitled. If at any time my bahy requires more nutrition than can be offered with breast milk alone, I am not eligible for the study.

245 3. Discomforts There could be risks of venipuncture (taking blood from a vein) including discomfort and/or bruising at the site of the puncture. Less commonly, a small clot, swelling of the vein, or bleeding may occur at the puncture.

4. Benefits I will receive the benefit of a personal in-deptb carotene profile. The overall benefit of the study is the possible improvement in the design of recommendations regarding nutrient intake while breast feeding. I understand that I will be compensated 50.00 at the successful completion of the study, le. 4 different sampling days. If greater than half the study has been completed, ie. 2 different sampling days, the amount of payment can be prorated to 25.00. 5. Anticipated duration of subjects participation (including number of visits): I will be asked to come to the data collection site 4 times at specific intervals (total duration to complete participation would be approximately 6 weeks).

I hereby acknowledge that ______has provided information about the procedure described above, about my rights as a subject, and be/she answered ail questions to my satisfaction. I understand that I may contact him/her at phone #614-292-4069 should I have additional questions. He/she has explained the risks described above and I understand them; be/she has offered to explain ail possible risks or complications. I understand that, where appropriate, the U.S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during my participation in this study that may contain my name or other personal identifiers may be made available to the sponsor of this study. Beyond this, I understand that my participation will remain confidential. I understand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing future care. No guarantee has been given to me concerning this treatment or procedure. I understand in signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and I am not releasing the investigator, the sponsor, the institution, or its agents from any legal iiabiiity for damages that they might otherwise have. In the event of Injury resulting from participation in this study, I also understand that immediate medical treatment is available at University Hospitals of The Ohio State Univenity and that the costs of such treatment will be at my expense; financial compensation beyond that required by law is not available. Questions about this should be directed to the Office of Research Risks Protection at 292-5958.

1 have read and fully understand the consent form. I sign it freely and voiuntariiy. A copy has been given to me.

Date:______Time Signed ______(subject) Witness (es)______If (person authorized to consent for Required subject if required)

I certify that I have personaiiy completed ail blanks in this form and explained them to the subject or his/her representative before requesting the subject or his/her representative to sign it.

Date:______Signed: ______(Signature of project director or his/her authorized representative)

246 RESEARCH PROTOCOL: 97H0461

Requested Corrections -

1. Discuss the possibility of prorating payment if subject drops out before completion.

Prorating payment is possible if subjects have compieted greater than half the study. For half the study to be completed, the subject must have donated one set of samples, consumed the food provided, and then donated another set of samples. The amount of payment will be $25.00.

2. Address buccal mucosal cell sampling compared with blood serum sampling.

Human buccal mucosal cells are easily and routinely accessible and can be collected by a non-invasive method. Only a limited number of cells can be collected at a time, so a more sensitive method of detection must be used. Blood serum sampling has been the accepted standard for determination of carotenoid content for many years. This study involves following dietary intake from the mother to the mother’s blood, as well as following maternal intake to the infant through the consumption of breast milk. The lack of published data on how maternal intake of carotenoid containing food influences the content of infant blood serum made the sampling of blood serum required for acceptability of published data. Knowing the difficulties involved in sampling blood from infants, the alternative of buccal mucosal cell sampling from infants is preferred over not receiving consent for any sample from the infant.

Comparison values in adults from a published study are listed below: (Peng, Y-S and Y-M Peng. 1992. Cancer Epidemiology, Biomarkers & Prevention. 1:375- 382.)

Blood Buccal cells Lycopene 315 ng/ml 15 ng/1.6 x 10‘ cells P-Carotene 137 ng/ml 5 ng/1.6 x 10‘ cells

3. Revise the consent form: see attached.

247 THE OHIO STATE UNIVERSITY Protocol No.

CONSENT TO INVESTIGATIONAL TREATMENT OR PROCEDURE

I,______, hereby authorize or direct Steve Schwartz. Ph.D.. associates or assistants of his/her choosing, to perform the following treatment or procedure (describe in general terms):In this study, the subjects are supposed to be nonsmokers, be totally breast feeding (infant receives no other food), be no more than 6 weeks post delivery, and be in generally good health without chronic diseases.

As part of the study, 1 agree to be asked to do the following things; On the same day 1 visit the data collection site, 1 will bring my baby to have a blood sample drawn from a vein of no more than 1 teaspoon or 1/10 of an ounce. The blood will be drawn from my baby by a trained phlebotomist. 1 agree that 1 will not give my baby any foods other than my own breast milk during the study. Four separate visits to the data collection center will be required. They will coincide with my own visits.

upon______. (baby’s name) The experimental (research) portion of the treatment or procedure is: In order to study the effect of consuming fresh versus processed food products on the carotene content of breast milk and blood, carotene levels of breast milk and blood will be measured at different stages of lactation in totally breast feeding women. There is no experimental treatment involved. This is done as part of an investigation entitled: Human breast milk carotenoid levels.

1. Purpose of the procedure or treatment: The purpose of the study is to provide information about how changing carotene levels in lactating women effect blood serum levels in their breast fed infants.

2. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): I may choose not to participate or to withdraw my infant at any time without loss of any benefits to which I am otherwise entitled. If at any time my baby requires more nutrition than can be offered with breast milk alone, I am not eligible for the study.

3. Discomforts There could be risks of venipuncture (taking blood from a vein) including discomfort and/or bruising at the site of the puncture. Less commonly, a small clot, swelling of the vein, or bleeding may occur at the puncture.

248 4. Benefits My infant will receive the benefit of a personal in-depth carotene profile. The overall benefit of the study is the possible improvement in the design of recommendations regarding nutrient intake while breast feeding.

5. Anticipated duration of subjects participation (including number of visits): I will be asked to come to the data collection site 4 times at specific intervals (total duration to complete participation would be approximately S weeks). These visits will coincide with my own.

I hereby acknowledge that Steve Schwartz and/or Charlotte Moxley has provided information about the procedure described above, about my rights as a subject, and he/she answered all questions to my satisfaction. I understand that I may contact him/her at phone # 614-292-4069 should I have additional questions. He/she has explained the risla described almve and I undentand them; he/she has offered to explain all possible risks or complications.

I understand that, where appropriate, the U S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during my participation in this study that may contain my name or other personal identifiers may lie made available to the sponsor of this study. Beyond this, I understand that my participation will remain confidential.

I undentand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing future care. No guarantee has been given to me concerning this treatment or procedure.

I undentand in signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and I am not releasing the investigator, the sponsor, the institution, or its agents from any legal liability for damages that they might otherwise have.

In the event of injury resulting from participation in this study, I also understand that immediate medical treatment is available at University Hospitals of The Ohio State University and that the costs of such treatment will be at my expense; financial compensation beyond that required by law is not available. Questions about this should be directed to the Office of Research Risks Protection at 292-5958.

1 have read and fiilly understand the consent form. I sign it freely and voluntarily. A copy has been given tome.

Date:______Time______Signed ______(subject) Wimess (es)______If (person authorized to consent for Required subject if required)

I certify that I have personally completed all blanks in this form and explained them to the subject or his/her representative before requesting the subject or his/her representative to sign it.

Date:______Signed: ______(Signature of project director or his/her authorized representative)

249 THE OHIO STATE UNIVERSITY Protocol No.

CONSENT TO INVESTIGATIONAL TREATMENT OR PROCEDURE

I,______, hereby authorize or direct Steve Schwartz. Ph D., associates or assistants of his/her choosing, to perform the following treatment or procedure (describe in general terms):In this study, the subjects are supposed to be nonsmokers, be totally breast feeding (infant receives no other food), be no more than 6 weeks post delivery, and be in generally good health without chronic diseases. As part of the study, I agree to be asked to do the following things: On the same day I visit the data collection site, 1 will bring my baby to have a sample of his/her buccal mucosal cells taken. Buccal mucosal cells are those surface cells that are on the inside of the mouth. 1 understand that sampling involves swabbing the inside of my baby’s cheeks with a cotton tipped applicator and then collecting the saliva. 1 agree that I will not give my baby any foods other than my own breast milk during the study. Four separate visits to the data collection center will be required. They will coincide with my own visits.

upon______. (baby’s name) The experimental (research) portion of the treatment or procedure is: In order to study the effect of consuming fresh versus processed food products on the carotene content of breast milk and blood, carotene levels of breast milk and blood will be measured at different stages of lactation in totally breast feeding women. There is no experimental treatment involved. This is done as part of an investigation entitled: Human breast milk carotenoid levels.

1. Purpose of the procedure or treatment: The purpose of the study is to provide information about how changing carotene levels in lactating women effect buccal mucosal cell carotene content in their breast fed infants.

2. Possible appropriate alternative procedure or treatment (not to participate in the study is always an option): I may choose not to participate or to withdraw my infant at any time without loss of any benefits to which I am otherwise entitled. If at any time my baby requires more nutrition than can he offered with breast milk alone, I am not eliÿble for the study.

3. Discomforts There should be minimal discomfort to my baby during the swabbing of his/her cheeks.

250 4. Benefits My infant will receive the benefit of a personal in-depth carotene profile. The overall benefit of the study is the possible improvement in the design of recommendations regarding nutrient intake while breast feeding.

5. Anticipated duration of subjects participation (including number of visits): I will be asked to bring my infant to the data collection site 4 times at specific intervals (total duration to complete participation would be approximately 5 weeks). These visits will coincide with my own.

I hereby acknowledge that ______has provided information about the procedure described above, about my rights as a subject, and he/she answered all questions to my satisfaction. I understand that I may contact him/her at phone # 614-292-4069 should I have additional questions. He/she has explained the risks described above and I understand them; he/she has offered to explain all possible risks or complications.

I understand that, where appropriate, the U.S. Food and Drug Administration may inspect records pertaining to this study. I understand further that records obtained during my participation in this study that may contain my name or other personal identifiers may be made available to the sponsor of this study. Beyond this, 1 understand that my participation will remain confidential.

1 understand that I am free to withdraw my consent and participation in this project at any time after notifying the project director without prejudicing fhture care. No guarantee has been given to me concerning this treatment or procedure.

I understand in signing this form that, beyond giving consent, I am not waiving any legal rights that I might otherwise have, and 1 am not releasing the investigator, the sponsor, the institution, or its agents from any legal liability for damages that they might otherwise have.

In the event of injury resulting from participation in this study, I also understand that immediate medical treatment is available at University Hospitals of The Ohio State University and that the costs of such treatment will be at my expense; financial compensation beyond that required by law is not available. Questions about this should be directed to the Office of Research Risks Protection at 292-5958.

1 have read and fully understand the consent form. 1 sign it fieely and voluntarily. A copy has been given to me. Date:______Time______Signed ______(subject) Wimess (es)______(person authorized to consent for subject if required)

1 certify that 1 have personally completed all blanks in this form and explained them to the subject or his/her representative before requesting the subject or his/her representative to sign it

Date:______Signed:_ (Signature of project director or his/her authorized representative) 251