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Pain Perception in Humans Is Altered by the Presence of Foods in the Mouth

Permanent Link: http://ufdc.ufl.edu/UFE0021773/00001

Material Information

Title: Pain Perception in Humans Is Altered by the Presence of Foods in the Mouth
Physical Description: 1 online resource (56 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: pain, perception, tas2r38, taste, thermal
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The main reason that patients seek a doctor is pain. Pain is not only costly, but it can sometimes be difficult to control and interferes with daily activities. This research investigated the effect of six various tastes on thermal pain perception on sixty healthy adults (30 males, 30 females, age ranged from 18-63). When the taste was present in the mouth, the participants experienced a higher thermal pain threshold. The results from this research study contribute to a new theory of the inhibitory effects tastes may play. However, the mechanism behind this has yet to be discovered. The unknown mechanism might be related to endogenous opioids releases as our natural body painkillers. If various tastes do have inhibitory effects on pain, pain-killers might at least in part, be replaced with different food items. Individuals can be less dependent on over-the-counter drugs and rely on healthy 'pain killers.' This theory may also explain the reasons for our diet choices.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Sims, Charles A.
Local: Co-adviser: Sitren, Harry S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021773:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021773/00001

Material Information

Title: Pain Perception in Humans Is Altered by the Presence of Foods in the Mouth
Physical Description: 1 online resource (56 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: pain, perception, tas2r38, taste, thermal
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The main reason that patients seek a doctor is pain. Pain is not only costly, but it can sometimes be difficult to control and interferes with daily activities. This research investigated the effect of six various tastes on thermal pain perception on sixty healthy adults (30 males, 30 females, age ranged from 18-63). When the taste was present in the mouth, the participants experienced a higher thermal pain threshold. The results from this research study contribute to a new theory of the inhibitory effects tastes may play. However, the mechanism behind this has yet to be discovered. The unknown mechanism might be related to endogenous opioids releases as our natural body painkillers. If various tastes do have inhibitory effects on pain, pain-killers might at least in part, be replaced with different food items. Individuals can be less dependent on over-the-counter drugs and rely on healthy 'pain killers.' This theory may also explain the reasons for our diet choices.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Sims, Charles A.
Local: Co-adviser: Sitren, Harry S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021773:00001


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PAIN PERCEPTION IN HUMANS IS ALTERED BY THE PRESENCE OF FOODS INT THE
MOUTH



















By
WENDY SZE MING WAI




















A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA INT PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008

































O 2008 Wendy Sze Ming Wai









ACKNOWLEDGMENTS

I would like to thank Dr. Harry Sitren and Dr. Charles Sims for permitting my thesis

research proj ect to be conducted in the Department of Community Dentistry and Behavioral

Sciences. I would also like to thank Dr. Roger Fillingim for introducing me to Dr. Henrietta

Logan. I have learned a lot through this research experience, and I am really grateful for having

this great opportunity. I am really appreciative of Dr. Bartoshuk, Dr. Fillingim, Dr. Logan, Dr.

Sims, and Dr. Sitren for serving on my committee and for being my mentors. In addition, I

would like to thank Dr. Taimour Langaee for helping me with genotyping and haplotyping the

saliva samples.

I would like to especially thank Alesia Apana, Myrnelle Damas, and Issa Issa for

assisting me in conducting this proj ect. Not only were they my co-workers, they are also my

friends who were very supportive. The experiment would not have been possible without their

help, and this experience would not have been as enj oyable. I would also like to thank all my

friends and family for their support and helping me through my education. I am very thankful for

everyone who assisted me in this study.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............3.....


LIST OF TABLES ................ ...............6............ ....


LIST OF FIGURES .............. ...............7.....


AB S TRAC T ......_ ................. ............_........8


CHAPTER


1 INTRODUCTION ................. ...............10.......... ......


2 LITERATURE REVIEW ................. ...............12................


3 STUDY DESIGN AND METHODS .............. ...............18....


Subj ects ................. ...............18.................
Study Design............... ...............18.
Materials .............. ...... .... .............2

Psychological Questionnaires............... .............2
Dependent Scales............... ...............22.
Taste Stimuli ................. ...............23.................
Heat Stimuli ................. ...............23.................
Genotyping/ Haplotyping .............. ...............24....
Statistical Analyses ................. ...............24.................

4 RE SULT S .............. ...............26....


Subj ects .................. ...............26.................
Baseline Temperature ................. ...............27.......... .....
Taste Stimuli Trials............... ...............28.
Non-Stimuli Trial -Trial 16............... ...............30...
Delta Temperature .............. ...............3 0....
Intensity of Taste Stimuli............... ...............3. 1
Palatability of Taste Stimuli .............. ...............32....
T AS 2R3 8 H apl oty pes ................. ...............3.. 3......... ....
6-n-Propylthiouracil1 Te sting ................. ...............35........... ..

5 DI SCUS SSION ................. ...............36................


APPENDIX


A CHARACTERISTICS OF PARTICIPANTS (AGE, SEX, AND CLASSIFICATION
OF ETHNICITY)............... ...............4











B CHARACTERISTICS OF PARTICIPANTS (EMPLOYMENT STATUS, HIGHEST
EDUCATION LEVEL COMPLETED, AND MARITAL STATUS) ................. ................42

C CHARACTERISTICS OF PARTICIPANTS (HEALTH HISTORY/PROBLEMS, EAR
INFECTIONS, AND TONSILLECTOMY) .............. ...............43....

D CHARACTERISTICS OF PARTICIPANTS (SMOKING HABIT AND EATING
HABIT) ................. ...............44.......... ......

E PSYCHOLOGICAL VARIABLES................ ..............4

F DELTA TEMPERATURE OF TASTE STIMULI .............. ...............46....

G COMPARISONS OF THE TEMPERATURE OF TASTE STIMULI TRIALS ...................47

H INTENSITY AND PALATABILITY OF TASTE STIMULI ................ ........_ .........48

I PROP INTENSITY RATING .............. ...............49....

J CORRELATIONS OF PROP VERSUS INTENSITY AND PALATABILITY OF
TASTE STIMULI................ ...............50

LIST OF REFERENCES ................. ...............51................

BIOGRAPHICAL SKETCH .............. ...............56....










LIST OF TABLES


Table page

A-1 Characteristics of participants age, sex, and classification of ethnicity ................... .......41

B-1 Characteristics of participants employment status, highest education level
completed, and marital status............... ...............42.

C-1 Characteristics of participants health history/problems, ear infections, and
tonsillectomy ........._ ...... .___ ...............43....

D-1 Characteristics of participants smoking habit and eating habit ................. ................. 44

E-1 Means and standard deviation of psychological variables............_.._. .......................45

F-1 Means and standard deviation of delta temperature of taste stimuli ................. ...............46

G-1 Paired t-tests for comparisons of the temperature of taste stimuli trials ................... .........47

H-1. Means and standard deviation of intensity and palatability of taste stimuli ................... .......48

I-1 Means and standard deviation of PROP intensity rating ................. ........................49

J-1 Correlations of PROP versus intensity and palatability of taste stimuli ................... .........50










LIST OF FIGURES


Figure page

4-1 Temperature of Non-Taste Stimuli Trials. ............... ...............27...............

4-2 Comparison of baseline temperature between males and females at which
participants report moderate pain............... ...............28..

4-3 Comparison of averaged temperature of six taste stimuli trials between males and
females at which participants report moderate pain. ............. ...............29.....

4-4 Temperature of Taste Stimuli .. ............... ...............29........... .

4-5 Temperature Comparisons for Selected Sample of Subj ects ................ ............... .....30O

4-6 Delta Temperature of Taste Stimuli.. .......................... .......31

4-7 Intensity of Taste Stimuli.. ............. ...............32.....

4-8 Palatabilty of Taste Stimuli............... ...............32

4-9 TAS2R3 8 Haplotypes versus Intensity Ratings of Taste Stimulus. ................ ...............34

4-10 TAS2R3 8 Haplotypes versus Delta Temperature of Taste Stimulus ................ .. .............34

4-11 Comparison of PROP Intensity. ................. ...............35.._._._ ....









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PAINT PERCEPTION IN HUMANS IS ALTERED BY THE PRESENCE OF FOODS INT THE
MOUTH

By

Wendy Sze Ming Wai

May 2008

Chair: Charles Sims
Maj or: Food Science and Human Nutrition

Published studies suggest that taste influences pain perception. However, the mechanism

has not been established. Most research has been concentrated on sweet taste analgesia;

conversely, very little research has investigated bitter taste analgesia. The aim of this present

study was to verify the results from a previous pilot study, Exploring the Bidirectional Influence

of Taste and Pain (IRB# 166-2006). The effects of different tastes on pain were examined in

sixty healthy adults, split equally between males and females. The mean age was 23 years old,

ranging from 18-63 years old. A thermal stimulus in testing was used to determine whether taste

plays a role in pain inhibition. Each participant reported their moderate pain level with and

without the presence of taste stimuli. Repeated Measures Analysis of Variance (ANOVA)

showed that at baseline temperature, males and females reported perception of moderate pain at

similar temperatures. The temperature rated as moderate pain was significantly higher for all

taste stimuli compared to baseline temperature (all p-values <.001) with no significant difference

between sexes. However, there was a significant difference between sexes (p = 0.046) for the

average temperature for all six-taste stimuli trials. In addition, no significant difference was

found among the temperatures of the taste stimuli trials except for 1.) trial with sucrose yogurt










compared to trial with anise-flavored yogurt (t = -2. 1, p = .039) and 2.) trial with anise-flavored

yogurt compared to trial with caffeine yogurt (t = 2. 182, p = .033). Genetic variation in taste

might be a factor that affects the thermal responses between individuals. Individuals who possess

TAS2R3 8 homozygous AVI/AVI haplotypes (non-tasters) rated the taste stimuli as a lower

intensity; individuals who possess TAS2R3 8 homozygous PAV/PAV haplotypes (tasters) rated

the taste stimuli as a higher intensity. No significant difference was found between sexes in

intensity and palatability ratings. No significant correlation was observed between the TAS2R3 8

gene and the temperature at which the participants indicated moderate pain. Comparison between

sexes of PROP intensity rating was not significantly different (T = .85, p = .40). In conclusion,

regardless of the intensity or palatability of the taste stimuli, pain inhibition still resulted. Similar

to preliminary results, the data showed that pain response is altered in the presence of food, but

distraction and the olfactory organ may play a role in this effect.









CHAPTER 1
INTTRODUCTION

A recent pilot study, Exploring the Bidirectional Influence of Taste and Pain (IRB# 166-

2006) conducted by Dr. Henrietta Logan and colleagues, was presented at the American

Association for Dental Research (AADR) Conference in New Orleans, Louisiana. The study had

shown there is pain inhibition in the presence of taste. Twenty-four males and 26 females were

recruited on the University of Florida campus and paid for their participation. The mean age was

24 years and ranged from 18 to 37 years. Seventy-two percent (n=36) of the participants

considered themselves as white, 6% (n=3) black, and the remainder of the sample categorized

themselves as others. Questionnaires of medical history and likes and dislikes of food were given

to subjects before testing. A thermal machine called Medoc Thermal Sensory Analyzer (TSA-

2001, Ramat Yushai, Israel) was used to deliver brief heat stimuli. During the first part of the

thermal testing, a brief heat pulse was applied to non-repetitive sites on the subj ect' s left

forearm. The thermal stimulus reached its target temperature and remained at the target

temperature for 5 seconds. The subj ects were asked to rate the peak intensity of the temperature

by using a scale from 0 to 100, where 0 means no sensation and 100 means the strongest

experienced sensation of any kind. Various temperature degrees were used and given in random

order. After each temperature, the subj ects were asked to give an intensity rating on the

temperature they experienced. For the second part of the thermal testing, the thermal stimulus for

each trial was set at different specific temperature for each trial ranging from 35 degrees Celsius

to 52 degrees Celsius, and the food item was administered simultaneously with the thermal

stimulus. The food items, administered in random order, were plain non-flavored fat-free yogurt,

plain non-flavored fat-free yogurt + sugar (12.5 g/ 100 g of yogurt), plain non-flavored fat-free

yogurt + sugar (12.5 g/ 100 g of yogurt) + McCormick's strawberry extract (3 ml/ 100 g of










yogurt), plain non-flavored fat-free yogurt + sugar (12.5 g/ 100 g of yogurt) + McCormick' s

anise extract (1 ml), plain non-flavored fat-free yogurt + caffeine (0.15 g/ 100 g of yogurt), and

strawberry j am. The subj ects swished the food item around in their mouths for 5-6 seconds then

swallowed it. Simultaneously, the thermal stimulus was administered, and the subj ects rated the

temperature using the intensity scale and the food item using the intensity and palatability scales.

The results of this study were that both sweet and bitter had an effect in inhibiting pain, but the

bitter taste was more effective. To analyze the data, paired t-tests with Bonferonni corrections

was used. The data showed pain sensory ratings associated with all six food items were

significantly lower than pain ratings without food items. All t-values were greater than 4.4 and p-

values were less than 0.0001. The mean differences for ratings with and without food items

ranged from -6.7 for strawberry-flavored yogurt to -13.1 for caffeine yogurt. This suggests that

all flavors were effective in reducing pain. Repeated Measures Analysis of Variance (ANOVA)

and post hoc testing showed that strawberry jam, anise-flavored yogurt, and caffeine yogurt were

the more effective taste stimulus in inhibiting pain than were sucrose yogurt and strawberry-

flavored yogurt. Results support the hypothesis that there is pain inhibition in presence of taste.

However, it is not clear whether the observed phenomenon is distraction or the result of a

complex interaction between the cranial nerves V, VII and IX.

The aim of this thesis research was to verify the results from the prior pilot study. In

addition, PROP paper testing and genetic testing of taste sensitivity was also generated to further

investigate the outcome of the study. The following hypotheses were tested in this research:

1. At baseline, males will report moderate pain at a significantly higher temperature than
females.

2. The temperature rated as moderate pain will be significantly higher for all food stimuli
compared to baseline.









CHAPTER 2
LITERATURE REVIEW

A main reason that patients seek a doctor is pain. According to the International

Association for the Study of Pain (IASP), pain costs an estimated $100 billion a year in medical

costs, lost working days, and workers compensation. Pain is not only costly, but it can sometimes

be difficult to control and interferes with daily activities. Pain stimuli travel through the nerves

from three parts of the nervous system: the peripheral nerves, the spinal cord and the brain.

Although pain is universal, there are differences in an individual's perception, expression

and tolerance of pain. Many factors influence an individual's perception of pain such as age

(Riley et al. 1998), gender (Jensen et al. 1992), blood pressure (Bruehl et al. 1999) and menstrual

cycle (Riley et al. 1999, Fillingim and Ness 2000).

Among men and women, women are at greater risk for several chronic painful disorders,

including temporomandibular disorders (LeResche et al. 1999), interstitial cystitis (Jones and

Nyberg 1997), arthritis (Buckwalter and Lappin 2000), and trigeminal neuralgia (Unruh 1996).

Women are also at greater risk for autoimmune disorders that have a pain component, such as

rheumatoid arthritis, lupus, and scleroderma (Buckwalter and Lappin 2000). Men are at greater

risk for some pain disorders, including cluster headache (Dodick et al. 2000) and pancreatitis

(Lin et al. 2000).

There are many factors that may contribute to the differences in pain perception between

sexes. Developmental factors influence the structural and functional sex differences in nervous

system development (McEwen 2001). In addition, brain function and activation during pain can

differ between males and females (Paulson et al. 1998). Sex hormone receptors can also

influence nociceptive activity through genomic and nongenomic effects (Aloisi 2000). Sex

differences in immune responses (Da Silva 1999, Gregory et al. 2000), biological factors










(Fillingim et al. 2000) such as genetics (Mogil et al. 2000), and psychosocial factors (Fillingim et

al. 2000) such as anxiety (Edwards et al. 2000), abuse history (Spertus et al. 1999), coping

(Affleck et al. 1999, Keefe et al. 2000), gender roles (Robinson et al. 2001), and family history

(Fillingim et al. 2000) can also contribute to sex differences in pain sensitivity. Exogenous

hormones have also been related to clinical pain (LeResche et al. 1997, Wise et al. 2000,

Musgrave et al. 2001) and to experimental pain sensitivity (Fillingim and Edwards 2001).

Analgesic drugs help to relieve pain by acting on the peripheral system and the central

nervous system. The analgesia system is mediated by three maj or components: the

periaquaductal grey matter (in the midbrain), the nucleus raphe magnus (in the medulla), and the

nociception inhibitory neurons within the dorsal horns of the spinal cord, which act to inhibit

nociception transmitting neurons also located in the spinal dorsal horn.

Nonhuman animal models show sex differences in nociceptive responses, but the results

vary across pain assays. For example, female rats are more sensitive to electrical stimuli and to

chemical such as formalin (Mogil 2000), but greater opioid analgesia in males than females are

found in nonhuman animal models, primarily in rodents (Fillingim and Ness 2000, Kest et al.

2000). Among humans, women report lower pain thresholds and tolerances than men, and that

ratings of suprathreshold stimuli are often higher among women than men across a wide range of

painful stimuli (Fillingim and Maixner 1995, Riley et al. 1998). Research suggests that sex

differences among humans can be measured between mu- and kappa-opioid agonists. For

example, following oral surgery, females experienced more prolonged analgesia than males

using kappa- (or weak mu-) opioid analgesics (Miaskowski et al. 2000). Analgesia effects from

nonsteroidal anti-inflammatory drugs vary in response may be Sex-related such that females









demonstrate less effect than males (Walker and Carmody 1998), although findings of no sex

difference have also emerged (Averbuch and Katzper 2000).

Studies have provided evidence for the relationship between a sweet tasting solution such

as sucrose, fructose or glucose to the tongue and analgesia in rats (de Vasconcellos et al. 2006)

as well as human infants (Blass and Hoffmeyer 1991, Blass and Watt 1999, Ramenghi et al.

2002). The sweet-analgesia effect discovered by Blass appears to disappear around age 3 months

in human infants. However, recent studies appear to find evidence for effects at later ages (Miller

et al. 1994; Pepino and Mennella 2005). The analgesic effects of sweet tasting solutions may also

seem to have an effect on adults (Lewkowski et al. 2003, Mercer and Holder 1997); some might

disagree (Pepino and Mennella 2005).

Different palatable substances do not have equivalent effects on relieving pain; nutritive

solutions such as polycose or sucrose may be more effective than nonnutritive solutions such as

saccharin (D'Anci et al. 1997). Another example is lactose, which does not have equivalent

analgesic effects to sucrose, fructose or glucose (Blass and Shide 1994). This indicates analgesic

effects are not based solely on sweet tasting stimuli. However, there are not many studies that

have been done on the effect of other tastant stimuli such as quinine, which has been shown to be

effective in calming crying newborns (Graillon et al. 1997).

The mechanism of analgesia consequent to intraoral administration of sugars is still

unknown, but it is known that if sugar is administered through the nasogastric tube, the analgesic

reaction is absent. When newborns were given sugar through the intraoral route rather than the

nasogastric route both the crying time and behavior score were dramatically decreased

(Ramenghi et al. 1999). This study provides evidence that only intraoral administration of some

taste stimuli initiates analgesia.









Comfort foods might play a role in the analgesia effect (Wansink et al. 2003). Comfort

foods are foods that evoke a psychologically comfortable and pleasurable state for an individual.

Comfort foods are also preferred based on a physiological need. Some individuals receive

addictive qualities from certain food. This effect may due to the fact that the body releases trace

amounts of opiates when palatable foods are consumed (Wansink et al. 2003). As a result, both

mood and satisfaction are elevated. Although only a small amount of opiates are released, this

might reinforce a preference for foods that are associated with these feelings.

Tastes can be confused with odor, such as the perception of sweet and sour (Stevenson et

al. 1999). Some research studies have shown odors are able to determine tastants such as sucrose.

When such odors are presented with a sweet-taste solution, the solution is given a higher

sweetness rating from participants (Prescott et al. 2004). Odors help an individual interpret

flavors of different tastes (Prescott et al. 2004). When concentrations of peach (Cliff and Noble

1990) and strawberry (Schifferstein and Verlegh 1996) are increased, sweetness is enhanced.

There are thousands of taste buds on the tongue's surface. In the mouth, food stimulates taste

receptors in the taste buds. The food molecules stimulate specialized sites on the microvilli

extending from cells in the taste buds. The cells trigger nerve impulses in nerve fibers

innervating the taste buds. Impulses travel along the taste cranial nerves to the brain, which

interprets the impulses as a distinct taste. Taste buds detect sweetness, saltiness, sourness, and

bitterness. Taste is mediated by three cranial nerves: CN VII, IX, and X. CN VII, the facial nerve

innervates taste buds in the fungiform papillae on the anterior two-thirds of the tongue. CN IX,

glossopharyngeal nerve, innervates taste buds in the circumvallate papillae at the one-third rear

of the tongue. The foliate papillae (rear edges of the tongue) are dually innervated by CN VII and

IX. Unfortunately, the role of CN X, the vagus nerve, in the taste is not fully understood. Each









taste bud is surrounded by a basket-like group of nerve fibers that mediate pain sensation. These

fibers are part of CN V, the trigeminal nerve.

CN VII normally inhibits CN IX. When CN VII is damaged or anesthetized, taste from IX

is intensified (Yanagisawa et al. 1998). Similarly, CN VII normally inhibits CN V. When CN

VII is damaged or anesthetized, pain from V is intensified (Tie et al. 1999). Specifically,

anesthesia significantly alters the perceived bum of 10 ppm capsaicin applied to tongue; the burn

was intensified on the contralateral side and reduced on the ipsilateral side. There is speculation

that taste has the general function of inhibiting many activities that are incompatible with eating

(Bartoshuk et al. 2005). Thus, studies showing inhibition of pain by sweet tasting substances (as

well as some other taste stimuli) are compatible with studies that suggest taste inhibition of pain

at multiple body sites.

TAS2R3 8 gene is associated with taste ability. A region of chromosome 7 in the TAS2R3 8

gene has shown an association with the ability to taste bitter (Kim et al. 2003). There is a

physiological link between genetic variation and bitter tasting, which might affects diet choices

and related health (Duffy et al. 2004, Basson et al. 2005). With regard to haplotypes of

TAS2R3 8, those who are homozygous for the recessive allele (AVI/AVI) are nontasters, those

who are heterozygous (AVI/PAV) or homozygous (PAV/PAV) for the dominant allele are tasters

(Kim et al. 2003). The perceived bitterness of PROP divides tasters into two subgroups:

supertasters (those who perceive the most bitterness) and medium tasters (those who perceive

less bitterness). There is a strong relationship between TAS2R3 8 haplotype variation and bitter

tasting compounds such as 6-n-propylthiouracil (PROP), which also determines whether an

individual is a taster or non-taster. Non-tasters do not perceive the PROP solution as bitter as

tasters do. The tasting variation associates with oral sensation and tobacco or alcohol behaviors.









Generally, individuals who tasted the least bitterness from PROP or who were homozygous

AVI/AVI are more likely to consume more alcohol than those who tasted the most bitterness

from PROP or who were homozygous PAV/PAV (Duffy et al. 2004). Individuals who tasted

the least bitterness from PROP consumed alcoholic beverages five to six times more per week on

average compared to those who tasted the most bitterness from PROP averaged consuming

alcoholic beverages two to three times per week (Duffy et al. 2004).









CHAPTER 3
STUDY DESIGN AND IVETHODS

Subj ects

Candidates were screened by telephone to assess eligibility. Participants were included if

they did not have a history of treatment for depression, schizophrenia, bipolar or personality

disorder, if they were not currently receiving treatment for hypertension, or if they did not have

any history of food allergies or lactose intolerance.

Participants were recruited from advertisements posted on University of Florida campus

and around Gainesville Shands hospital. The risks and possible side effects were explained in

detail to each subj ect, and testing only proceeded after a signed consent form has been obtained.

The subj ects were free to withdraw from the experiment at any time without consequences. All

testing took place in a research laboratory room with the subj ect seated in an adjustable dental

chair in an upright position. The testing was completed in a single session, which lasted

approximately an hour and a half and was conducted by three experimenters. At the end of the

session, all subj ects were compensated with a $3 5 Publix supermarket gift card for participating.

The Institutional Review Board at the University of Florida approved this study (IRB# 53 8-

2006). They also approved the advertisements, questionnaires, and data collection sheets.

Study Design

Each participant was scheduled for a one testing session, which lasted up to 90 minutes at a

research lab located on the 2nd flOOr of the dental tower of Shands hospital. After the participants

consented to participate in the study, they were asked to rinse their mouths with water. A

minimum of 5 minutes elapsed before saliva was collected. During the waiting period after

rinsing, participants completed a questionnaire packet containing the following standardized

measures: Health and Well-being, McGill Pain questionnaire, Pain Catastrophizing Scale (PCS),









State-Trait Anxiety Inventory (STAI), Life Orientation Test-Revised (LOT-R), and Perceived

Stress Scale (PSC). When the questionnaires were completed, saliva was collected with Oragene

DNA Self Collection Kits (DNA Genotek Inc. 2006) and the FRID questionnaire was given to

participants to complete. Before testing began, intensity and likes/dislikes scales were explained

to the participants and two magnitude matching forms were given to the participants to

familiarize them with the two scales.

Each participant was given a series of contact heat stimuli using a computer controlled

Medoc Pathway Pain and Sensory Evaluating System (TSA-2001, Ramat Yushai, Israel). All

trials were conducted on participant's left forearm, and the thermode was placed an inch above

the wrist and an inch below the elbow. There were a total of 16 trials. Half way through the

study, an additional trial, Trial 16, was added at the end after the taste stimuli trials to establish

that baseline had not changed. Trials 1-9 and 16 were the non-stimuli trials, and trials 10-15 were

with the taste stimuli. During the trials, the temperature thermode increased at 0.5 oC/second

until the participant stopped the thermode by pressing a button. When the thermode was stopped,

it indicated that the participant had reached his/her moderate pain threshold. The question "Was

that moderate pain?" was asked to ensure the temperature was stopped at the moderate pain

level. Between each thermal trial, there was a 30 seconds rest period to minimize the effects of

prolonged heat on the skin surface. After the non-stimuli trials, the trials with stimuli were

conducted in the same manner. The taste stimuli were administered in randomized order between

participants to avoid possible order effects of the stimuli. When the thermode was at its baseline

of 35oC, the taste stimulus was given to the participant. The participant was asked to move the

taste stimulus around his/her mouth for 5 to 6 seconds prior to swallowing. When the participant

had reached his/her moderate pain threshold, the question "Was that moderate pain?" was asked.










The participant also had to rate the intensity and the palatability of the taste stimulus. After the

ratings were given, the participant rinsed his/her mouth with water to remove any residual taste.

The same procedure was repeated with the remaining fiye taste stimuli. At the end of the taste

stimuli trials, a non-stimuli trial was conducted. After the thermal testing, the participant was

asked to place a PROP fi1ter paper on top of the tongue and to keep the fi1ter paper moistened

with saliva for 15 seconds. At the end of the 15 seconds, the participant gave a rating of the

intensity of the taste. An extra copy of the informed consent form and the Publix gift card were

given to the participant at the end of the session.

Materials

Psychological Questionnaires

The Health and Well-being questionnaire consists of brief questions about health (health

problems (Fan et al. 2002), smoking habit and eating habit) and demographics (age, ethnicity,

race, education level, employment status and marital status).

The Pain Catastrophizing Scale (PCS) consists of 13 items related to thoughts and feelings

toward a painful situation. Participants were asked to indicate the degree to which each item

reflects their thoughts and feelings on a 4-point scale when they are experiencing pain. PCS is

divided into three subscales of catastrophizing based on content: rumination (items 8, 9, 10, and

11), magnifieation (items 6, 7, and 13), and helplessness (items 1, 2, 3, 4, 5, and 12) (Sullivan

and Bishop 1995). Scoring for each item is 0 4 (0 = Not at all, 1 = to a slight degree, 2 = to a

moderate degree, 3 = to a great degree, 4 = all the time). The scoring for each sub scale is the sum

of the items.

The State-Trait Anxiety Inventory (STAI) is a 40 items questionnaire relating to anxiety

level (Spielberger et al. 1970). The first 20 items convey present anxiety level (state), and the last

20 items compute general anxiety level (trait). Scoring for each item is 1 to 4 (1 = Not at all, 2 =









somewhat, 3= Moderately so, 4= Very much so). The scores for the positive feeling items 1, 2, 5,

8, 10, 11, 15, 16, 19, and 20 are reversed (4 = Not at all, 3 = somewhat, etc). After the scores are

reversed, the sum of the state of anxiety can be calculated. Scores ranging from 20 thru 65 are

considered 'low state anxiety', and scores from 66 and up are defined as 'high state anxiety'

(Tenenbaum et al. 1985). To calculate trait anxiety level, scores of items 1, 6, 7, 10, 13, 16, and

19 have to be reversed. After the scores are reversed, the sum of the trait anxiety can be

calculated. Scores ranging from 20 thru 56 are considered 'low trait anxiety', and scores from 57

and up are defined as 'high trait anxiety'(Tenenbaum et al. 1985).

The Life Orientation Test Revised (LOT-R) consists of 6 items relating to optimism and

pessimism. Participants were asked to indicate the degree to which each item reflects their

general feelings and expectations in life. This questionnaire has been revised to differentiate

optimism from neuroticism (Carver et al. 1989). Items 1, 3, and 6 measure the degree of

optimism. Items 2, 4, and 5 measure the degree of pessimism. Scoring for each item is 0 4 (0 =

I disagree a lot, 1 = I disagree a little, 2 = I neither agree or disagree, 3 = I agree a little, 4 = I

agree a lot). The scoring for the pessimistic statements will be reversed (4 = I disagree a lot, 3 = I

disagree a little, etc.). To obtain the score of LOT-R, the sum of the items is calculated.

The McGill Pain questionnaire (MPQ) consists of 20 groups of single words describing

pain. The words in each group increases in intensity order.

The Perceived Stress Scale (PSS) consists of 14 items relating to thoughts and feelings of

different situations. It is suggested for "examining the role of nonspecific appraised stress in the

etiology of disease and behavioral disorders and as an outcome measure of experienced levels of

stress" (Cohen et al. 1983). Participants were asked to indicate the degree to which each item

reflects their thoughts and feelings in the last month. Scoring for each item is 0 to 4 (0=Never, 1=









Almost never, 2=Sometimes, 3=Fairly often, and 4=Very often). The scoring of positive items

(4, 5, 6, 7, 9, 10, and 13) scoring will be reversed (0= Very often, 1=Fairly often, etc.). To obtain

the score of PSS, the sum of the items will be calculated.

The FRID questionnaire consists of 10 items relating to thoughts and feelings toward the

experimental pain procedure. Participants were asked to indicate the degree to which each item

reflects their current thoughts and feelings. FRID is divided into three subscales of

catastrophizing based on content: high Psychological Involvement (items 3, 7, and 9), high

Negative Expectancies (items 1, 4, and 8), and high Efficacy and Control beliefs (items 2, 5, and

10). Scoring for each item is 1 to 5 (1 = Not at all and 5= Very much so). The scoring for each

subscale is the sum of the items.

The Magnitude Matching forms consist of 14 items relating to the intensity level of every

day life experiences (e.g. Brightness of the light in this room, loudness of a whisper, sweetness

of a coke, etc). Before each item was rated, the participants were asked to specify their strongest

sensation that they have ever experienced in their lifetime. The strongest sensation was their 100

mark on the 0 100 intensity scale. Participants were asked to rate the intensity level of the 14

items consistently based on their own individual 0 100 intensity scale. The second part of the

magnitude matching forms consists of 11 everyday food items. Before the participants

proceeded, they were asked to think of disliking and liking of any sort that would be their -100

mark and their +100 mark. Then the participants were asked to rate their degree of liking/

disliking of the 11 items consistently based on their own individual -100 +100 intensity scale.

Dependent Scales

After each thermal trial with taste stimulus, participants were instructed to verbally rate the

intensity and the palatability of the taste stimuli on a 0-100 numerical scales. Two scales were

used in the study: Intensity scale and Likes/ Dislikes Scale. The intensity scale is a scale of how










intense something is. It ranges from 0 to 100, O being no sensation at all and 100 being the

strongest experienced intensity of any kind. The likes/ dislikes scale is a scale of palatability. The

scale ranges from -100 to + 100. -100 is the strongest disliking of any kind; 0 is neutral; + 100 is

the strongest liking of any kind.

Taste Stimuli

All food products were available in retail stores with the exception of the caffeine.

Caffeine was obtained from the Department of Food Science and Human Nutrition, where all the

food stimuli were prepared to assure consistency. Prepared yogurt stimuli were discarded after

48 hours to avoid food spoiling and contamination. There were six different taste stimuli: plain

non-flavored fat-free yogurt, plain non-flavored fat-free yogurt + sucrose (12.5 g/100 g of

yogurt), plain non-flavored fat-free yogurt + sucrose (12.5 g/100 g of yogurt) + McCormick' s

strawberry extract (3 ml/ 100 g of yogurt), plain non-flavored fat-free yogurt + sucrose (12.5

g/100 g of yogurt) + McCormick' s anise extract (1 ml), plain non-flavored fat-free yogurt +

caffeine (0.15 g/ 100 g of yogurt), and Welch's strawberry preserve. The order of the six taste

stimuli was randomized. Randomization was determined by the Flash Action Script program.

After each taste stimulus trial, tap water was used to rinse participants' mouths to remove any

residual tastes.

Heat Stimuli

Thermal test was performed with a Medoc Thermal Sensory Analyzer (TSA-2001, Ramat

Yishai, Israel). A 3 x 3 cm square thermode was placed an inch above the wrist and an inch

below the elbow due to mid-forearm appears more sensitive to responses (Meyer et al. 1992).

The thermode is set at 3 5oC as a baseline. The device delivers a brief thermal stimulus to the skin

using an increasing scale. The probe temperature increases at 0.5 oC/second. A button on a

handheld device held by the subj ect is used to signal the device to stop. When activated, the









device decreases at 8 oC/second. The thermode does not exceed 52 oC in this study to prevent

any injury to the skin surface.

Genotyping/ Haplotyping

Oragene DNA self-collection kits (DNA Genotek Inc. 2006) were used to collect DNA

from saliva. The bitter taste receptor gene, TAS2R3 8, was investigated to determine the

relationship of the participant' s taste status and various tastes playing a role in pain inhibition.

With regard to haplotypes of TAS2R3 8, those who are homozygous for the recessive allele

(AVI/AVI) are nontasters, those who are heterozygous (AVI/PAV) or homozygous (PAV/PAV)

for the dominant allele are tasters (Kim et al. 2003). The perceived bitterness of PROP divides

tasters into two subgroups: supertasters (those who perceive the most bitterness) and medium

tasters (those who perceive less bitterness). Whatman #1 filter papers were saturated in a 6-n-

propylthiouracil (PROP) solution, which is a medicine for hyperthyroidism, was used to measure

the individual's taste status (Duffy et al. 2004). Individuals who are non-tasters give a lower

intensity rating for the PROP paper and vice versa (Duffy et al. 2004). PROP paper contained

about 1.6 mg of PROP. The maintenance dosage for this medication varies from 50 200

mg/day (Solomon et al. 1986). Thus, the dosage, which is very minimal in this study, is not

expected to elicit any clinical effect.

Statistical Analyses

Data were analyzed using SPSS (Windows version 15.0). Only differences of p < .05 are

reported as statistically significant. Repeated Measures Analysis of Variance was used to

compare the temperature of each trial with and without the taste stimuli. One-Way ANOVA was

conducted with the factor being haplotypes and the dependent variable being likes/dislikes rating

of taste stimuli, PROP intensity. Independent t-tests were also conducted to compare two










variables of interest, and correlations tests were performed to determine the relationship between

variables.









CHAPTER 4
RESULTS

Subj ects

Sixty normal, healthy individuals were recruited. There were 30 males (mean age = 22.4,

median age = 22.0, SD = 4.1i, age ranged from 18 37; 1 participant refused to give his age) and

30 females (mean age = 22.4, median age = 21.0, SD = 8.0, age ranged from 18 63) (Table A-

1). Within the 30 males, there were 80.0% non-Hispanic white, 3.3% non-Hispanic black, 3.3%

Hispanic white, 3.3% Hispanic black, 6.7% Asian, and 3.3% mixed (Table A-1). Within the 30

females, 60% were non-Hispanic white, 20% were non-Hispanic black, 13.3% were Hispanic

white and 6.7% were Asian (Table A-1).

Twelve of the thirty male participants and ten out of the thirty females participants were

currently employed (Table B-1). Most of the participants were single and completed some

college level courses. The maj ority of the participants had no history of health problems and

never reported cancer, chronic pain, or cardiac disease. Only nine participants had a history of

asthma or bronchitis, seven participants had a history of pneumonia, one had a history of

diabetes, and one had a history of head trauma (Table C-1).

Most of the participants were non-smokers (Table D-1). According to the United States

Department of Agriculture Food Guide Pyramid, each individual has his/ her own food pyramid

guide to follow. However, 2-4 servings of 4 ounces of meat, 2-4 servings of 2-4 cup of fruits and

3-5 servings of 1 cup of vegetables are recommended. Compared to the USDA Food Guide

Pyramid, the participants' average consumption of protein, fruits, and vegetables are well below

the recommended amount (Table D-1). Based on the psychological questionnaires, all

participants scored within the normal limits. They were emotionally stable, not excessively

optimistic, pessimistic, or too anxious and nervous (Table E-1). For all the temperature trials,










one male participant was eliminated from data analysis. The participant had reached the safety

level, 52 OC, of the Pathway thermode machine without reporting moderate pain.


Temperature of Non-Taste Stimuli Trials Temperature of Non-Taste Stimuli Trials













Tnal Tnal Tnal Trial Tnal Tnal Trial Trial Tnal
123456789
Tnal 1 Tnal 2 Tnal 3 Tnal 4 Tnal 5 Tnal 6 Tnal 7 Tnal 8 Tnal 9
A B

Figure 4-1.Temperature of Non-Taste Stimuli Trials. A) Means and standard errors of
temperature during the non-stimuli trials for all participants' response at which they
report moderate pain. Asterisks indicate values that are significantly (P < 0.05)
compared to Trial 9. B) Means and standard errors of temperature during the non-
stimuli trials at which participants report moderate pain, separately for both males and
females.

Baseline Temperature

All the non-taste stimuli trials were compared to trial 9, which was the last non-taste

stimuli trial before the six taste-stimuli trials. It is assumed that the participants have familiarized

with the thermode and have recognized their moderate pain level by trial 9. When each non-taste

stimulus trial was compared to trial 9, it can be concluded that there were no significant

differences among trials 6, 7, 8, and 9 (Figure 4-1A). However, the p-value for trial 6 is 0.063,

which is marginally significant. Unlike trial 6, trial 7 has a p-value of .223 and trial 8 has a p-

value of .155. As a result, the average of the temperature at which the participants reported

moderate pain during trials 7, 8 and 9 were used to determine the baseline temperature. There

was also no significant difference between males and females when the non-taste stimuli trials








were compared (p = 0.063) (Figure 4-1B). When the baseline temperature is determined with the

average of Trial 7, 8, and 9 for the males and females, the averages between sexes are compared.

As shown in Figure 4-2, the average for the males is 44.7 and for females is 43.3. Males have a

higher average of the baseline temperature than females, but it was not significant (p = .11i).

Otnrparison Ehive\enS~eest
Eheel ine Ten~perdtue(Trials7,849)

465
^45.5




I' 41.5
40.5I
MieFemdae


Figure 4-2. Comparison of baseline temperature between males and females at which
participants report moderate pain.
Taste Stimuli Trials

When the temperature at which the participants reported moderate pain during the six taste

stimuli trials were averaged and compared between sexes, the average temperature for males was

45.7 degrees Celsius and 44.1 degrees Celsius for females. The difference between males and

females was significant (p = .046) (Figure 3). When taste stimuli trials were sorted by taste

stimuli, the temperatures at which the participants reported moderate pain were compared. The

temperatures were significantly different from baseline temperature (Figure 4-4A). The p-values

of all the six taste stimuli compared to baseline are below .005. However, similar to the non-taste

stimuli trials, there was no significant difference between the sexes (Figure 4-4B) (p = .051).

When the temperatures of the six taste stimuli trials were compared among each other, there was





Temperature of Taste Stimuli


IL

e~ai~~c~04)i~~q~ 10~ ~da~ ~ ~a"
,"" ,~:ab pCP~ C~\~b~y2~0~~0~.~~
,j"`


no significant difference among each other except for 1.) trial with sucrose yogurt compared to

trial with anise-flavored yogurt (t = -2.1i, p = .039) and 2.) trial with anise-flavored yogurt


compared to trial with caffeine yogurt (t = 2. 182, p = .033) (Table G-1).


Comparison Between Sexes at
Average Temperature of Taste
Stimuli Trials (10-15)


46.5
46
S45.5
45
S44.5
S44
43.5
43
42.5
42


Female


Figure 4-3. Comparison of averaged temperature of six taste stimuli trials between males and
females at which participants report moderate pain.


Temperature of Taste Stimuli


46s

4

4i


-4--Male
---m--- Female


B~C~ ~lO~,aU~ ~C~,6U~ ,d~C~ P
~~w~~~~
8


Figure 4-4. Temperature of Taste Stimuli. A) Means and standard errors of temperature during
the stimuli trials at which participants report moderate pain. Asterisks indicate values
that are significantly (p < 0.05) compared to baseline temperature. The number 1
shows the significant difference between the sucrose trial and anise trial. The number
2 shows the significant difference between the anise trial and caffeine trial. B) Means
and standard errors of temperature during the stimuli trials at which participants
report moderate pain, separately for both males and female.


,f~ii-t.










Non-Stimuli Trial -Trial 16


Temperature Comparisons for Selected Sample of
Subjects
















Figure 4-5. Temperature Comparisons for Selected Sample of Subj ects. Means and standard
errors of temperature during the stimuli trials at which participants report moderate
pain. Asterisks indicate values that are significantly (P < 0.05) compared to baseline
temperature.

Trial 16 was added half way through the study. The results of Trial 16 were obtained from

20 females and 9 males. When Trial 16 was compared to the baseline temperature, the

temperature at which participants reported moderate pain during Trial 16 was not significantly

different from baseline temperature (F = .281, p = .60) (Figure 4-5). There was no significant

difference between sexes.

Delta Temperature

Delta temperature is the difference of the temperature at which the participants reported

moderate pain during the taste stimulus trial and the baseline temperature (average of Trials 7, 8,

9). A positive delta temperature represents a pain inhibition, and a negative temperature

represents the taste stimuli caused more pain, therefore, no pain inhibition. The delta

temperatures for all taste stimuli were positive (Figure 4-6A). This indicates all taste stimuli

resulted in pain response modulation; temperatures at which the participants reported moderate










pain during tastestimuli trials were at least 0.5 degrees Celsius higher than baseline temperature

(Table F-1). There were no significant difference between males and females (Figure 4-6B).

Della Tempenitaire of Tilste Stimuli Trials Deita Temperature of Taste Stimuli







iiYogwt


A : B~ ,.
Figur 4-.DlaTmeaur fTseSiui.A en n tndr rosoet


B)Rogr SIMean Sand taPndardg ~ierroirs fdlatmeauedrn h at tmlrasa



amog reac 46 tast stiuiecpfo1.temperature of taste stimuli A en d stadria withr scos e your

comare totemperature ofig h taste stimuli trial wt an wise flavred ipt yogu rt (t -.1 p .39 aind



2mn ahtsesiuiecp o .) temperature of taste stimuli trial with anis-flaore youtcmprdt tmeaure ftat



stimuli trial with caffeine yogurt (t = 2. 182, p = .033) (Table G-1).

Intensity of Taste Stimuli

From the six taste stimuli that were used in testing, both males and females rated caffeine

yogurt as the taste stimulus with the highest intensity; the overall mean was 54.32 (Figure 4-7A).

Both sexes rated strawberry jam as the second most intense; the overall mean was 50. 15 (Figure

4-7B). Anise-flavored yogurt was rated the third most intense; the overall mean was 40.22

(Figure 4-7A). There was no significant difference between sexes (p = .539) (Figure 4-7B).











Intensity of Taste Stinlull


Intensity orTaste Stimuli


65 -1 60

mMale
(n= 29)



PlsScoeSrwer kie CfeeSrwer
Your Your xtac wlrt Ygut
A B'
Fiue4-7 Inenit ofTseSiui ) en n tnad roso nestyo attml
at0- whc patciat repoted B) Men an stnaderosonesiyoat
stml at whic patciat reotd seprael fo mae an emls


Palatability of Taste Stimuli


Palatability of Taste Stimuli


H Male (n=29)
I Female (n=30)


Z


Plain Sucrose Strawberry Anise Caffeine Strawbery Jam
AYogurt Yogurt Yogudt Yogudt Yogut Yogud B


Figure 4-8.Palatabilty of Taste Stimuli. A) Means and standard errors of palatability of taste
stimuli. B) Means and standard errors of intensity of taste stimuli, separately for
males and females.

Palatability of Taste Stimuli

Between the six taste stimuli, caffeine yogurt was rated as the least palatable taste

stimuli; the overall mean was -50.37 (Table H-1). Strawberry-flavored yogurt was rated as the

most palatable; the overall mean was 39.92 (Table H-1). Both females and males rated caffeine









yogurt as the least palatable taste stimulus. The mean unpalatability rating given by males for

caffeine yogurt was -46.55, and the females' mean was -54.07 (Figure 4-8A). Males rated

strawberry jam as the most palatable; the mean was 36.41 (Figure 4-8B). Females rated

strawberry-flavored yogurt as the most palatable; the mean was 39.33 (Figure 4-8B). There was

no significant sex difference (p = .541).

TAS2R38 Haplotypes

Of the 60 participants, genotypes of seven DNA samples were not obtained due to

experimental errors. Four categories of TAS2R3 8, AVI/AVI, AVI/PAV, PAV/PAV, and

miscellaneous, can be defined in this study. There were thirty-two percent (n=17) of the

participants' TAS2R38 gene were homozygous AVI/AVI, 34% (n=18) heterozygous AVI/PAV,

23% (n=12) homozygous PAV/PAV, 3.7% (n=2) heterozygous AAV/PAV, 3.7% (n=2)

AVI/AAV, 1.8% (n=1) AVI/AAI, and 1.8% (n=1) AAI/PAV.

The intensity ratings of PROP were compared among the TAS2R3 8 haplotypes. There was

a significant association between the TAS2R3 8 haplotypes and PROP intensity rating. However,

there was no significant association between the TAS2R3 8 haplotypes and sex, ethnicity,

palatability and intensity ratings of taste. From the TAS2R3 8 haplotypes, homozygous AVI/AVI

individuals showed lower intensity ratings on both the caffeine yogurt and the strawberry jam,

and homozygous PAV/PAV individuals showed higher intensity ratings on both taste stimuli

(Figures 4-9A and B). The differences in the intensity ratings of the caffeine yogurt among the

TAS2R3 8 haplotypes were not significant (p = 0.30) (Figure 4-9A). Similar to caffeine, the

differences in the intensity ratings of the strawberry j am among the TAS2R3 8 haplotypes were

not significant (p = 0.057) (Figure 4-9B). The delta temperatures during the trial with caffeine

yogurt were not significantly different among the TAS2R3 8 haplotypes (p = 0.91) (Figure 4-














0.36) (Figure 4-10B).


TAS2R38 Haplotypes vs. Intensity of Strawberry Jam


Figure 4-9. TAS2R3 8 Haplotypes versus Intensity Ratings of Taste Stimulus A) The mean and
standard error of the intensity of caffeine yogurt among TAS2R3 8 haplotypes. B) The
mean and standard error of the intensity of strawberry j am among the different
TAS2R3 8 haplotypes stimuli, baseline temperature and delta temperature.


TAS2R38 Haplotypes vs. Delta Temperature of TAS2R38 Haplotypes vs. Delta Temperature of
Caffeine Yogurt Strawberry Jam




1.21 1.2 -





PAVIAVI AAIA I. PAIV AAVIA-

A B
Figue 410. AS238 aploype vesus elt Teperaureof asteStiulu. A)Themea
an stnaderro hedlatmeaue fcfen out mn h A23
haltps B)-1 Th mea an stadar ero of th det epeaueosrwer


jmaogteTAS2R38 htpoye JIPVTSR8raplotypes.AIIA


10A). Similarly, during the taste stimuli trial with strawberry jam, there were no significant


difference among the TAS2R3 8 haplotypes (p


TAS2R38 Haplotypes vs. Intensity of Caffeine Yogurt


AVl/AVI AVl/PAV, PAVIPAV AAVIPAV,
PAV/AVI AAV/AVI,
TAS2R38 Haplotypes AA


AVl/AVI AVl/PAV, PAVIPAV AAVIPAV,
PAV/AVI AAV/AVI,
TAS2R38 Haplotypes A










6-n-Propylthiouracil Testing

The female participants reported a higher mean rating of PROP compared to males (Table

iI-), but the difference was not significant (t = -.852, p = .398) (Figure 4-11A). Among the

TAS2R3 8 haplotypes, individuals who were homozygous AVI/AVI had the lowest rating on

PROP paper, heterozygous AVI/PAV rated as intermediate, and homozygous PAV/PAV rated it

the most intense. There were significant associations between the PROP ratings and the

TAS2R3 8 haplotypes (Figure 4-1 1B). However, there was also no significant correlation

between the intensity ratings of the taste stimuli and the PROP ratings except with the intensity

of caffeine yogurt (p = 0.33) (Table J-1). In addition, among males, there were significant

correlation between the intensity of PROP and intensity of anise-flavored yogurt and caffeine

yogurt (Table J-1).


Comparison Between Sexes at PROP TAS2R38 Hlplorypes us. PROP Intensity
Intensity Ratings

45 I. 0






O5 PAVAV AAVIAVI

A B
Fiur 4-1 oprsno RO nest.A opaio fItniyRaig ewe ee
~Measo nest ftsesiuia hc atcpnsrpresprtl o ae




genotypes.loype









CHAPTER 5
DISCUSSION

The purpose of this study was to determine the thermal pain responses with and without

the consumption of taste stimuli on healthy individuals and to conclude whether genetic variation

in taste plays a role. The first hypothesis that was proposed in this study was rej ected because

males did not report at a significantly higher mean pain threshold than females. However, the

second hypothesis failed to be rej ected because the temperatures at which the participants

reported moderate pain during the taste stimuli were significantly different when compared to the

baseline temperature. This study revealed an additional finding. There was a significant

association between the TAS2R3 8 genetic variations and PROP paper ratings.

During the non-taste stimuli, males reported moderate pain at a higher temperature than

females, although the difference is not significant. Previous studies have shown that there is a

sex difference in the perception of pain (Jensen et al. 1992, Fillingim and Maxiner 1995, Riley et

al. 1998). The average of the temperature at which participants reported moderate pain during

trials 1 through 5 were significantly different from trial 9. Trial 6 was marginally significantly

different; therefore, it wasn't used as part of the baseline temperature. Only the average

temperatures of trials 7, 8, and 9 were used to determine baseline temperature. It is assumed that

the participants recognize what moderate pain is after practice and result in an increase of

temperature throughout the trials. This might have resulted because participants were afraid the

temperature of the thermal would reach beyond their moderate pain.

Similar to the non-taste stimuli trials, males also reported in a higher pain threshold than

females during the taste stimuli trials, but the difference was not significant. This indicates that

taste stimuli have an effect on thermal responses. All taste stimuli resulted in an increase in

temperature at which was reported moderate pain. In other words, various tastes have pain









inhibition effects. Similar to Blass et al. 1991, sweet resulted in pain inhibition. Some have

suggested that palatability might play a role. However, our data show that palatability of the taste

stimuli was not linked to pain modulation. Plain non-flavored fat-free yogurt, anise-flavored

yogurt, and caffeine yogurt, which participants rated in the negatives on the palatability scale,

also have an effect on pain inhibition (Table E-1). When the temperature of the taste stimuli

trials were compared to each other, the only significant differences were found for 1.)

temperature of taste stimuli trial with sucrose yogurt compared to temperature of taste stimuli

trial with anise-flavored yogurt (t = -2.1i, p = .039) and 2.) temperature of taste stimuli trial with

anise-flavored yogurt compared to temperature of taste stimuli trial with caffeine yogurt (t =

2. 182, p = .033). These results further show that palatability of taste stimuli do not play a role in

pain inhibition. Studies on physiological responses toward comfort food have shown when

palatable foods are consumed, the body releases trace amounts of opiates (Wansink et al. 2003),

which elevate both mood and satisfaction. The result in this study shows that pain inhibition is

not only caused by palatable tastes, but also unpalatable tastes which suggests the taste stimuli

were not perceived as a comfort food by the participants. This can be concluded because the food

rated as unpalatable also had a pain inhibition effect. The exact reason why and how the tastes

have inhibitory effects is still unknown, but the unknown mechanism could be triggered from

endogenous opioids. There are three common endogenous opioids that inhibit pain: leu-

enkephalin, beta-endorphins, and dynorphins (Akil et al. 1984). Enkephalin is found throughout

the endocrine system, and is produced by the adrenal medulla and in the gastrointestinal tract

(Akil et al. 1984). When the taste stimulus is in the mouth, it might have triggered the production

of endogenous opioids. As a result, the participants experienced pain inhibition during the taste

stimuli trials.










Apparently, pain inhibition is not based on palatability, but the mechanism that is

responsible remains unknown. The participant' s pain tolerance for the thermal stimulus increased

about one degree Celsius (Table F-1). In Lilleso et al. 2000, the heat pain threshold was

increased by one degree Celsius with the usage of morphine. This suggests the taste stimuli used

in this study can represent one dosage of morphine.

An additional trial, Trial 16, was added at the end of the 15 thermal trials. The purpose of

the additional trial was to determine whether the temperature at which participants report

moderate pain during a non-taste stimuli trial after taste stimuli trials is similar to baseline

temperature, i.e. that trial 16 confirms the baseline temperature and the pain inhibitory effect of

the taste stimuli. Trial 16 was added to be part of the study design halfway throughout the study;

therefore, results were only gathered from 29 participants. Twenty of the 29 participants were

males and nine were females. Due to the small sample size for trial 16, the temperature at which

the participants reported moderate pain resulted in a huge variability. When the temperature of

trial 16 was compared to baseline temperature, it is not significantly different. This shows the

order of the trials did not play a role in pain inhibition. Pain modulation occurs only during the

taste-stimuli trials.

Genetics might play a role in the pain inhibition effects of the tastes stimuli. Genetic

variation in taste was gathered from collecting saliva to measure the bitter receptor gene,

TAS2R3 8. Homozygous AVI/AVI haplotypes are non-tasters, heterozygous AVI/PAV

haplotypes are intermediate tasters, and homozygous PAV/PAV haplotypes are tasters. Bitter

taste was measured by using 6-n-propylthiouracil (PROP). Depending on the intensity rating of

PROP, it distinguishes non-tasters and medium tasters (Fisher et al. 1939). Individuals who rate

PROP a higher intensity are tasters, and individuals who rate it a lower intensity are non-tasters.









The ratings of PROP and the TAS2R3 8 haplotypes have a significant correlation; however, the

correlation between the delta temperature and the TAS2R3 8 haploptypes is not significant.

From saliva collection, the TAS2R3 8 bitter taste receptor gene was analyzed. There was no

significant correlation between the TAS2R3 8 haplotypes and palatability of the taste stimuli,

intensity of the taste stimuli, baseline temperature, or delta temperature. There is no significant

correlation between PROP intensity ratings and the intensity ratings of the six taste stimuli

except with caffeine yogurt (p = 0.33) (Table J-1). However, among males, there were significant

correlation between the intensity of PROP and intensity of anise-flavored yogurt and caffeine

yogurt (Table J-1). In addition, there is no strong correlation between PROP intensity ratings and

the palatability of the six taste stimuli (Table J-1).

These results contribute to a new theory of the inhibitory effects the intensity of the taste

stimuli play. There is an unidentified mechanism in our body system for the pain modulation

during the taste stimuli trials. The unknown mechanism might be related to endogenous opioid

release. If various tastes do have inhibitory effects on pain, pain-killers might at least in part, be

replaced with different food items. Individuals can be less dependent on over-the-counter drugs

and rely on healthier 'pain killers'. This might also suggest the reason individuals have

preferences in their daily diet.









CHAPTER 6
SUMMARY AND CONCLUSIONS

Researchers have reported that food tastes may have inhibitory effects on perception of

pain. The cause of this effect remains unknown, but it may partly be due to an endogenous

inhibitory mechanism in our body. The main focus of this study was to determine the effect of

various tastes on the response to a pain stimulus, the thermal.

This study showed that all taste stimuli tested had a pain inhibitory effect on when the

participants reported moderate pain from the thermal stimulus. Males did not report moderate

pain significantly higher than females. Similar to other studies, sweet taste was shown to have a

pain inhibitory effect. In addition, other tastes such as caffeine and anise also have an inhibitory

effect compared to baseline. This demonstrates that unpalatable tastes also possess pain

modulation properties.

TAS2R3 8 haplotypes distinguish the individuals who are non-tasters and tasters. However,

no significant relationship was found between the genotypes and the level of pain modulation.

This result is maintained regardless of palatability of the food items, i.e. pain modulation still

remains. The effect that is observed in this study might be caused by a more complicated

phenomenon than an individual's taste ability. There may be a more complex, unknown

mechanism. Further studies are needed to determine whether distraction or the olfactory organ

play a role in to trigger this unknown mechanism that is causing pain inhibition.









APPENDIX A
CHARACTERISTICS OF PARTICIPANTS (AGE, SEX, AND CLASSIFICATION OF
ETHNICITY)

Table A-1. Characteristics of participants age, sex, and classification of ethnicity
Male (n = 30) Female (n = 30)
Age (years)
Mean + SD 22.4 + 4.1 22.4 + 8.0
Median 22.0 21.0
Range 18-37 18-63
Ethnicity
Non- Hispanic White 24 18
Non- Hispanic Black 1 6
Hispanic White 1 4
Hispanic Black 1 0
Asian 2 2
Mixed 1 0
* Males (n=29) for age









APPENDIX B
CHARACTERISTICS OF PARTICIPANTS (EMPLOYMENT STATUS, HIGHEST
EDUCATION LEVEL COMPLETED, AND MARITAL STATUS)

Table B-1. Characteristics of participants employment status, highest education level
completed, and marital status
Male (n = 30) Female (n = 30)
Currently Employed
Yes 12 10
140 18 20
Education Level
Some College 25 25
College Graduate 4 2
Postgraduate/Professional 1 3
Marital Status
Single 28 28
Married 2 2









APPENDIX C
CHARACTERISTICS OF PARTICIPANTS (HEALTH HISTORY/PROBLEMS, EAR
INTFECTIONS, AND TONSILLECTOMY)

Table C-1. Characteristics of participants health history/problems, ear infections, and
tonsillectomy
Male (n = 30) Female (n = 30)*
Health Problems
Cancer 0 0
Chronic lung disease, Emphysema 0 0
Asthma, or bronchitis 6 3
Congestive heart failure 0 0
Diabetes 0 1
Heart attack 0 0
Pneumonia 4 3
Stroke 0 0
Head Trauma or Injury 0 1
Ear Infection
No 17 17
Yes, but not serious 8 5
Yes, required antibiotics more than once 2 5
Yes, required tubes in ears 3 2
Tonsillectomy
Yes 4 5
No 26 24










APPENDIX D
CHARACTERISTICS OF PARTICIPANTS SMOKINGG HABIT AND EATINTG HABIT)

Table D-1. Characteristics of participants smoking habit and eating habit
Male (n = 30) Female (n = 30)


Smoking Habit
Never Smoked
Past Smoker
Current Smoker
Eating Habit (average/day)
4 oz serving of protein
Mean + SD
Median
1/2 cup serving of vegetables
Mean + SD
Median
1/2 cup serving of fruits
Mean + SD
Median


2.9 + 1.48
3.0


2.2 + 1.48
2.0


2.1 + 1.50 2.5 + .937
2.0 2.0


2.2 + 1.69
2.0


2.5 + 1.41
2.0










APPENDIX E
PSYCHOLOGICAL VARIABLES

Table E-1. Means and standard deviation of psychological variables
Male (n=30) Female (n=30)


Questionnaires
Pain Catastrophizing Scale
Helplessness
Rumination
Magnification
State Anxiety*
Trait
LOTR
PSS
FRID
Ne ative Ex ectancies
Efficacy Control
Ps cholo 'cal Involvement
* Females (n=28) for state anxiety


2.87 + 2.70
4.27 + 2.95
2.00 + 1.53
28.93 + 5.61
32.43 + 6.07
16.70 + 5.61
16.60 + 5.76

4.50 + 1.22
13.07 + 1.82
10.87 + 2.19


4.00 + 3.26
4.67 + 2.68
2.27 + 2.03
29.25 + 6.58
32.97 + 4.77
18.97 + 3.70
20.03 + 6.69

5.07 + 1.62
12.09 + 1.80
10.63 + 2.06









APPENDIX F
DELTA TEMPERATURE OF TASTE STIMULI

Table F-1. Means and standard deviation of delta temperature of taste stimuli
Taste Stimuli a Temperature Male (n=29) Female (n=30)
Plain Yo urt .88 + 1.42 1.04 + 1.34 .71 + 1.49
Sucrose Yo urt .77 + 1.49 .66 + 1.57 .89 + 1.42
Strawberry Yo urt .87 + 1.31 .88 + 1.14 .85 + 1.47
Anise Y gurt 1.15 + 1.35 1.04 + 1.13 1.25 + 1.54
Caffeine Y gurt .79 + 1.51 .94 + 1.22 .64 + 1.76
Strawberry Jam 1.01 + 1.13 1.15 + .965 .86 + 1.28










APPENDIX G
COMPARISONS OF THE TEMPERATURE OF TASTE STIMULI TRIALS

Table G-1. Paired t-tests for comparisons of the temperature of taste stimuli trials
Taste Stimuli t Significance
Plain vs. Sucrose .556 .580
Plain vs. Strawberry .000 1.00
Plain vs. Anise -1.65 .105
Plain vs. Caffeine .726 .471
Plain vs. Strawberry Jam -.636 .527
Sucrose vs. Strawberry -.580 .564
Sucrose vs. Anise* -2.11 .039
Sucrose vs. Caffeine .197 .845
Sucrose vs. Strawberry Jam -1.16 .250
Strawberry vs. Anise -1.79 .078
Strawberry vs. Caffeine .732 .467
Strawberry vs. Strawberry Jam -.723 .472
Anise vs. Caffeine* 2.18 .033
Anise vs. Strawberry Jam .952 .345
Caffeine vs. Strawberry Jam -1.45 .154
* Difference is significant at the 0.05 level (2-tailed).










APPENDIX H
AND PALATABILITY OF TASTE STIMULI


INTENSITY


Table H-1. Means and standard deviation of intensity and palatability of taste stimuli


Male (n=29)

38.62 + 18.89
28.28 + 15.86
34.48 + 16.82
38.48 + 17.83
51.90 + 21.06
48.90 + 22.20

- 23.14 + 28.74
23.97 + 23.84
40.52 + 22. 13
-10.28 + 34.92
-46.55 + 20.53
42.93 + 23.32


Female (n=30)

39.27 + 22.39
31.30 +18.88
32.00 + 19.50
41.93 + 21.08
56.67 + 24.99
51.37 + 20.96

-25.33 + 34.66
30.00 + 31.18
39.33 + 22.58
-16.67 + 40.09
-54.07 + 30.41
30.10 + 40.27


Intensity
Plain Yogurt
Sucrose Yogurt
Strawberry Yogurt
Anise Yogurt
Caffeine Yogurt
Strawberry Jam
Palatability
Plain Yogurt
Sucrose Yogurt
Strawber Yo urt
Anise Yo urt
Caffeine Yogurt
Strawberry Jam









APPENDIX I
PROP INTENSITY RATINTG

Table I-1. Means and standard deviation of PROP intensity rating
Male (n=30) Female (n=30)
PROP Intensity 27.67 + 28.27 33.83 + 27.81









APPENDIX J
CORRELATIONS OF PROP VERSUS INTENSITY AND PALATABILITY OF TASTE
STIMULI

Table J-1. Correlations of PROP versus intensity and palatability of taste stimuli
PROP (n=59) Male (n=29) Female (n=30)
Intensity
Plain Yogurt .075 .072 .077
Sucrose Yogurt .189 .260 .117
Strawberry Yogurt .171 .320 .060
Anise Yogurt .135 .387 -.092
Caffeine Yogurt .330 .514 ** .164
Strawberry Jam .207 .289 .110
Palatability
Plain Yogurt .124 .135 .126
Sucrose Yogurt .202 .327 .094
Strawberry Yogurt .175 .264 .096
Anise Yogurt -.076 -.202 .048
Caffeine Yogurt -.186 -.283 -.107
Strawberry Jam .129 -.096 .306
**Correlation is significant at the 0.01 level (2-tailed).
*Correlation is significant at the 0.05 level (2-tailed).










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BIOGRAPHICAL SKETCH

Wendy Sze Ming was born in Hong Kong, China. At the age of eight, she moved to the

United States with her parents and older brother. In 2002, she graduated from St. Augustine High

School in St. Augustine, Florida, and began her undergraduate education at the University of

Florida. In 2006, she earned her bachelor' s degree in Nutritional Sciences and entered the

nutritional sciences master' s thesis program at the University of Florida. She did her thesis

research experiment at the Department of Community Dentistry and Behavioral Sciences under

the supervisor of Dr. Henrietta Logan. Wendy will be graduating with her master' s degree in

Spring 2008, and she will be attending Nova Southeastern University for the optometry program,

the Class of 2012.





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1 PAIN PERCEPTION IN HUMANS IS ALTERED BY THE PRESENCE OF FOODS IN THE MOUTH By WENDY SZE MING WAI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Wendy Sze Ming Wai

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3 ACKNOWLEDGMENTS I would lik e to thank Dr. Harry Sitren and Dr. Charles Sims for permitting my thesis research project to be conducte d in the Department of Commun ity Dentistry and Behavioral Sciences. I would also like to thank Dr. Roger Fillingim for in troducing me to Dr. Henrietta Logan. I have learned a lot through this research experience, and I am really grateful for having this great opportunity. I am rea lly appreciative of Dr. Bartosh uk, Dr. Fillingim, Dr. Logan, Dr. Sims, and Dr. Sitren for serving on my committee and for being my mentors. In addition, I would like to thank Dr. Taimour Langaee for he lping me with genotyping and haplotyping the saliva samples. I would like to especi ally thank Alesia Apana, Myrnelle Damas, and Issa Issa for assisting me in conducting this project. Not only were they my co-workers, they are also my friends who were very supportive. The experiment would not have been possible without their help, and this experience would not have been as enjoyable. I would also like to thank all my friends and family for their support and helping me through my education. I am very thankful for everyone who assisted me in this study.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES.........................................................................................................................7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INTRODUCTION..................................................................................................................10 2 LITERATURE REVIEW.......................................................................................................12 3 STUDY DESIGN AND METHODS..................................................................................... 18 Subjects...................................................................................................................................18 Study Design................................................................................................................... ........18 Materials.................................................................................................................................20 Psychological Questionnaires.......................................................................................... 20 Dependent Scales............................................................................................................. 22 Taste Stimuli....................................................................................................................23 Heat Stimuli................................................................................................................... ..23 Genotyping/ Haplotyping.......................................................................................................24 Statistical Analyses........................................................................................................... ......24 4 RESULTS...............................................................................................................................26 Subjects...................................................................................................................................26 Baseline Temperature........................................................................................................... ..27 Taste Stimuli Trials........................................................................................................... ......28 Non-Stimuli Trial Trial 16.................................................................................................... 30 Delta Temperature.............................................................................................................. ....30 Intensity of Taste Stimuli..................................................................................................... ...31 Palatability of Taste Stimuli.................................................................................................. .32 TAS2R38 Haplotypes............................................................................................................. 33 6-n-Propylthiouracil Testing...................................................................................................35 5 DISCUSSION.........................................................................................................................36 APPENDIX A CHARACTERISTICS OF PARTICIPANTS (AGE, SEX, AND CL ASSIFICATION OF ETHNICITY)....................................................................................................................41

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5 B CHARACTERISTICS OF PARTICIPANTS (EMPLOYMENT STATUS, HIGHEST EDUCATION LEVEL COMPLETED, AND M ARITAL STATUS)................................... 42 C CHARACTERISTICS OF PARTICIPANTS (HEALTH HISTORY/PROBLEMS, EAR INFECTIONS, AND TONSILLECTOMY) .......................................................................... 43 D CHARACTERISTICS OF PARTICIP ANTS (S MOKING HABIT AND EATING HABIT)...................................................................................................................................44 E PSYCHOLOGICAL VARIABLES........................................................................................ 45 F DELTA TEMPERATURE OF TASTE STIMULI................................................................ 46 G COMPARISONS OF THE TEMPERATURE OF TASTE STIMULI TRIALS ................... 47 H INTENSITY AND PALATABILITY OF TASTE STIMULI...............................................48 I PROP INTENSITY RATING................................................................................................49 J CORRELATIONS OF PROP VERSUS IN TENSITY AND PALATABI LITY OF TASTE STIMULI...................................................................................................................50 LIST OF REFERENCES...............................................................................................................51 BIOGRAPHICAL SKETCH.........................................................................................................56

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6 LIST OF TABLES Table page A-1 Characteristics of participants ag e, sex, and classifi cation of ethnicity .......................... 41B-1 Characteristics of participants em ployment status, highe st education level completed, and marital status............................................................................................. 42C-1 Characteristics of participants hea lth history/problems, ear infections, and tonsillectomy......................................................................................................................43D-1 Characteristics of participants smoking habit and eating habit....................................... 44E-1 Means and standard deviati on of psychological variables.................................................45F-1 Means and standard deviation of delta temperature of taste stimuli.................................. 46G-1 Paired t-tests for comparisons of the temperature of ta ste stimuli trials............................ 47H-1. Means and standard deviation of intensity and palatability of taste stimuli.......................... 48I-1 Means and standard deviation of PROP intensity rating...................................................49J-1 Correlations of PROP versus intensity and palatability of taste stimuli............................ 50

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7 LIST OF FIGURES Figure page 4-1 Temperature of Non-Taste Stimuli Trials.............................................................. 274-2 Comparison of baseline temperature between males and females at which participants report moderate pain....................................................................................... 284-3 Comparison of averaged temperature of six taste stimuli trials between males and females at which participants report moderate pain.......................................................... 294-4 Temperature of Taste Stimuli............................................................................................ 294-5 Temperature Comparisons for Se lected Sample of Subjects..................................... 304-6 Delta Temperature of Taste Stimuli................................................................................... 314-7 Intensity of Taste Stimuli................................................................................................. ..324-8 Palatabilty of Taste Stimuli............................................................................................... .324-9 TAS2R38 Haplotypes ve rsus Intensity Ratings of Taste Stimulus................................... 344-10 TAS2R38 Haplotypes versus Delta Temperature of Taste Stimulus................................. 344-11 Comparison of PROP Intensity.......................................................................................... 35

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PAIN PERCEPTION IN HUMANS IS ALTERED BY THE PRESENCE OF FOODS IN THE MOUTH By Wendy Sze Ming Wai May 2008 Chair: Charles Sims Major: Food Science and Human Nutrition Published studies suggest that taste influences pain perception. However, the mechanism has not been established. Most research has been concentrat ed on sweet taste analgesia; conversely, very little research ha s investigated bitter taste anal gesia. The aim of this present study was to verify the results from a previous pilot study, Exploring the Bidirectional Influence of Taste and Pain (IRB# 166-2006). The effects of different tastes on pain were examined in sixty healthy adults, split equally between males and females. The mean age was 23 years old, ranging from 18-63 years old. A thermal stimulus in testing was used to determine whether taste plays a role in pain inhibition. Each participant repor ted their moderate pa in level with and without the presence of tast e stimuli. Repeated Measures Analysis of Variance (ANOVA) showed that at baseline temperature, males and females reported perception of moderate pain at similar temperatures. The temperature rated as moderate pain was significantly higher for all taste stimuli compared to baseline temperature (all p-values <.001) with no significant difference between sexes. However, there was a significant difference between sexes ( p = 0.046) for the average temperature for all sixtaste stimuli trials. In additi on, no significant difference was found among the temperatures of the taste stimu li trials except for 1.) trial with sucrose yogurt

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9 compared to trial with an ise-flavored yogurt (t = -2.1, p = .039) and 2.) trial with anise-flavored yogurt compared to trial with caffeine yogurt (t = 2.182, p = .033). Genetic variation in taste might be a factor that affects the thermal responses between indi viduals. Individuals who possess TAS2R38 homozygous AVI/AVI haplotypes (non-tas ters) rated the taste stimuli as a lower intensity; individuals who possess TAS2R38 homozygous PAV/ PAV haplotypes (tasters) rated the taste stimuli as a higher intensity. No significant difference was found between sexes in intensity and palatability ratings. No signifi cant correlation was observe d between the TAS2R38 gene and the temperature at which the participan ts indicated moderate pain. Comparison between sexes of PROP intensity rating was not significantly different (T = .85, p = .40). In conclusion, regardless of the intensity or palatability of the ta ste stimuli, pain inhibition still resulted. Similar to preliminary results, th e data showed that pain response is altered in the pres ence of food, but distraction and the olfactory organ may play a role in this effect.

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10 CHAPTER 1 INTRODUCTION A recent pilot study, Exploring the Bidirecti onal Influence of Taste and Pain (IRB# 1662006) conducted by Dr. Henrietta Logan and co lleagues, was presented at the American Association for Dental Research (AADR) Confer ence in New Orleans, Louisiana. The study had shown there is pain inhibition in the presence of taste. Twenty-four ma les and 26 females were recruited on the University of Florida campus and paid for their participation. The mean age was 24 years and ranged from 18 to 37 years. Sevent y-two percent (n=36) of the participants considered themselves as white, 6% (n=3) black and the remainder of the sample categorized themselves as others. Questionnai res of medical history and likes and dislikes of food were given to subjects before testing. A thermal mach ine called Medoc Thermal Sensory Analyzer (TSA2001, Ramat Yushai, Israel) was used to deliver brie f heat stimuli. During the first part of the thermal testing, a brief heat pulse was applied to non-repetitive sites on the subjects left forearm. The thermal stimulus reached its targ et temperature and remained at the target temperature for 5 seconds. The subj ects were asked to rate the p eak intensity of the temperature by using a scale from 0 to 100, where 0 mean s no sensation and 100 means the strongest experienced sensation of any kind. Various temper ature degrees were used and given in random order. After each temperature, the subjects we re asked to give an intensity rating on the temperature they experienced. For the second part of the thermal testing, the thermal stimulus for each trial was set at different specific temperat ure for each trial ranging from 35 degrees Celsius to 52 degrees Celsius, and the food item was administered simultaneously with the thermal stimulus. The food items, administered in random order, were plain non-flavored fat-free yogurt, plain non-flavored fat-free yogurt + sugar (12.5 g/ 100 g of yogurt) plain non-flavored fat-free yogurt + sugar (12.5 g/ 100 g of yogurt) + McCorm icks strawberry extract (3 ml/ 100 g of

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11 yogurt), plain non-flavored fat-free yogurt + s ugar (12.5 g/ 100 g of yogurt) + McCormicks anise extract (1 ml), plain non-flavored fat-fr ee yogurt + caffeine (0.15 g/ 100 g of yogurt), and strawberry jam. The subjects swished the food item around in their mouths for 5-6 seconds then swallowed it. Simultaneously, the thermal stimulus was administered, and the subjects rated the temperature using the intensity scale and the food item using the intensity and palatability scales. The results of this study were that both sweet and bitter had an effect in inhibiting pain, but the bitter taste was more effective. To analyze the data, paired t-tests with Bonferonni corrections was used. The data showed pain sensory ratin gs associated with all six food items were significantly lower than pain ra tings without food items. All t-values were greater than 4.4 and pvalues were less than 0.0001. The mean differenc es for ratings with and without food items ranged from -6.7 for strawberry-flavored yogurt to -13.1 for caffeine yogurt. This suggests that all flavors were effective in reducing pain. Repeated Measures Analysis of Variance (ANOVA) and post hoc testing showed that strawberry ja m, anise-flavored yogurt, and caffeine yogurt were the more effective taste stimulus in inhibiting pain than were sucrose yogurt and strawberryflavored yogurt. Results support th e hypothesis that there is pain in hibition in presence of taste. However, it is not clear whethe r the observed phenomenon is dist raction or the result of a complex interaction between the cranial nerves V, VII and IX. The aim of this thesis res earch was to verify the results from the prior pilot study. In addition, PROP paper testing and ge netic testing of taste sensitivity was also generated to further investigate the outcome of the study. The follo wing hypotheses were tested in this research: 1. At baseline, males will report moderate pain at a significantly higher temperature than females. 2. The temperature rated as moderate pain will be significantly higher for all food stimuli compared to baseline.

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12 CHAPTER 2 LITERATURE REVIEW A m ain reason that patients seek a doctor is pain. According to the International Association for the Study of Pain (IASP), pain costs an estimate d $100 billion a year in medical costs, lost working days, and workers compensati on. Pain is not only costly, but it can sometimes be difficult to control and interf eres with daily activities. Pain stimuli travel through the nerves from three parts of the nervous system: the peri pheral nerves, the spinal cord and the brain. Although pain is universal, there are differences in an individuals perception, expression and tolerance of pain. Many factors influence an individuals perception of pain such as age (Riley et al. 1998), gender (Jense n et al. 1992), blood pressure (Bru ehl et al. 1999) and menstrual cycle (Riley et al. 1999, F illingim and Ness 2000). Among men and women, women are at greater ri sk for several chronic painful disorders, including temporomandibular diso rders (LeResche et al. 1999), interstitial cystitis (Jones and Nyberg 1997), arthritis (Buckw alter and Lappin 2000), and trig eminal neuralgia (Unruh 1996). Women are also at greater risk for autoimmune disorders that have a pain component, such as rheumatoid arthritis, lupus, and scleroderma (Buckwalter and Lappin 2000). Men are at greater risk for some pain disorders, including cluster headache (Dodick et al. 2000) and pancreatitis (Lin et al. 2000). There are many factors that may contribute to the differences in pain perception between sexes. Developmental factors influence the struct ural and functional sex differences in nervous system development (McEwen 2001). In addition, brain function and activation during pain can differ between males and females (Paulson et al. 1998). Sex hormone receptors can also influence nociceptive activity through genom ic and nongenomic effects (Aloisi 2000). Sex differences in immune responses (Da Silv a 1999, Gregory et al. 2000) biological factors

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13 (Fillingim et al. 2000) such as genetics (Mogil et al. 2000), and psychosocia l factors (Fillingim et al. 2000) such as anxiety (Edwards et al. 2000), abuse history (Spert us et al. 1999), coping (Affleck et al. 1999, Keefe et al. 2000), gender ro les (Robinson et al. 2001), and family history (Fillingim et al. 2000) can also contribute to se x differences in pain sensitivity. Exogenous hormones have also been related to clinical pain (LeResche et al. 1997, Wise et al. 2000, Musgrave et al. 2001) and to experimental pain sensitivity (Fillingim and Edwards 2001). Analgesic drugs help to relieve pain by ac ting on the peripheral system and the central nervous system. The analgesia system is mediated by three major components: the periaquaductal grey matter (in the midbrain), the nucleus raphe magnus (in the medulla), and the nociception inhibitory neurons with in the dorsal horns of the spin al cord, which act to inhibit nocicep tion transmitting neurons also located in the spinal dorsal horn. Nonhuman animal models show sex differences in nociceptive responses, but the results vary across pain assays. For example, female rats are more sensitive to electrical stimuli and to chemical such as formalin (Mogil 2000), but greater opioid analgesia in males than females are found in nonhuman animal models, primarily in rodents (Fillingim and Ness 2000, Kest et al. 2000). Among humans, women report lower pain thre sholds and tolerances than men, and that ratings of suprathreshold stimuli are often highe r among women than men across a wide range of painful stimuli (Fillingim and Maixner 1995, Rile y et al. 1998). Research suggests that sex differences among humans can be measured between muand kappa-opioid agonists. For example, following oral surgery, females expe rienced more prolonged analgesia than males using kappa(or weak mu-) opioid analgesics (Miaskowski et al. 2000). Analgesia effects from nonsteroidal anti-inflammatory drugs vary in re sponse may be Sex-related such that females

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14 demonstrate less effect than males (Walker and Carmody 1998), althoug h findings of no sex difference have also emerged (Averbuch and Katzper 2000). Studies have provided evidence for the relatio nship between a sweet tasting solution such as sucrose, fructose or glucose to the tongue a nd analgesia in rats (de Vasconcellos et al. 2006) as well as human infants (Blass and Hoffmey er 1991, Blass and Watt 1999, Ramenghi et al. 2002). The sweet-analgesia effect discovered by Bl ass appears to disappear around age 3 months in human infants. However, recent studies appear to find evidence for effects at later ages (Miller et al. 1994; Pepino and Mennella 20 05). The analgesic effects of sweet tasting solutions may also seem to have an effect on adults (Lewkowski et al. 2003, Mercer and Ho lder 1997); some might disagree (Pepino and Mennella 2005). Different palatable substances do not have e quivalent effects on reli eving pain; nutritive solutions such as polycose or sucrose may be mo re effective than nonnutritive solutions such as saccharin (DAnci et al. 1997). Another example is lactose, which does not have equivalent analgesic effects to sucrose, fructose or glucose (Blass and Shid e 1994). This indicates analgesic effects are not based solely on sw eet tasting stimuli. However, there are not many studies that have been done on the effect of other tastant stimuli such as quini ne, which has been shown to be effective in calming crying newbor ns (Graillon et al. 1997). The mechanism of analgesia consequent to in traoral administration of sugars is still unknown, but it is known that if sugar is administer ed through the nasogast ric tube, the analgesic reaction is absent. When newborns were given su gar through the intraoral route rather than the nasogastric route both the cryi ng time and behavior score we re dramatically decreased (Ramenghi et al. 1999). This st udy provides evidence that only intr aoral administration of some taste stimuli initiates analgesia.

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15 Comfort foods might play a role in the analgesia effect (Wansink et al. 2003). Comfort foods are foods that evoke a psyc hologically comfortable and pleasur able state for an individual. Comfort foods are also preferred based on a physiological need. Some individuals receive addictive qualities from certain food. This effect may due to the fact that the body releases trace amounts of opiates when palatable foods are cons umed (Wansink et al. 2003). As a result, both mood and satisfaction are elevated. Although only a small amount of opiate s are released, this might reinforce a preference for foods that are associated with these feelings. Tastes can be confused with odor, such as th e perception of sweet and sour (Stevenson et al. 1999). Some research studies ha ve shown odors are able to determ ine tastants such as sucrose. When such odors are presented with a sweet-tas te solution, the solution is given a higher sweetness rating from participants (Prescott et al. 2004). Odors help an individual interpret flavors of different tastes (Prescott et al. 2004). When concentrations of peach (Cliff and Noble 1990) and strawberry (Schifferstein and Verl egh 1996) are increased, sweetness is enhanced. There are thousands of taste buds on the tongues surface. In the mouth, food stimulates taste receptors in the taste buds. The food molecule s stimulate specialized sites on the microvilli extending from cells in the taste buds. The cel ls trigger nerve impulses in nerve fibers innervating the taste buds. Impul ses travel along the taste crania l nerves to the brain, which interprets the impulses as a distinct taste. Taste buds detect sweetness, saltiness, sourness, and bitterness. Taste is mediated by three cranial nerves : CN VII, IX, and X. CN VII, the facial nerve innervates taste buds in the fungiform papillae on the anterior two-thirds of the tongue. CN IX, glossopharyngeal nerve, innervates taste buds in the circumvallate papillae at the one-third rear of the tongue. The foliate papillae (rear edges of the tongue) are dually innerv ated by CN VII and IX. Unfortunately, the role of CN X, the vagus nerve, in the taste is not fully understood. Each

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16 taste bud is surrounded by a basket-l ike group of nerve fibers that mediate pain sensation. These fibers are part of CN V, the trigeminal nerve. CN VII normally inhibits CN IX When CN VII is damaged or anesthetized, taste from IX is intensified (Yanagisawa et al. 1998). Similarl y, CN VII normally inhibits CN V. When CN VII is damaged or anesthetized, pain from V is intensified (Tie et al. 1999). Specifically, anesthesia significantly alters th e perceived burn of 10 ppm capsai cin applied to tongue; the burn was intensified on the contralateral side and reduced on the ipsilateral side. There is speculation that taste has the general functi on of inhibiting many activities that are incompatible with eating (Bartoshuk et al. 2005). Thus, studi es showing inhibition of pain by sweet tasting substances (as well as some other taste stimuli) are compatible w ith studies that suggest taste inhibition of pain at multiple body sites. TAS2R38 gene is associated with taste abil ity. A region of chromosome 7 in the TAS2R38 gene has shown an association with the ability to taste bitter (Kim et al. 2003). There is a physiological link between genetic variation and bitt er tasting, which might affects diet choices and related health (Duffy et al. 2004, Basson et al. 2005). With regard to haplotypes of TAS2R38, those who are homozygous for the recessi ve allele (AVI/AVI) are nontasters, those who are heterozygous (AVI/PAV) or homozygous (P AV/PAV) for the dominant allele are tasters (Kim et al. 2003). The perceived bitterness of PROP divides tasters into two subgroups: supertasters (those who perceive the most bitt erness) and medium tast ers (those who perceive less bitterness). There is a strong relationship between TA S2R38 haplotype variation and bitter tasting compounds such as 6-npropylthiouracil (PROP), which also determines whether an individual is a taster or non-ta ster. Non-tasters do not perceive the PROP solution as bitter as tasters do. The tasting variation as sociates with oral sensation and tobacco or alcohol behaviors.

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17 Generally, individuals who tast ed the least bitterness from PROP or who were homozygous AVI/AVI are more likely to consume more alcohol than those who tasted the most bitterness from PROP or who were homozygous PAV/PAV (D uffy et al. 2004). Individuals who tasted the least bitterness from PROP consumed alcoholic beverages five to six times more per week on average compared to those who tasted the most bitterness from PROP averaged consuming alcoholic beverages two to three ti mes per week (Duffy et al. 2004).

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18 CHAPTER 3 STUDY DESIGN AND METHODS Subjects Candidates were screened by teleph one to assess eligibility. Participants were included if they did not have a history of treatment for depression, schizophrenia, bipolar or personality disorder, if they were not currently receiving trea tment for hypertension, or if they did not have any history of food allergies or lactose intolerance. Participants were recruited from advertisem ents posted on University of Florida campus and around Gainesville Shands hosp ital. The risks and possible side effects were explained in detail to each subject, and testing only proceed ed after a signed consent form has been obtained. The subjects were free to withdr aw from the experiment at an y time without consequences. All testing took place in a research la boratory room with the subject seated in an adjustable dental chair in an upright position. The testing was completed in a single session, which lasted approximately an hour and a half and was conducte d by three experimenters. At the end of the session, all subjects were compensated with a $35 Publix supermarket gift card for participating. The Institutional Review Board at the Univer sity of Florida appr oved this study (IRB# 5382006). They also approved the advertisements, questionnaires, and data collection sheets. Study Design Each participant was scheduled for a one tes ting session, which lasted up to 90 m inutes at a research lab located on the 2nd floor of the dental tower of Sha nds hospital. After the participants consented to participate in th e study, they were asked to rins e their mouths with water. A minimum of 5 minutes elapsed before saliva was collected. During the waiting period after rinsing, participants completed a questionnaire packet containing the following standardized measures: Health and Well-being, McGill Pain qu estionnaire, Pain Catastrophizing Scale (PCS),

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19 State-Trait Anxiety Inventory (S TAI), Life Orientation Test-Revised (LOT-R), and Perceived Stress Scale (PSC). When the que stionnaires were completed, saliva was collected with Oragene DNA Self Collection Kits (DNA Genotek Inc. 20 06) and the FRID questionnaire was given to participants to complete. Before testing began, in tensity and likes/dislikes scales were explained to the participants and two magnitude matching forms were given to the participants to familiarize them with the two scales. Each participant was given a series of contact heat stimuli using a computer controlled Medoc Pathway Pain and Sensory Evaluating Sy stem (TSA-2001, Ramat Yushai, Israel). All trials were conducted on particip ants left forearm, and the thermode was placed an inch above the wrist and an inch below th e elbow. There were a total of 16 trials. Half way through the study, an additional trial, Trial 16, was added at th e end after the taste stimu li trials to establish that baseline had not changed. Trials 1-9 and 16 were the non-stim uli trials, and trials 10-15 were with the taste stimuli. During the trials, the temperature ther mode increased at 0.5 C/second until the participant stopped th e thermode by pressing a button. When the thermode was stopped, it indicated that the participant had reached his/ her moderate pain threshold. The question Was that moderate pain? was asked to ensure the temperature was stopped at the moderate pain level. Between each thermal trial, there was a 30 seconds rest period to mi nimize the effects of prolonged heat on the skin surface. After the non-stimuli trials, the trials with stimuli were conducted in the same manner. The taste stimuli were administered in randomized order between participants to avoid possible orde r effects of the stimuli. When th e thermode was at its baseline of 35C, the taste stimulus was given to the participant. The pa rticipant was asked to move the taste stimulus around his/her mouth for 5 to 6 sec onds prior to swallowing. When the participant had reached his/her moderate pain threshold, the question Was that moderate pain? was asked.

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20 The participant also had to rate the intensity and the palatability of the taste stimulus. After the ratings were given, the participant rinsed his/her mouth with water to rem ove any residual taste. The same procedure was repeated with the remaini ng five taste stimuli. At the end of the taste stimuli trials, a non-stimuli tria l was conducted. After the thermal testing, the participant was asked to place a PROP filter paper on top of the tongue and to keep the filter paper moistened with saliva for 15 seconds. At the end of the 15 seconds, the participant gave a rating of the intensity of the taste. An extra copy of the inform ed consent form and the Publix gift card were given to the participant at the end of the session. Materials Psychological Questionnaires The Health and W ell-being questionnaire consis ts of brief questions about health (health problems (Fan et al. 2002), smoking habit and eat ing habit) and demographics (age, ethnicity, race, education level, employment status and marital status). The Pain Catastrophizing Scale (PCS) consists of 13 items related to thoughts and feelings toward a painful situation. Participants were asked to indicate the degree to which each item reflects their thoughts and feelings on a 4-point scale when they are experiencing pain. PCS is divided into three subscales of catastrophizing based on content: rumination (items 8, 9, 10, and 11), magnification (items 6, 7, and 13), and helple ssness (items 1, 2, 3, 4, 5, and 12) (Sullivan and Bishop 1995). Scoring for each item is 0 4 (0 = Not at all, 1 = to a sl ight degree, 2 = to a moderate degree, 3 = to a great de gree, 4 = all the time). The scori ng for each subscale is the sum of the items. The State-Trait Anxiety Inventory (STAI) is a 40 items questionnaire relating to anxiety level (Spielberger et al 1970). The first 20 items convey present anxiety level (state), and the last 20 items compute general anxiety level (trait). Scori ng for each item is 1 to 4 (1 = Not at all, 2 =

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21 somewhat, 3= Moderately so, 4=Very much so). The scores for the positive feeling items 1, 2, 5, 8, 10, 11, 15, 16, 19, and 20 are reversed (4 = Not at al l, 3 = somewhat, etc). After the scores are reversed, the sum of the state of anxiety can be calculated. Scores ranging from 20 thru 65 are considered low state anxiety, and scores from 66 and up are defined as high state anxiety (Tenenbaum et al. 1985). To calculate trait anxiety level, scores of items 1, 6, 7, 10, 13, 16, and 19 have to be reversed. After the scores are reversed, the sum of th e trait anxiety can be calculated. Scores ranging from 20 thru 56 are cons idered low trait anxiet y, and scores from 57 and up are defined as high trait anxiety(Tenenbaum et al. 1985). The Life Orientation Test Re vised (LOT-R) consists of 6 items relating to optimism and pessimism. Participants were asked to indicate the degree to which each item reflects their general feelings and expectations in life. This questionnaire has been revised to differentiate optimism from neuroticism (Carver et al. 1989 ). Items 1, 3, and 6 measure the degree of optimism. Items 2, 4, and 5 measure the degree of pessimism. Scoring for each item is 0 4 (0 = I disagree a lot, 1 = I disagree a little, 2 = I neit her agree or disagree, 3 = I agree a little, 4 = I agree a lot). The scoring for the pessimistic statemen ts will be reversed (4 = I disagree a lot, 3 = I disagree a little, etc.). To obt ain the score of LOT-R, the su m of the items is calculated. The McGill Pain questionnaire (MPQ) consists of 20 groups of single words describing pain. The words in each group increases in intensity order. The Perceived Stress Scale (PSS) consists of 14 items relating to thoughts and feelings of different situations. It is suggested for examini ng the role of nonspecific appraised stress in the etiology of disease and behavioral disorders and as an outcome me asure of experienced levels of stress (Cohen et al. 1983). Participants were asked to indicate the degree to which each item reflects their thoughts and feelings in the last mo nth. Scoring for each item is 0 to 4 (0=Never,1=

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22 Almost never, 2=Sometimes, 3=Fairly often, and 4=Very often). The scoring of positive items (4, 5, 6, 7, 9, 10, and 13) scoring wi ll be reversed (0=Very often, 1= Fairly often, etc.). To obtain the score of PSS, the sum of th e items will be calculated. The FRID questionnaire consists of 10 items relating to thoughts and feelings toward the experimental pain procedure. Part icipants were asked to indicate the degree to which each item reflects their current thoughts and feelings. FRID is divided into three subscales of catastrophizing based on content: high Psychological Involveme nt (items 3, 7, and 9), high Negative Expectancies (items 1, 4, and 8), and hi gh Efficacy and Control beliefs (items 2, 5, and 10). Scoring for each item is 1 to 5 (1 = Not at all and 5=Very much so). The scoring for each subscale is the sum of the items. The Magnitude Matching forms consist of 14 items relating to the intensity level of every day life experiences (e.g. Brightne ss of the light in this room, loudness of a whisper, sweetness of a coke, etc). Before each item was rated, the pa rticipants were asked to specify their strongest sensation that they have ever experienced in th eir lifetime. The strongest sensation was their 100 mark on the 0 100 intensity scale. Participants were asked to ra te the intensity level of the 14 items consistently based on their own individual 0 100 intensity scale. The second part of the magnitude matching forms consists of 11 ev eryday food items. Before the participants proceeded, they were asked to think of disliking and liking of any sort that would be their -100 mark and their +100 mark. Then the participants were asked to rate their degree of liking/ disliking of the 11 items consistently based on th eir own individual -100 +100 intensity scale. Dependent Scales After each therm al trial with taste stimulus, partic ipants were instructed to verbally rate the intensity and the palatability of the taste stimuli on a 0-100 nume rical scales. Two scales were used in the study: Intensity scale and Likes/ Dislikes Scale. The in tensity scale is a scale of how

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23 intense something is. It ranges from 0 to 100, 0 being no sensation at all and 100 being the strongest experienced intensity of any kind. The likes/ dislikes scale is a s cale of palatability. The scale ranges from to + 100. is the strongest disliking of any kind; 0 is neutral; + 100 is the strongest liking of any kind. Taste Stimuli All food products were availabl e in retail stores with th e exception of the caffeine. Caffeine wa s obtained from the Department of Food Science and Human Nu trition, where all the food stimuli were prepared to assure consistenc y. Prepared yogurt stimu li were discarded after 48 hours to avoid food spoiling and contamination. There were six different taste stimuli: plain non-flavored fat-free yogurt, pl ain non-flavored fat-free yogur t + sucrose (12.5 g/100 g of yogurt), plain non-flavored fat-free yogurt + su crose (12.5 g/100 g of yogurt) + McCormicks strawberry extract (3 ml/ 100 g of yogurt), pl ain non-flavored fat-fr ee yogurt + sucrose (12.5 g/100 g of yogurt) + McCormicks anise extract (1 ml), plain non-flavored fat-free yogurt + caffeine (0.15 g/ 100 g of yogurt), and Welchs strawberry preserve The order of the six taste stimuli was randomized. Randomization was dete rmined by the Flash Action Script program. After each taste stimulus trial, tap water was used to rinse participants mouths to remove any residual tastes. Heat Stimuli Therm al test was performed with a Medoc Thermal Sensory Analyzer (TSA-2001, Ramat Yishai, Israel). A 3 x 3 cm square thermode wa s placed an inch above the wrist and an inch below the elbow due to mid-forearm appears more sensitive to responses (Meyer et al. 1992). The thermode is set at 35C as a baseline. The device delivers a brief thermal stimulus to the skin using an increasing scale. The probe temper ature increases at 0.5 C/second. A button on a handheld device held by the subject is used to signal the devi ce to stop. When activated, the

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24 device decreases at 8 C/second. The thermode does not exceed 52 C in this study to prevent any injury to the skin surface. Genotyping/ Haplotyping Oragene DNA self-collection k its (DNA Genotek Inc. 2006) were used to collect DNA from saliva. The bitter tast e receptor gene, TAS2R38, was i nvestigated to determine the relationship of the participants ta ste status and various tastes playing a role in pain inhibition. With regard to haplotypes of TAS2R38, thos e who are homozygous for the recessive allele (AVI/AVI) are nontasters, those who are hete rozygous (AVI/PAV) or homozygous (PAV/PAV) for the dominant allele are tasters (Kim et al 2003). The perceived bitte rness of PROP divides tasters into two subgroups: supertasters (those w ho perceive the most bitterness) and medium tasters (those who perceive less bitterness). Whatman #1 filter papers were saturated in a 6-npropylthiouracil (PROP) solution, wh ich is a medicine for hyperthyr oidism, was used to measure the individuals taste status (Duffy et al. 2004). Individuals who are non-tasters give a lower intensity rating for the PROP paper and vice ve rsa (Duffy et al. 2004). PROP paper contained about 1.6 mg of PROP. The maintenance dosage for this medication varies from 50 200 mg/day (Solomon et al. 1986). Thus, the dosage, which is very minimal in this study, is not expected to elicit any clinical effect. Statistical Analyses Data were analyzed using SPSS ( Windows version 15.0). Only differences of p .05 are reported as statistically significant. Repeated Measures Analysis of Variance was used to compare the temperature of each trial with a nd without the taste stimuli. One-Way ANOVA was conducted with the factor being haplotypes and th e dependent variable be ing likes/dislikes rating of taste stimuli, PROP intensity. Independent t-tests were also conducted to compare two

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25 variables of interest, and correla tions tests were performed to de termine the relationship between variables.

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26 CHAPTER 4 RESULTS Subjects Sixty norm al, healthy individua ls were recruited. There were 30 males (mean age = 22.4, median age = 22.0, SD = 4.1, age ranged from 18 37; 1 participant refused to give his age) and 30 females (mean age = 22.4, median age = 21.0, SD = 8.0, age ranged from 18 63) (Table A1). Within the 30 males, there were 80.0% nonHispanic white, 3.3% no n-Hispanic black, 3.3% Hispanic white, 3.3% Hispanic black, 6.7% Asia n, and 3.3% mixed (Table A-1). Within the 30 females, 60% were non-Hispanic white, 20% we re non-Hispanic black, 13.3% were Hispanic white and 6.7% were Asian (Table A-1). Twelve of the thirty male participants and te n out of the thirty females participants were currently employed (Table B-1). Most of the participants were singl e and completed some college level courses. The majority of the participants had no history of health problems and never reported cancer, chronic pai n, or cardiac disease. Only nine participants had a history of asthma or bronchitis, seven participants had a history of pneumonia, one had a history of diabetes, and one had a history of head trauma (Table C-1). Most of the participants we re non-smokers (Table D-1). Ac cording to the United States Department of Agriculture Food Guide Pyramid, each individual has his/ her own food pyramid guide to follow. However, 2-4 servings of 4 ounces of meat, 2-4 servings of 2-4 cup of fruits and 3-5 servings of 1 cup of vege tables are recommended. Compar ed to the USDA Food Guide Pyramid, the participants averag e consumption of protein, fruits and vegetables are well below the recommended amount (Table D-1). Base d on the psychological questionnaires, all participants scored within the normal limits. Th ey were emotionally stable, not excessively optimistic, pessimistic, or too anxious and nervous (Table E-1). For all the temperature trials,

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27 one male participant was eliminated from data analysis. The participant had reached the safety level, 52 C, of the Pathway thermode m achine without reporting moderate pain. A Temperature of Non-Taste Stimuli Trials40 41 42 43 44 45 46 Trial 1Trial 2Trial 3Trial 4Trial 5Trial 6Trial 7Trial 8Trial 9Temperature (C ) * *B Temperature of Non-Taste Stimuli Trials40 41 42 43 44 45 46 Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7 Trial 8 Trial 9Temperature (C ) Male Female Figure 4-1.Temperature of Non-Taste Stimuli Trials. A) Means and standard errors of temperature during the non-stimuli trials fo r all participants response at which they report moderate pain. Asterisks indicate values that are sign ificantly (P < 0.05) compared to Trial 9. B) Means and standa rd errors of temp erature during the nonstimuli trials at which participants report moderate pain, separately for both males and females. Baseline Temperature A ll the non-taste stimuli trials were compar ed to trial 9, which was the last non-taste stimuli trial before the six taste-stimuli trials. It is assumed that the participants have familiarized with the thermode and have recognized their moderate pain level by trial 9. When each non-taste stimulus trial was compared to trial 9, it can be concluded that th ere were no significant differences among trials 6, 7, 8, and 9 (Figure 41A). However, the p-value for trial 6 is 0.063, which is marginally significant. Unlike trial 6, trial 7 has a p -value of .223 and trial 8 has a pvalue of .155. As a result, the average of the te mperature at which the participants reported moderate pain during trials 7, 8 and 9 were used to determine the baseline temperature. There was also no significant difference between males and females when the non-taste stimuli trials

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28 were compared ( p = 0.063) (Figure 4-1B). Wh en the baseline temperature is determined with the average of Trial 7, 8, and 9 for the males and fema les, the averages between sexes are compared. As shown in Figure 4-2, the average for the ma les is 44.7 and for females is 43.3. Males have a higher average of the baseline temperature th an females, but it was not significant ( p = .11). Comparison Between Sexes at Baseline Tem perature (Trials 7,8,9)40.5 41.5 42.5 43.5 44.5 45.5 46.5 MaleFemaleTem p erature ( C ) Figure 4-2. Comparison of baseline temperat ure between males and females at which participants report moderate pain. Taste Stimuli Trials When the temperature at which the p articipants reported moderate pain during the six taste stimuli trials were averaged and compared betwee n sexes, the average temperature for males was 45.7 degrees Celsius and 44.1 degrees Celsius for females. The difference between males and females was significant ( p = .046) (Figure 3). When taste stim uli trials were sorted by taste stimuli, the temperatures at which the particip ants reported moderate pain were compared. The temperatures were significantly different from baseline temperature (Figure 4-4A). The p-values of all the six taste stimuli compared to baseline are below .005. However, similar to the non-taste stimuli trials, there was no significant difference between the sexes (Figure 4-4B) ( p = .051). When the temperatures of the si x taste stimuli trials were comp ared among each other, there was

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29 no significant difference among each other except for 1.) trial with sucrose yogurt compared to trial with anise-flavored yogurt (t = -2.1, p = .039) and 2.) trial with anise-flavored yogurt compared to trial with caffeine yogurt (t = 2.182, p = .033) (Table G-1). Comparison Between Sexes at Avera g e Temperature of Taste Stimuli Trials (10-15)42 42.5 43 43.5 44 44.5 45 45.5 46 46.5 MaleFemale Temperature (C ) Figure 4-3. Comparison of averaged temperature of six taste stimuli trials between males and females at which participan ts report moderate pain. A Temperature of Taste Stimuli 42 42.5 43 43.5 44 44.5 45 45.5 46 46.5Baseline P l a in Yog urt S u c ros e Yog urt S t r a wberry E xtra c t Yog u rt Anise Yogurt Caf fei n e Yog urt S t ra w be rry Jam * 1 2Temperature (C )B Temperature of Taste Stimuli 42 42.5 43 43.5 44 44.5 45 45.5 46 46.5B a s e l i n e P l a i n Y o g u r t S u c r o s e Y o g u r t S t r a w b e r r y E x t r a c t Y o g u r t A n is e Y o g u r t C a f f e i n e Y o g u r t S t r a w b e r r y J a m Male Female Temperature (C ) Figure 4-4. Temperature of Taste Stimuli. A) Means and standard errors of temperature during the stimuli trials at which participants report moderate pain. Asterisks indicate values that are significantly (p < 0.05) compared to baseline temperature. The number 1 shows the significant difference between the sucrose trial and anise trial. The number 2 shows the significant difference between the anise trial and caffeine trial. B) Means and standard errors of temperature during the stimuli trials at which participants report moderate pain, separately for both males and female.

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30 Non-Stimuli Trial Trial 16 Temperature Comparisons for Selected Sample of Subjects 42 42.5 43 43.5 44 44.5 45 45.5 46 46.5Baseline Plain Yogurt Sucrose Yogurt Strawberry Extract Yogurt Anise Yogurt Caffeine Yogurt Strawberry Jam Trial 16 Temperature (C ) * Figure 4-5. Temperature Comparisons for Selected Sample of Subjects. Means and standard errors of temperature during the stimuli trials at which participants report moderate pain. Asterisks indicate values that are significantly (P < 0.05) compared to baseline temperature. Trial 16 was added half way through the study. The results of Trial 16 were obtained from 20 females and 9 males. When Trial 16 was co mpared to the baseline temperature, the temperature at which participants reported moderate pain duri ng Trial 16 was not significantly different from baseline temperature (F = .281, p = .60) (Figure 4-5). There was no significant difference between sexes. Delta Temperature Delta tem perature is the difference of the te mperature at which the participants reported moderate pain during the taste stimulus trial and the baseline temperature (average of Trials 7, 8, 9). A positive delta temperature represents a pain inhibition, and a negative temperature represents the taste stimuli caused more pai n, therefore, no pain inhibition. The delta temperatures for all taste stimuli were positive (Figure 4-6A). This indicates all taste stimuli resulted in pain response modulation; temperatur es at which the particip ants reported moderate

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31 pain during tastestimuli trials we re at least 0.5 degrees Celsius hi gher than baseline temperature (Table F-1). There were no signi ficant difference between males and females (Figure 4-6B). A B Figure 4-6. Delta Temperature of Taste Stimuli. A) Means and standard errors of delta temperature during the taste stimuli trials at which participants report moderate pain. B) Means and standard errors of delta temp erature during the taste stimuli trials at which participants report moderate pain, separately for males and females. There was no significant difference between sexes ( p .05). There was no significant difference among each taste stimuli except for 1.) temperatur e of taste stimuli trial with sucrose yogurt compared to temperature of taste stimuli trial with anise flav ored yogurt (t = -2.1, p = .039) and 2.) temperature of taste stimuli trial with aniseflavored yogurt compared to temperature of taste stimuli trial with caffeine yogurt (t = 2.182, p = .033) (Table G-1). Intensity of Taste Stimuli From the six taste stimuli that were used in testing, both males and females rated caffeine yogurt as the taste stimulus with the highest intensity; the over all mean was 54.32 (Figure 4-7A). Both sexes rated strawberry jam as the second most intense; the overall mean was 50.15 (Figure 4-7B). Anise-flavored yogurt was rated the third most inte nse; the overall mean was 40.22 (Figure 4-7A). There was no significant difference between sexes ( p = .539) (Figure 4-7B).

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32 A B Figure 4-7. Intensity of Taste Stimuli. A) Means a nd standard errors of in tensity of taste stimuli at which participants reported. B) Means a nd standard errors of intensity of taste stimuli at which participants reported, separately for males and females. A Palatability of Taste Stimuli-60 -40 -20 0 20 40 601 2 3 4 5 6Palatability Ratings Strawberry Jam Yogurt Caffeine Yogurt Anise Yogurt Strawberry Yogurt Sucrose Yogurt Plain YogurtB Palatability of Taste Stimuli-60 -40 -20 0 20 40 60 Palatabilit y Ratin g s Male (n=29) Female (n=30) Plain Strawberry Sucrose A nise Caffeine Strawberry Jam Figure 4-8.Palatabilty of Taste Stimuli. A) Means and standard errors of palatability of taste stimuli. B) Means and standard errors of intensity of taste stimuli, separately for males and females. Palatability of Taste Stimuli Between the six taste stimuli, caffeine yogurt was rated as the l east palatable taste stim uli; the overall mean was -50.37 (Table H-1) Strawberry-flavored yogurt was rated as the most palatable; the overall mean was 39.92 (Table H-1). Both females and males rated caffeine

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33 yogurt as the least palatable taste stimulus. The mean unpalatability rating given by males for caffeine yogurt was .55, and the females me an was .07 (Figure 4-8A). Males rated strawberry jam as the most palatable; the mean was 36.41 (Figure 4-8B). Females rated strawberry-flavored yogurt as the most palatabl e; the mean was 39.33 (Figure 4-8B). There was no significant sex difference ( p = .541). TAS2R38 Haplotypes Of the 60 participants, genotypes of seve n DNA sam ples were not obtained due to experimental errors. Four categories of TAS2R38, AVI/AVI, AVI /PAV, PAV/PAV, and miscellaneous, can be defined in this study. Th ere were thirty-two percent (n=17) of the participants TAS2R38 gene were homozygous AVI/AVI, 34% (n=18) heterozygous AVI/PAV, 23% (n=12) homozygous PAV/PAV, 3.7% (n=2) heterozygous AAV/PAV, 3.7% (n=2) AVI/AAV, 1.8% (n=1) AVI/AAI, and 1.8% (n=1) AAI/PAV. The intensity ratings of PROP were compar ed among the TAS2R38 haplotypes. There was a significant association between the TAS2R38 haplotypes and PR OP intensity rating. However, there was no significant association between the TAS2R38 haplotype s and sex, ethnicity, palatability and intensity ratings of taste. From the TAS2R38 haplot ypes, homozygous AVI/AVI individuals showed lower intensity ratings on bo th the caffeine yogurt and the strawberry jam, and homozygous PAV/PAV individua ls showed higher intensity ratings on both taste stimuli (Figures 4-9A and B). The differences in the intensity ratings of the caffeine yogurt among the TAS2R38 haplotypes were not significant ( p = 0.30) (Figure 4-9A). Similar to caffeine, the differences in the intensity rati ngs of the strawberry jam among the TAS2R38 haplotypes were not significant (p = 0.057) (Figure 4-9B). The delta temp eratures during the trial with caffeine yogurt were not significantly differe nt among the TAS2R38 haplotypes ( p = 0.91) (Figure 4-

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34 10A). Similarly, during the taste stimuli trial with strawberry jam, there were no significant difference among the TAS2R38 haplotypes ( p = 0.36) (Figure 4-10B). A TAS2R38 Haplotypes vs. Intensity of Caffeine Yogurt35 40 45 50 55 60 65 70 75 AVI/AVIAVI/PAV, PAV/AVI PAV/PAVAAV/PAV, AAV/AVI, AAI/AVI, AAI/PAV TAS2R38 HaplotypesIntensity Ratings of Caffeine Yogurt B TAS2R38 Haplotypes vs. Intensity of Strawberry Jam35 40 45 50 55 60 65 70 75 AVI/AVIAVI/PAV, PAV/AVI PAV/PAVAAV/PAV, AAV/AVI, AAI/AVI, AAI/PAV TAS2R38 HaplotypesIntensity Ratings of Strawberry Jam Yogurt Figure 4-9. TAS2R38 Haplotypes vers us Intensity Ratings of Taste Stimulus A) The mean and standard error of the intensity of caffe ine yogurt among TAS2R38 haplotypes. B) The mean and standard error of the intensity of strawberry jam among the different TAS2R38 haplotypes stimuli, baseline temperature and delta temperature. A B Figure 4-10. TAS2R38 Haplotypes versus Delta Temperature of Taste Stimulus. A) The mean and standard error of th e delta temperature of ca ffeine yogurt among the TAS2R38 haplotypes. B) The mean and standard erro r of the delta temperature of strawberry jam among the TAS2R38 haplotypes.

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35 6-n-Propylthiouracil Testing The fe male participants reported a higher mean rating of PROP compared to males (Table I-1), but the difference was not significant (t = -.852, p = .398) (Figure 4-11A). Among the TAS2R38 haplotypes, individuals who were homozygous AVI/AVI had the lowest rating on PROP paper, heterozygous AVI/PAV rated as intermediate, and homozygous PAV/PAV rated it the most intense. There were significant asso ciations between the PROP ratings and the TAS2R38 haplotypes (Figure 4-11B). However, there was also no significant correlation between the intensity ratings of the taste stimuli and the PROP ra tings except with the intensity of caffeine yogurt ( p = 0.33) (Table J-1). In addition, among males, there were significant correlation between the intensity of PROP and intensity of anise-flavored yogurt and caffeine yogurt (Table J-1). A B Figure 4-11. Comparison of PROP Intensity. A) Comparison of In tensity Ratings Between Sexes Means of intensity of taste stimuli at whic h participants reported, separately for males and females. B) TAS2R38 Haplotypes ve rsus PROP Intensity. The mean and standard error of the intensity of PROP paper among the different TAS2R38 genotypes.

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36 CHAPTER 5 DISCUSSION The purpose of this study was to determ ine the thermal pain responses with and without the consumption of taste stimuli on healthy indivi duals and to conclude wh ether genetic variation in taste plays a role. The first hypothesis that was proposed in this study was rejected because males did not report at a significantly higher mean pain threshold than females. However, the second hypothesis failed to be rejected because the temperatures at which the participants reported moderate pain during the taste stimuli were significantly different when compared to the baseline temperature. This study revealed an additional finding. There was a significant association between the TA S2R38 genetic variations and PROP paper ratings. During the non-taste stimuli, males reported moderate pain at a higher temperature than females, although the difference is not significant. Previous studies have shown that there is a sex difference in the perception of pain (Jensen et al. 1992, Fill ingim and Maxiner 1995, Riley et al. 1998). The average of the temperature at whic h participants reported moderate pain during trials 1 through 5 were significan tly different from trial 9. Trial 6 was marginally significantly different; therefore, it wasnt used as part of the baseline temperature. Only the average temperatures of trials 7, 8, and 9 were used to de termine baseline temperature. It is assumed that the participants recognize what moderate pain is after practice and result in an increase of temperature throughout the trials. Th is might have resulted because participants were afraid the temperature of the thermal would reach beyond their moderate pain. Similar to the non-taste stimuli trials, males al so reported in a higher pain threshold than females during the taste stimuli trials, but the di fference was not significant. This indicates that taste stimuli have an effect on thermal responses All taste stimuli resulted in an increase in temperature at which was reported moderate pain In other words, various tastes have pain

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37 inhibition effects. Similar to Blass et al. 1991, sweet resulted in pain inhibition. Some have suggested that palatability might pl ay a role. However, our data show that palatability of the taste stimuli was not linked to pain modulation. Plai n non-flavored fat-free yo gurt, anise-flavored yogurt, and caffeine yogurt, which participants rate d in the negatives on the palatability scale, also have an effect on pain i nhibition (Table E-1). When the temperature of the taste stimuli trials were compared to each other, the onl y significant differences were found for 1.) temperature of taste stimuli trial with sucrose yogurt compared to temperature of taste stimuli trial with anise-flavored yogurt (t = -2.1, p = .039) and 2.) temperature of taste stimuli trial with anise-flavored yogurt compared to temperature of taste stimuli trial with caffeine yogurt (t = 2.182, p = .033). These results further show that palata bility of taste stimuli do not play a role in pain inhibition. Studies on physio logical responses toward co mfort food have shown when palatable foods are consumed, the body releases trace amounts of opiates (Wansink et al. 2003), which elevate both mood and satisfaction. The resu lt in this study shows th at pain inhibition is not only caused by palatable tastes but also unpalatable tastes which suggests the taste stimuli were not perceived as a comfort food by the partic ipants. This can be concluded because the food rated as unpalatable also had a pain inhibition effect. The exac t reason why and how the tastes have inhibitory effects is still unknown, but the unknown mechanism could be triggered from endogenous opioids. There are three common en dogenous opioids that inhibit pain: leuenkephalin, beta-endorphins, and dynorphins (Ak il et al. 1984). Enkephalin is found throughout the endocrine system, and is produced by the adre nal medulla and in the gastrointestinal tract (Akil et al. 1984). When the taste stimulus is in the mouth, it might have triggered the production of endogenous opioids. As a result, the participan ts experienced pain in hibition during the taste stimuli trials.

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38 Apparently, pain inhibition is not based on palatability, but the mechanism that is responsible remains unknown. The part icipants pain tolera nce for the thermal stimulus increased about one degree Celsius (Table F-1). In Lill eso et al. 2000, the heat pain threshold was increased by one degree Celsius with the usage of morphine. This suggests the taste stimuli used in this study can represent one dosage of morphine. An additional trial, Trial 16, was added at the end of the 15 thermal trials. The purpose of the additional trial was to determine whether th e temperature at which participants report moderate pain during a non-taste stimuli trial after taste stimuli trials is similar to baseline temperature, i.e. that trial 16 confirms the baseline temperature and the pain inhibitory effect of the taste stimuli. Trial 16 was added to be pa rt of the study design halfway throughout the study; therefore, results were only gathered from 29 pa rticipants. Twenty of the 29 participants were males and nine were females. Due to the small sample size for trial 16, th e temperature at which the participants reported modera te pain resulted in a huge variab ility. When the temperature of trial 16 was compared to baseline temperature, it is not significantly different. This shows the order of the trials did not play a role in pain inhibition. Pain modulati on occurs only during the taste-stimuli trials. Genetics might play a role in the pain inhibi tion effects of the tastes stimuli. Genetic variation in taste was gathered from collecti ng saliva to measure the bitter receptor gene, TAS2R38. Homozygous AVI/AVI haplotypes are non-tasters, heterozygous AVI/PAV haplotypes are intermediate tasters, and homo zygous PAV/PAV haplotypes are tasters. Bitter taste was measured by using 6-n-propylthiourac il (PROP). Depending on the intensity rating of PROP, it distinguishes non-tasters and medium tasters (Fisher et al. 1939). Indivi duals who rate PROP a higher intensity are taster s, and individuals who rate it a lower intensity are non-tasters.

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39 The ratings of PROP and the TAS2R38 haplotypes have a signifi cant correlation; however, the correlation between the delta temperature and the TAS2R38 haploptypes is not significant. From saliva collection, the TAS2R38 bitter taste receptor gene was analyzed. There was no significant correlation between the TAS2R38 haplot ypes and palatability of the taste stimuli, intensity of the taste stimuli, baseline temperature, or delta temperature. There is no significant correlation between PROP intensity ratings and the intensity ratings of the six taste stimuli except with caffeine yogurt (p = 0.33) (Table J-1) However, among males, there were significant correlation between the intensity of PROP and intensity of anise-flavored yogurt and caffeine yogurt (Table J-1). In addition, there is no strong correlation between PROP intensity ratings and the palatability of the six taste stimuli (Table J-1). These results contribute to a new theory of the inhibitory effects the intensity of the taste stimuli play. There is an unidentified mechan ism in our body system for the pain modulation during the taste stimuli trials The unknown mechanism might be related to endogenous opioid release. If various tastes do have inhibitory effects on pain, pain-k illers might at least in part, be replaced with different food items. Individuals can be less dependent on over-the-counter drugs and rely on healthier pain killers. This mi ght also suggest the r eason individuals have preferences in th eir daily diet.

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40 CHAPTER 6 SUMMARY AND CONCLUSIONS Researchers have reported that food tastes may have inhibitory effects on perception of pain. The cause of this effect remains unknow n, but it may partly be due to an endogenous inhibitory mechanism in our body. The main focus of this study was to de termine the effect of various tastes on the response to a pain stimulus, the thermal. This study showed that all taste stimuli tested had a pain inhibitory effect on when the participants reported moderate pain from the thermal stimulus. Males did not report moderate pain significantly higher than females. Similar to other studies, sweet tast e was shown to have a pain inhibitory effect. In additi on, other tastes such as caffeine a nd anise also have an inhibitory effect compared to baseline. This demonstrat es that unpalatable tastes also possess pain modulation properties. TAS2R38 haplotypes distinguish the individual s who are non-tasters and tasters. However, no significant relationship was found between th e genotypes and the level of pain modulation. This result is maintained regard less of palatability of the food items, i.e. pain modulation still remains. The effect that is observed in th is study might be caused by a more complicated phenomenon than an individua ls taste ability. There may be a more complex, unknown mechanism. Further studies are needed to dete rmine whether distraction or the olfactory organ play a role in to trigger this unknown m echanism that is causing pain inhibition.

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41 APPENDIX A CHARACTERISTICS OF PARTICIPANTS (A GE, SEX, AND CL ASSIFICATION OF ETHNICITY) Table A-1. Characteristics of participants age, sex, and classi fication of ethnicity Male (n = 30) *Female (n = 30) Age (years) Mean + SD 22.4 + 4.1 22.4 + 8.0 Median 22.0 21.0 Range 18-37 18-63 Ethnicity NonHispanic White 24 18 NonHispanic Black 1 6 Hispanic White 1 4 Hispanic Black 1 0 Asian 2 2 Mixed 1 0 Males (n=29) for age

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42 APPENDIX B CHARACTERISTICS OF PARTICIPANTS (EMPLOYMENT STATUS, HIGHEST EDUCATION LEVEL COMPLETED, AND M ARITAL STATUS) Table B-1. Characteristics of participants employment status, hi ghest education level completed, and marital status Male (n = 30) Female (n = 30) Currently Employed Yes 12 10 No 18 20 Education Level Some College 25 25 College Graduate 4 2 Postgraduate/Professional 1 3 Marital Status Single 28 28 Married 2 2

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43 APPENDIX C CHARACTERISTICS OF PARTICIPANTS (HEALTH HISTORY/PROBLEMS, EAR INFECTIONS, AND T ONSILLECTOMY) Table C-1. Characteristics of participants health history/problems, ear infections, and tonsillectomy Male (n = 30) Female (n = 30)* Health Problems Cancer 0 0 Chronic lung disease, Emphysema 0 0 Asthma, or bronchitis 6 3 Congestive heart failure 0 0 Diabetes 0 1 Heart attack 0 0 Pneumonia 4 3 Stroke 0 0 Head Trauma or Injury 0 1 Ear Infection No 17 17 Yes, but not serious 8 5 Yes, required antibiotics more than once2 5 Yes, required tubes in ears 3 2 Tonsillectomy Yes 4 5 No 26 24

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44 APPENDIX D CHARACTERISTICS OF PARTICIPANTS (S MOKING HABIT AN D EATING HABIT) Table D-1. Characteristics of particip ants smoking habit and eating habit Male (n = 30) Female (n = 30) Smoking Habit Never Smoked 27 25 Past Smoker 3 3 Current Smoker 0 2 Eating Habit (average/day) 4 oz serving of protein Mean + SD 2.9 + 1.48 2.2 + 1.48 Median 3.0 2.0 1/2 cup serving of vegetables Mean + SD 2.1 + 1.50 2.5 + .937 Median 2.0 2.0 1/2 cup serving of fruits Mean + SD 2.2 + 1.69 2.5 + 1.41 Median 2.0 2.0

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45 APPENDIX E PSYCHOLOGICAL VARIABLES Table E-1. Means and standard devi ation of psychological variables Male (n=30) Female (n=30) Questionnaires Pain Catastrophizing Scale Helplessness 2.87 + 2.70 4.00 + 3.26 Rumination 4.27 + 2.95 4.67 + 2.68 Magnification 2.00 + 1.53 2.27 + 2.03 State Anxiety* 28.93 + 5.61 29.25 + 6.58 Trait 32.43 + 6.07 32.97 + 4.77 LOTR 16.70 + 5.61 18.97 + 3.70 PSS 16.60 + 5.76 20.03 + 6.69 FRID Negative Expectancies 4.50 + 1.22 5.07 + 1.62 Efficacy Control 13.07 + 1.82 12.09 + 1.80 Psychological Involvement 10.87 + 2.19 10.63 + 2.06 Females (n=28) for state anxiety

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46 APPENDIX F DELTA TEMPERATURE OF TASTE STIMULI Table F-1. Means and standard deviation of delta tem perature of taste stimuli Taste Stimuli Temperature Male (n=29)Female (n=30) Plain Yogurt .88 + 1.42 1.04 + 1.34 .71 + 1.49 Sucrose Yogurt .77 + 1.49 .66 + 1.57 .89 + 1.42 Strawberry Yogurt .87 + 1.31 .88 + 1.14 .85 + 1.47 Anise Yogurt 1.15 + 1.35 1.04 + 1.13 1.25 + 1.54 Caffeine Yogurt .79 + 1.51 .94 + 1.22 .64 + 1.76 Strawberry Jam 1.01 + 1.13 1.15 + .965 .86 + 1.28

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47 APPENDIX G COMPARISONS OF THE TEMPERATURE OF TASTE STIMULI TRIALS Table G-1. Paired t-tests for com parisons of the temperature of taste stimuli trials Taste Stimuli t Significance Plain vs. Sucrose .556 .580 Plain vs. Strawberry .000 1.00 Plain vs. Anise -1.65 .105 Plain vs. Caffeine .726 .471 Plain vs. Strawberry Jam -.636 .527 Sucrose vs. Strawberry -.580 .564 Sucrose vs. Anise* -2.11 .039 Sucrose vs. Caffeine .197 .845 Sucrose vs. Strawberry Jam -1.16 .250 Strawberry vs. Anise -1.79 .078 Strawberry vs. Caffeine .732 .467 Strawberry vs. Strawberry Jam -.723 .472 Anise vs. Caffeine* 2.18 .033 Anise vs. Strawberry Jam .952 .345 Caffeine vs. Strawberry Jam -1.45 .154 Difference is significant at the 0.05 level (2-tailed).

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48 APPENDIX H INTENSITY AND PALATABILITY OF TASTE STIMULI Table H-1. Means and standard deviation of in te nsity and palatability of taste stimuli Male (n=29) Female (n=30) Intensity Plain Yogurt 38.62 + 18.89 39.27 + 22.39 Sucrose Yogurt 28.28 + 15.86 31.30 + 18.88 Strawberry Yogurt 34.48 + 16.82 32.00 + 19.50 Anise Yogurt 38.48 + 17.83 41.93 + 21.08 Caffeine Yogurt 51.90 + 21.06 56.67 + 24.99 Strawberry Jam 48.90 + 22.20 51.37 + 20.96 Palatability Plain Yogurt 23.14 + 28.74 -25.33 + 34.66 Sucrose Yogurt 23.97 + 23.84 30.00 + 31.18 Strawbery Yogurt 40.52 + 22.13 39.33 + 22.58 Anise Yogurt -10.28 + 34.92 -16.67 + 40.09 Caffeine Yogurt -46.55 + 20.53 -54.07 + 30.41 Strawberry Jam 42.93 + 23.32 30.10 + 40.27

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49 APPENDIX I PROP INTENSITY RATING Table I-1. Means and standard devi ation of PROP intensity rating Male (n=30) Female (n=30) PROP Intensity 27.67 + 28.27 33.83 + 27.81

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50 APPENDIX J CORRELATIONS OF PROP VERSUS INTE NSITY AND PALATABI LITY OF TASTE STIMULI Table J-1. Correlations of PROP versus intensity and palatability of taste stimuli **Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed). PROP (n=59) Male (n=29) Female (n=30) Intensity Plain Yogurt .075 .072 .077 Sucrose Yogurt .189 .260 .117 Strawberry Yogurt .171 .320 .060 Anise Yogurt .135 .387 -.092 Caffeine Yogurt .330 .514 ** .164 Strawberry Jam .207 .289 .110 Palatability Plain Yogurt .124 .135 .126 Sucrose Yogurt .202 .327 .094 Strawberry Yogurt .175 .264 .096 Anise Yogurt -.076 -.202 .048 Caffeine Yogurt -.186 -.283 -.107 Strawberry Jam .129 -.096 .306

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56 BIOGRAPHICAL SKETCH Wendy Sze Ming was born in Hong Kong, China. At the age of eight, she m oved to the United States with her parents and older brother. In 2002, she gr aduated from St. Augustine High School in St. Augustine, Florida, and began her undergraduate education at the University of Florida. In 2006, she earned her bachelors de gree in Nutritional Sciences and entered the nutritional sciences masters thesis program at the University of Florid a. She did her thesis research experiment at the Department of Co mmunity Dentistry and Behavioral Sciences under the supervisor of Dr. Henrietta Logan. Wendy w ill be graduating with her masters degree in Spring 2008, and she will be attending Nova Sout heastern University for the optometry program, the Class of 2012.