<%BANNER%>

Wheat Bioactives and Antioxidant Activity

Permanent Link: http://ufdc.ufl.edu/UFE0043494/00001

Material Information

Title: Wheat Bioactives and Antioxidant Activity
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Smith, Michelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: antioxidant -- bioactive -- bran -- dna -- wheat
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 objectives of this pilot study were (1) to show that consumption of wheat bran cereal by normal, healthy human subjects will provide antioxidant protection as measured by DNA strand breaks and (2) to determine if there is a dose response in cereal consumption. Following a baseline blood draw, 44 volunteers were randomized to consume either one serving (50g) or two servings of wheat bran cereal for 21 days at which time a second blood draw was performed. The antioxidant protection of the phenolic compounds was determined in peripheral blood mononuclear cells by measuring DNA strand breaks using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. If the antioxidants in the cereal were bioavailable, then the DNA will show fewer strand breaks in the form of a smaller percentage of apoptotic cells when the cells are cultured with hydrogen peroxide. The results showed that there was no apparent protection of lymphocyte DNA from oxidative stress. Reasons for this may be poor bioavailability, however, the study was not designed to observe lack of bioavailability. Phenolic acids are bound to large amounts of fiber found in the cereal product. This binding could prevent absorption of the phenolic acids, keeping them from exerting their antioxidant effects in the body. Other reasons may include variability in DNA strand breaks among subjects.
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.
Statement of Responsibility: by Michelle Smith.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043494:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043494/00001

Material Information

Title: Wheat Bioactives and Antioxidant Activity
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Smith, Michelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: antioxidant -- bioactive -- bran -- dna -- wheat
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 objectives of this pilot study were (1) to show that consumption of wheat bran cereal by normal, healthy human subjects will provide antioxidant protection as measured by DNA strand breaks and (2) to determine if there is a dose response in cereal consumption. Following a baseline blood draw, 44 volunteers were randomized to consume either one serving (50g) or two servings of wheat bran cereal for 21 days at which time a second blood draw was performed. The antioxidant protection of the phenolic compounds was determined in peripheral blood mononuclear cells by measuring DNA strand breaks using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. If the antioxidants in the cereal were bioavailable, then the DNA will show fewer strand breaks in the form of a smaller percentage of apoptotic cells when the cells are cultured with hydrogen peroxide. The results showed that there was no apparent protection of lymphocyte DNA from oxidative stress. Reasons for this may be poor bioavailability, however, the study was not designed to observe lack of bioavailability. Phenolic acids are bound to large amounts of fiber found in the cereal product. This binding could prevent absorption of the phenolic acids, keeping them from exerting their antioxidant effects in the body. Other reasons may include variability in DNA strand breaks among subjects.
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.
Statement of Responsibility: by Michelle Smith.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043494:00001


This item has the following downloads:


Full Text

PAGE 1

1 WHEAT BIOACTIVES AND ANTIOXIDANT ACTIVITY By MICHELLE SMITH 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 2011

PAGE 2

2 2011 Michelle Smith

PAGE 3

3 To my mother Kathleen Johnson, and my grandparents, Patricia and Robert Johnson

PAGE 4

4 ACKNOWLEDGMENTS I thank my personal advisor Dr. Susan Percival who gui ded me from the start. I thank my committee mem bers Dr. Wendy Dahl and Dr. Lisa McElwee White for sharing their time and knowledge I thank my labmates for their assistance whenever I needed it I thank my mom for always being there to listen and offer advice I tha nk my grandparents for their un yielding support financially and emotionally.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Background ................................ ................................ ................................ ............. 13 Specific Aims ................................ ................................ ................................ .......... 13 H ypothesis ................................ ................................ ................................ .............. 14 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Whole Grains ................................ ................................ ................................ .......... 15 Disease Prev ention ................................ ................................ .......................... 15 Bound Phytochemicals ................................ ................................ ..................... 16 Antioxidants ................................ ................................ ................................ ............ 16 Disease Preven tion ................................ ................................ .......................... 17 Antioxidant Location in Wheat ................................ ................................ .......... 18 Fiber and Disease Prevention ................................ ................................ ................. 18 Food Processing and Antioxidant Activity ................................ ............................... 20 Studies Measuring Bioavailability ................................ ................................ ........... 20 3 MATERIALS AND METHODS ................................ ................................ ................ 26 Composition of Study Materials ................................ ................................ .............. 26 DPPH Assay ................................ ................................ ................................ ........... 26 Subject Description ................................ ................................ ................................ 27 Study Design ................................ ................................ ................................ .......... 27 Blood Collection ................................ ................................ ................................ ...... 28 Serum Collection ................................ ................................ ................................ .... 28 Blood Cell Separation ................................ ................................ ............................. 28 Culture of PBMC for DNA Strand Breaks ................................ ................................ 29 Flow Cytom etry ................................ ................................ ................................ ....... 31 Statistical Analysis ................................ ................................ ................................ .. 31

PAGE 6

6 4 RESULTS ................................ ................................ ................................ ............... 33 5 DISCUSSION ................................ ................................ ................................ ......... 39 APPENDIX A COMPOSITION OF STUDY MATERIALS ................................ .............................. 41 B FLYER FOR RECRUITING PARTICIPANTS ................................ ......................... 42 C INCLUSION/EXCLUSION EVALUATION TO DETERMINE PARTICIPANT ELIGIBILITY ................................ ................................ ................................ ............ 43 D STUDY EMAIL ................................ ................................ ................................ ........ 44 E INFORMED CONSENT ................................ ................................ .......................... 45 F FINAL QUESTIONNAIRE ................................ ................................ ....................... 56 LIST OF REFERENCES ................................ ................................ ............................... 57 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 62

PAGE 7

7 LIST OF TABLES Table page 3 1 Volume of ascorbic acid, Milli Q water, and DPPH added to each tube and final ascorb ic acid concentration in mM. ................................ ............................. 32 4 1 Subject demographics. ................................ ................................ ....................... 35 4 2 Side effects and effects on satiety seen in participants after consuming wheat bran cereal for 21 days. ................................ ................................ ........... 36 4 3 Number of subjects with side effects in group A, group B, total and percent of the total. ................................ ................................ ................................ .............. 37

PAGE 8

8 LIST OF FIGURES Figure page 1 1 Breakdown of phytochemicals. ................................ ................................ ........... 24 1 2 Chemical structure of ferulic acid. ................................ ................................ ....... 25 3 1 Study design. ................................ ................................ ................................ ...... 32 4 1 Flow chart presenting the number of subjects at each stage of the study. ......... 35 4 2 Bar chart comparing percent apoptotic cells (mean SD) on day 21 seen in the groups consuming one (n = 21) or two (n = 20) servings of cereal. Statistical comparisons were determined using a t test (p = 0.255). .................. 37 4 3 Box plot comparing the median values of the percent of apoptotic cells seen in the baseline (n = 43) and day 21 (n = 41) blood draws. Statistical comparisons were determined using a Mann Whitney Rank Sum test on medians due to unequal variance (p = 0.837). ................................ ................... 38

PAGE 9

9 LIST OF ABBREVIATION S g Microgram L Microliter AA Ascorbic acid BMI Body mass index BSA Bovine serum albumin CO 2 Carbon dioxide DNA Deoxyribonucleic acid DPPH 2,2 dip henyl 1 picrylhydrazyl dUTP Deoxyuridine triphosphate EDTA Ethylenedidaminetetraacetic acid FITC Fluorescein isothiocyanate g Gram H 2 O 2 Hydrogen peroxide LPS Lipopolysaccharide mL Milliliter mM Millimolar NaCl Sodium chloride NSAIDS Non steroidal anti infl ammatory drugs PBMC P eripheral blood mononuclear cells PBS Phosphate buffered saline PI Propridium iodide RNase Ribonuclease TE Trolox equivalents TdT Terminal deoxynucleotidyl transferase

PAGE 10

10 TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling USDA United States Department of Agriculture

PAGE 11

11 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 WHEAT BIOAC TIVES AND ANTIOXIDANT ACTIVITY By Michelle Smith December 2011 Chair: Susan S. Percival Major: Food Science and Human Nutrition The objectives of this pilot study were (1) to show that consumption of wheat bran cereal by normal, healthy human subjects will provide antio xidant protection as measured by DNA strand breaks and (2) to determine if there is a dose response in cereal consumption. Following a baseline blood draw, 44 volunteers were randomized to consume either one serving (50g) or two servings of wheat bran cereal for 21 days at which time a second blood draw was performed. The antioxidant protection of the phenolic compounds was determined in peripheral blood mononuclear cells by measuring DNA strand breaks using the terminal deoxynucleotidyl t ransferase dUTP nick end labeling assay. If the antioxidants in the cereal were bioavailable, then the DNA will show fewer strand breaks in the form of a smaller percentage of apoptotic cells when the cells are cultured with H 2 O 2 The results showed that t here was no apparent protection of lymphocyte DNA from oxidative stress. Reasons for this may be poor bioavailability, however, the study was not designed to observe lack of bioava ilability. Phenolic acids are bound to large amounts of fiber found in the cereal product. This binding could prevent absorption of the phenolic acid s, keeping them from exerting their

PAGE 12

12 antioxidant effects in the body. Other reasons may include variability in DNA strand breaks among subjects.

PAGE 13

13 CHAPTER 1 INTRODUCTION Background Unti l recently, the phenolic compounds providing antioxidant activity in fruits and vegetables have received more attention than the phenolics in grains. This may be due to the underestimation of the amounts of phenolic compounds contained in grains. Most of t he phenolic compounds found in grains are bound to the cell wall. The refining process of wheat removes the wheat bran, therefore removing the phenolic compounds found there. The emphasis on eating whole grains has now been increased in the Guidelines but Americans continue to over consume refined grains and fall short on whole grain intake [1] Specific Aims Our proposed aim is to show that wheat bran cereal has important bioactive compound s that confer antioxidant protection to DNA in the f orm of reduced strand breaks. We will study two serving sizes of the cereal to determine if there is a dose response in the protection of DNA of the PMBCs Specifically, Our objective is to show that consumption of the wheat bioactives in the cereal in n ormal, healthy human subjects will provide that antioxidant protection to their DNA. The antioxidant protection will be determined by measuring DNA strand b reaks using the TUNEL assay. If the antioxidants in the cereal are bioavailable, then the DNA will s how fewer strand breaks when cells are cultured with H 2 O 2 Another objective is to determine if a greater amount of cereal consumption will have a greater impact on protecting against DNA stand breaks if the cereal does indeed provide antioxidant protectio n.

PAGE 14

14 Research on the bioavailability of the compounds in this cereal is limited. Positive results from this study could benefit consumers by knowing that their cereal is a source of antioxidants that can easily be consumed on a regular basis. Hypothesis Wh eat bran phenolics have been shown to have positive effects on health. If there are bioavailable phenolic compounds in the wheat bran cereal, then they may provide antioxidant protection in the form of protection against oxidatively induced DNA strand brea ks.

PAGE 15

15 CHAPTER 2 LITERATURE REVIEW Whole Grains The American Association of Cereal Chemists have defined a whole grain as components, the starchy endos perm, germ, and bran, are present in substantially the same relative proportions as they exist This means that for products such as flour to be considered whole grain, the three major components of bran, germ, and endosperm mu st be present in the same amounts that occur in t he 0 Dietary Guidelines for Americans have established that at least half of grain foods consum ed daily should be whole grain [3] Reasons for this include that the bran and germ fractions provide a majority of the biologically active compounds found in a grain, some of which are unique to grain products and are not foun d in other plant foods [4] Disease Prevention Epidemiological studies have shown associations between higher in takes of whole grain products and lower risk of chronic diseases such as heart disease [5, 6], diabetes [7 9], and cancer [5, 10 13] Intakes of refined grains have not shown these associations. One cause of this may be that refined grains do not contain t he bran and germ layers that are seen in whole grain products which are known to contain fiber, vitamins, minerals, and antioxidants [14] One study [15] looked at the antioxidant content of grain products, fruits, and vegetables purchased at a local groce ry store. Antioxidant content was determined using the DPPH reaction. They found that whole grain bread was 2000 Trolox equivalents (TE), compared to white bread at 1200 TE,

PAGE 16

16 indicating the contribution of bran and germ to antioxidant activity. The research ers believe that whole grain products contain biologically active antioxidants that could act independently or synergistically and/or additively with the fiber found in the whole grain provide health benefits beyond basic nutrition to reduce the risk of di seases. Bound Phytochemicals Foods commonly known to have antioxidant activity are fruits and vegetables, with little attention given to grains. This same study [15] found that the average antioxidant activity of cereal products is equal to or exceeds mos t vegetables or fruits. A possible reason for these findings is that this study took into account the bound phytochemicals producing antioxidant activity found in grains as well as the free phytochemicals producing antioxidant activity Studies before thi s did not measure bound phytochemicals and therefore it was reported that grains had lower antioxidant activity than fruits and vegetables. It has been found that in wheat, 90% of the antioxidants are bound [16] It is believed that bound wheat phenolics a ssociated with the cell walls may be resistant to upper gastrointestinal tract digestion and finally reach the colon, where colonic digestion by intestinal microflora may release the b ulk of the bound phenolics [17] These results show that while fruits an d vegetables have been found to be an important source of antioxidants, whole grains should be given more attention for their potential antioxidant activity. Antioxidants Antioxidants are molecules that react with and destroy free radicals, preventing the m from inflicting oxidative damage or mutation to vital components such as DNA or cell membranes [14, 18] Free radical compounds result from normal metabolic activity as well as from the diet and environment [1 4 ] Families of endogenous antioxidant

PAGE 17

17 enzyme s have evolved including superoxide dismutases for the elimination of the superoxide radical, and catalases and glutathione peroxidases for the elimination of hydrogen peroxide and organic peroxides [18] One example of an endogenous antioxidant is glutath ione. The most important antioxidant function of glutathione is to remove hydrogen peroxide and organic peroxides catalyzed by the selenium dependent enzyme glutathione peroxidase, forming water or alcohol, respectively [19]. E ndogenous antioxidant defense s can be supported by dietary antioxidants for maintaining health. Disease Prevention Several epidemiologic studies have shown that dietary antioxidants reduce the risk of many diseases and conditions including Parkinson disease [20 ] disease [2 1 ], myocardial infarctions [2 2 ], and coronary heart disease [2 3 ]. There are many individual compounds that contribute to the overall antioxidant activity of grains. They fall under the term phytochemical which is defined as a plan t based substance that ma y have a beneficial effect in the body but is non essential. Phytochemicals can be classified as nitrogen containing compounds, polyphenolics, terpenes, or organosulfur compounds as seen in Figure 1 1. N umerous studies [2 4 3 3 ] have found that ferulic aci d (Figur e 1 2 ) is the dominant phenolic acid in wheat accounting for 46 70% of the total phenolic acids in wheat on a per weight basis [24 ] As mentioned before, most of the antioxidants in wheat are in the bound form and ferulic acid is no exception. One study [17] found that bound ferulic acid contributed more than 97% of total ferulic acid for all wheat varieties tested. This was i n agreement with a later study [3 4 ] that found 95.8% of ferulic acid was found in the bound form and 4.2% in the free form in wheat bran.

PAGE 18

18 Other phenolic acids found in wheat are syringic, caffeic, vanillic and p coumaric acids [3 5 ] It is thought that there are other antioxidants in wheat that may act synergistically and/or additively with the phenolic acids to produce a strong er antioxidant activity of the wheat. These antioxidants are known to be carotenoids such as lutein [17, 2 4 2 5 ] and tocopherols [2 4 ] Antioxidant Location in Wheat Additional research [3 6 ] was done to further determine whether it was the bran, the germ, or the whole grain of wheat together that provided the most antioxidant activity. It was found that the polyphenolics in the bran/germ fraction was 15 to 18 fold higher when compared to the corresponding endosperm samples. They also looked at ferulic acid content and found that it was 50 to 70 fold higher in bran/germ fractions when compared to the endosperm fractions. This further shows that the processing of wheat is likely to be important when determining its antioxidant activity. Other research [2 5 2 8 3 5 3 7 3 8 ] found that the bran in particular had the most antioxidant activity than other products from the same species. They hypothesize this is due to the localization of the phenolic acids in the grain with the outer layers of the grain containing the greatest concentrations of total phenolic acids. Fiber and Disease Prevention Studies have shown that consuming foods such as whole grains that are high in fiber helps to reduce the risk of oral, pharyngeal, and esophageal cancers [3 9 ] and coronary he art disease [40 ] Fiber is believed to play a significant role in the utilization of the phenolic acids found in wheat bran as most of the antioxidants are associated with [3 7,41 ] It is th ought that the antioxidants in wheat bran can act independently or synergisticall y and/or additively

PAGE 19

19 with fiber to reduce disease [15] but it is difficult to separate the potential effect of fiber from that of the other components of the wheat bran. This c ould explain data sho wing that it is much better for the health of the human body to consume the dietary fiber as part of whole fiber rich foods with respect to the intake of only purified fiber, tablets, pills, and other medical pr eparations [37 ] It is because of the high amounts of fiber in bran that scientists question the bioavailability of the phenolics bound to this fiber. As stated before, it is possible that there is a synergistic and/or additive relationship with bound antioxidants and fiber. Ant ioxidants that are covalently bound to cell wall fiber can be transported to the large intestine to be released by fermentation in the presence of microbiota [4 2 ]. In fact, one in vitro study [4 3 ] using a model gut system to examine the release of ferulic acid from wheat bran shows that, over 95% of the total release of ferulic acid groups takes place during fermentation in the colon. Enzymatic hydrolysis in the colon can free phenolics such as ferulic acid to release the free acid for absorption into the e pithelium [4 2 4 3 ] This allows whole grain foods to provide antioxidant protection over a long time period through the entire digestive tract which provides unique protection that is not possible by any single component. Numerous researchers [2 9 3 7 4 4 ] believe that it is the complex mixture of phytochemicals in foods that provides better protective health benefits than individual isolated components through the combination of additive and/or synergistic effects. Previous studies [4 5 4 6 ] have even shown a detrimental effect on disease when using only the concentrated antioxidant to prevent disease. This further shows why for some

PAGE 20

20 antioxidants, it may be more beneficial to consume the dietary antioxidant in the food it is naturally found in so it can exer t the positive effects. Food Processing and Antioxidant Activity Although foods have been shown to have antioxidant activity, it is thought that the processing they go through to create foods such as breakfast cereals will reduce their antioxidant ac tivity According to one study [4 2 ] the reverse is true. Antioxidant activity increased gradually during cooking steps of breakfast cereal processing with the biggest increase coming from toasting. It appears that there may be no loss of natural antioxidants wh ile there is a format ion of new antioxidant activity, most likely Maillard reaction products. This is demonstrated by the comparison of crust antioxidant activity to that of crust free white bread. The crust was about double in antioxidant activity compare d to the crust free part showing little change in antioxidants in the interior of the bread loaf during baking. Maillard reaction products may explain the increase in crust antioxidant activity. Studies Measuring Bioavailability Few studies have examined the influence of the food matrix on the bioavailability of phenolic acids such as f erulic acid. One study by Adam [4 7 ] investigated the bioavailability of ferulic acid in rats in a complex wheat bran cereal matrix. The rats were fed the experimental diet f or 21 days. They found that the recovery of ferulic acid in urine was quite limited and they hypothesize that the cereal matrix appears to severely limit ferulic acid bioavailability. Another study [34 ] on the influence of the cell wall linkage on the bio availability of ferulic acid of an oral short term intake of wheat bran mixed with a standard diet in rats was evaluated There were three groups, the first received standard food, the second

PAGE 21

21 received standard food and pure ferulic acid, and the third rece ived standard food and bran. There was a fast appearance of ferulic acid in plasma after intake of pure ferulic acid. This could be explained by a fast absorbance of the compound in the jejunum or maybe in the stomach which was shown for other antioxidants found in grains. There was also an early appearance of ferulic acid in the bran group but only a small amount compared to the free ferulic acid group. This could be explained by the fact that only 4.2% of ferulic acid in bran is in the free form, the rest is bound. Over 24 hours, the amount of ferulic acid in the plasma remained constant in the bran group whereas it decreased to zero after only 4 hours in the free ferulic acid diet The antioxidant activity was better after consumption of the bran than the pure ferulic acid. This result emphasizes that ferulic acid alone cannot fully explain the antioxidant activity of plasma. This further indicates how the components of wheat bran may act synergistically and/or additively to produce antioxidant activity an d supplementation with wheat bran seems more efficient than a supplementation with pure ferulic acid. Kern [48 ] investigate d the bioavailability of ferulic acid from high bran wheat and determine if some of the covalently bound forms of this phenolic were absorbed. The study used six human volunteers that underwent a two day low polyphenol diet. Volunteers were asked to consume 100 grams of a commercial breakfast cereal and their blood was collected 1, 3, 6, 9, and 12 hours after the test meal. Volunteers were asked to collect urine for 24 hours on the day prior to the study day and throughout the study day. The results show a maximum absorption of ferulic acid between 1 and 3 hours after the test meal. This suggests that absorption of ferulic acid from the high bran breakfast cereal occurs primarily in the small intestine. In this study, the low levels

PAGE 22

22 of ferulic acid found in the plasma 6 hours after consumption of the cereal are indicative of little or no absorption from the large intestine. If ferulic ac id from cereal is released into the colon, it is more likely to be further metabolized by the microflora or excreted via feces indicating that bound ferulic acid is either not absorbed or absorbed only in very small amounts. A recent human study [4 9 ] look ed at the effect of bioprocessing the bran in whole wheat bread on the bioavailability of phenolic acids, the postprandial plasma antioxidant capacity, and ex vivo anti inflammatory properties. After consumption of a low phenolic diet for three days and an overnight fasting, eight healthy men consumed 300 grams of whole wheat bread containing native bran, the control, or bioprocessed bran, the enzymatically treating it with cell wa ll degrading enzymes. This was a randomized, blind cross over study. Their results showed the bioavailability of ferulic acid from the bioprocessed bread was about 3 fold higher than that of the control bread. They also found that the absorption of ferulic acid from the bioprocessed bread mainly took place in the small intestine although a large proportion of ferulic acid is known reach the colon bound to fiber. The researchers then looked to see if there were any immunomodulatory effects as a result of con suming the bioprocessed wheat bran. B lood samples were taken before bread ingestion and then at 1.25, 6, and 12 hours after bread ingestion. The blood was incubated with LPS from Escherichia coli in a final concentration of 1 g/L for 24 hours at 37C and 5% CO 2 to stimulate an inflammatory response. When looking at the pro /anti inflammatory cytokine ratios (IL 6:IL 10 and IL 10) the researchers found that compared with the control bread, the bioprocessed bread led to

PAGE 23

23 a lower ratio in the ex vivo L PS stimulated blood. Looking at these results, it can be concluded that the bioprocessing of bran increases the bioavailability of ferulic acid from whole wheat bran and it may have immunomodulatory effects. This type of study results could prove useful to optimize a food such as bread or cereal for the prevention of diet related diseases as well as other chronic diseases that could be prevented by increased phenolic acid absorption. To our knowledge, no study has been do ne to assess the effect of wheat br an cereal on oxidative stress in the human body. Wheat bran has high antioxidant activity in vitro so it is plausible that consuming wheat bran in the form of a ready to eat breakfast cereal may provide antioxidant protection for the cells of the body. The refore, the purpose of the proposed study is to demonstrate whether wheat bran cereal supplemented in the diet will lead to increased antioxidant protection in healthy human subjects.

PAGE 24

24 Figure 1 1. Breakdown of phytochemicals.

PAGE 25

25 Figure 1 2. Chemical structure of ferulic acid.

PAGE 26

26 CHAPTER 3 MATERIALS AND METHOD S Composition of Study Materials Wheat bran cere cereal used in this study. The Nutrition Label and ingredients can be found in Appendix A DPPH Assay A stock solution of 2,2 d iphenyl 2 picryl hydrazyl (DPPH) (Sigma, St. Louis, MO ) was prepa red in methanol (0.0049 g DPPH in 50 mL methanol at 0.25mM). A stock solution of ascorbic acid (Sigma, St. Louis, MO) was prepared in Milli Q water (0.011 g ascorbic acid in 50 mL Milli Q water). Tubes were lab eled 1 through 6. Milli Q water and ascorbic a cid were added to corresponding tubes in the amounts shown in Table 3 1. Fresh squeezed orange juice from Valencia oranges was centrifuged. The supernatant of the orange juice was collected and diluted in Milli Q water (30 L orange juice, 70 L Milli Q wa ter) and placed in a separate tube The wheat bran cereal was ground with a mortar and pestle. The ground cereal was weighed and 0.1 g was collected in a tube. Added to the cereal tube was 2 mL of methanol. The cereal tube was vortexed, sonicated, and cent rifuged (1600 rpm, 3 minutes). The supernatant was removed and place in a separate tube. From each tube (tubes labeled 1 6, the orange juice, and the wheat tubes) 10 L was transferred to a 96 well plate in triplicate. Next, 40 L of DPPH was added to the wells and mixed with the same pipette tip (50 L total volume). The plate was incubated for 20 minutes. Absorbance was read at 517 nm using a SPECTRAmax (M olecular Devices, Sunnyvale, CA) spectrophotometer.

PAGE 27

27 Subject Description Healthy adult males and fema les between the ages of 18 and 50 years were recruited to participate in a 21 day pilot study. Subjects were recruited via flyer (Appendix B ) word of mouth, and a listserv announcement from the University of Florida campus, and the Gainesville, Florida co mmunity during June of 2010. The study the study occurred by telephone and/or personal interviews. Exclusion criteria (Appendix C ) included the use of dietary supplements (not including multivitamins), strict vegetarian or vegan diet, use of antibiotics chronic use of NSAIDS, ongoing illness or infection, females that are pregnant or planning to become pregnant, and BMI greater than 3 5 A study email (Appendix D) was sent to those who were eligible with the w ritten informed consent (Appendix E ) attached so they may read it and discuss it with family and physicians if needed. Written consent was obtained from each subject Subjects were randomly placed into one of two treat ment grou ps. Group A subjects were instructed to consume one bag (50 g) of cereal per day and group B subjects were instructed to consum e two bags (100 g) of cereal per day. Subjects were told to return any uneaten bags of cereal at the end of the study. S tudy Design On day 1, the subjects were asked to come in to sign the informed consent and give a fasting baseline blood draw. They were then given their cereal and instructed to consume it corresponding with their g roup. Since the cereal is very fibrous, to prevent too much gastrointestinal distress, the subjects were told to eat the cereal throughout the day until their bodies adjusted.

PAGE 28

28 On day 21, when the study participants arrived for their final blood draw, a questionnaire (Appendix F ) was used to ev aluate compliance. Compliance was also assessed by the returned cereal bag count. The exit questionnaire also included questions to determine if the subjects experience any side effects, changes in weight or changes in appetite that would be attributed to the cereal. The study design can be seen in Figure 3 1 and includes the start and end dates. Blood Collection Fasting blood was collected into a serum tube and a PBMC tube on day 1 (baseline) and day 21. Blood was collected into one 10 mL sodium heparin t ube for PBMC separation, and one 10 mL SST TM tube (Vacutainer Becton Dickinson, Franklin Lakes, NJ) for serum. Tubes for PMBC were maintained at room temperature and the serum tubes were placed at 4 C. Both tubes were processed within one or two hours aft er collection under sterile conditions. Serum Collection Serum was removed from SST TM tubes after centrifugation (2000 g, 10 minutes 4 C ) and 400 L was added to 3.6 mL RPMI 1640 (Cellgro Mediatech, Herndon, VA) complete (100 U/mL Penicillin; 100 g/mL S treptomycin; 0.25 g/mL Fungizone; 50 g/mL Gentamycin; 2 mM 1 glutamine; 25 mM HEPES) to prepare culture medium with 10% autologous serum. This was stored at 4 C until use. Blood Cell Separation Whole blood was diluted and placed on a gradient to separate PBMC. The blood was diluted 1:1 with 0.9% NaCl. Diluted blood (6.5 mL) was layered over 3 mL of L ympholyte H cell separation medium (Cedarlane Diagnostics, Burlington, Ontario) and

PAGE 29

29 centrifuged (800 g, 20 minutes 20 C ). Using a fine tip sterile transfer pipette the PBMC band was removed from the gradient tube washed twice with RPMI 1640 complete by centrifugation ( 400 g 10 minutes 4 C ). Individual cell pellets were resuspended in 2 mL RPMI 1640 complete without serum and counted on a Z 1 S Particle Cou nter (Beckman Coulter Brea, CA) at 1:1000 dilution (10 L cells + 10 mL Isoton) An example of the PBMC band can be seen in Figure 3 2 Culture of PBMC for DNA Strand Break s On day 1, 2 .0 x 10 6 cells/mL in RPMI with 10% autologous serum were plated into a 12 well non treated plate. Added to each well was 5.5 L of 30% hydrogen peroxide to achieve a final concentration of 25 mmol/L The plate was incubated in a humidified 5% CO 2 atmosphere at 37 C for 2 hours. After incubation, the cells were transferred to a 15 mL conical tube. Each well was washed with 5 mL PBS twice, with the cells being transferred to the 15 mL tube. A last wash with 1 mL PBS was done and added to the 15 mL conical tube. This tube was centrifuged (800 g, 12 minutes, 4 C) and then aspirated being careful to avoid the cell pellet. The pellet was resuspended in 5 mL cold PBS and vortexed to ensure washing. The tube was again centrifuged (800 g, 12 minutes, 4 C), aspirated, resusp ended in 5 mL cold PBS, and vortexed. Another round of centrifuging (800 g, 12 minutes, 4 C) and aspirating occurred followed by resuspending the pellet in 1mL cold PBS and vortexing. The cells were then fixed by adding 1 mL cold 2% paraformaldehyde to the 1 mL suspension The tubes were incubated at 4 C for 20 minutes. After the incubation, the tubes were centrifuged (800 g, 12 minutes, 4 C), aspirated, resuspended in 5 mL cold PBS, and vortexed. This process was repeated two more times, with the second ti me using 1.5 mL cold PBS. After being

PAGE 30

30 vortexed, the cell membranes were permeabilized by adding 3.5 mL ice cold neat ethanol dropwise while vortexing each tube. The cell suspensions were stored at 20 C. The cells were processed using the ApoAlert TM DNA F ragmentation Assay Kit ( BD Biosciences Clonetech, Palo Alto, CA ) for flow cyto metric analysis This assay is based on the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling ( TUNEL) assay. Terminal deoxynucleotidyl transferase catalyzes t he incorporation of fluorescein hydroxyl ends of fragmented DNA. The tubes were removed from the freezer and centrifuged (800 g, 12 minutes, 4 C). The supernatant was aspirated, the cells were resuspended in 5 mL cold PBS, and then vortexed. The tubes were again centrifuged (800 g, 12 minutes, 4 C), aspirated, resuspended in 1 mL cold PBS, and vortexed again. These cell suspensions were transfer red to a 5 mL round mL conical tube was washed with 1 mL PBS again and transferred to the snap cap tube. From this point forward the cells were protected from light. The cells were centrifuged (800 g, 12 minutes, 4 C) aspirated, resuspended in 50 L equilibration buffer, and vortexed. The cells were equilibrated at room temperature for 5 minutes. Next, the cells were centrifuged (800 g, 12 minutes, 4 C), aspirated, resuspended with 26 L TdT incubation buffer, and v ortexed. The cells were incubated in a closed 37 C water bath for 60 minutes with gentle tapping every 15 minutes to mix. After incubation, 0.5 mL of 20 mM EDTA was added to terminate the reaction and then vortexed. The cells were centrifuged (800 g, 12 mi nutes, 4 C), aspirated, resuspended with 1 mL of 0.1% Triton X 100/BSA/PBS (5 mL PBS; 0.2% Triton X 100; 50 mg BSA) and vortexed. This was repeated first with the 1 mL of 0.1% Triton X 100/BSA/PBS, then with 0.25 mL propidium iodide /RNase/PBS

PAGE 31

31 (2.5 g/mL P I; 0.5 g/mL DNase free RNase) The tubes were incubated at room temperature for 30 minutes and then store in the dark at 4 C for same day flow cytometry analysis. Flow Cytometry The fluorescein labeled DNA was quantified by flow cytometry. This was done within 24 hours using the FACSort instrument located in the Flow Cytometry laboratory at the Interdisciplinary Center for Biotechnical Research. The populations were gated to omit debris and d ata was analyzed as a percent of apoptotic cells using FlowJo ve rsion 7.6.1 software. Apoptotic cells were determined by looking at the PI negative fluorescein isothiocyan a te positive (FITC+) quadrant. Statistical Analysis All statistics were performed using SigmaStat, version 11.0 Systat Software. A t test was perfo rmed on the data to look at the difference in the percent of apoptotic cells and serving size No ting no statistical difference in response between the two groups, all subjects were pooled together for further statistical analysis. An unpaired t test was r un to look at the difference in the percent of apoptotic cells from day 0 to day 21. When variances were unequal, the data was analyzed using a Mann Whitney Rank Sum test.

PAGE 32

32 Table 3 1. Volume of ascorbic acid, Milli Q water, and DPPH added to each tube and final ascorbic acid concentration in mM. Tube Ascorbic Acid (AA) Milli Q Water DPPH AA mM Concentration 1 0 L 100 L 40 L 0.000 2 10 L 90 L 40 L 0.089 3 20 L 80 L 40 L 0.178 4 30 L 70 L 40 L 0.268 5 40 L 60 L 40 L 0.357 6 50 L 50 L 40 L 0.446 Figure 3 1. Study design.

PAGE 33

33 C HAPTER 4 RESULTS The DPPH radical scavenging activity of the fresh squeezed orange juice was calculated to be 113.66 mM ascorbic acid equivalents (AAE). The DPPH radical scavenging activi ty of the ground wheat bran was calculated to be 0.366 mM AAE Fifty subjects were recruited and assessed for eligibility S ix participants were excluded for not meeting the age and illness inclusion criteria. Of those 44 subjects (18 males, 26 females) 4 3 showed up for the first blood draw, their demographics are listed in Table 4 1. Forty one of those 43 subjects cam e back for the final blood draw. One withdrew because of mild a dverse effects such as gastrointestinal distress. One did not show up to the final blood draw and was withdrawn. This is shown in Figure 4 1. The leftover bags were collected, counted, and compared with the total number of days missed eating the cereal as noted on the final questionnaire. A total of 41 participants consumed the wh eat bran cereal 810 out of the 861 total days This yields 94% compliance among the study participants. When looking at the responses on the final questionnaire, the participants listed five different side effects they experienced that they attributed to e ating the cereal. They were increased bowel movements, bloating, gas, stomach discomfort, and increased thirst. Increased bowel movements were listed by 19.5% of the subjects (4 people in group A, 4 people in group B). Bloating was listed by 12% of the sub jects (4 people in group A and 1 person in group B ). Gas was listed by 7% of the subjects (3 people in group A, none in group B) Stomach discomfort was listed 10% of the subjects (3 people in group A and 1 person in group B ). Increased thirst was listed b y 7% of the subjects. (3 people in group A none in group B) When asked if they noticed any changes in their

PAGE 34

34 satiety after consuming the cereal, 66% of the subjects (14 people in group A and 13 people in group B) said they felt full and ate less. There w as no significant difference observed when comparing the percent of apoptotic cells between group A consuming one serving of cereal and group B consuming two servings of cereal which is shown in Figure 4 2. After making this observation, both groups were p ooled together to compare the percent of apoptotic cells in the blood draws for day 0 and day 21. Figure 4 3 shows that the percent of apoptotic cells seen in the baseline and day 21 blood draws were not significantly different ( p = 0.837 ).

PAGE 35

35 Table 4 1. Subject demographics. Total 1 Serving 2 Servings Mean Age (years) 26.4 23 26.3 Age Range (years) 18 43 21 43 18 41 Males 18 9 9 Females 26 13 13 Figure 4 1. Flow chart presenting the number of subjects at each stage of the study.

PAGE 36

36 T able 4 2. Side effects and effects on satiety seen in participants after consuming wheat bran cereal for 21 days. Subject Side Effects Effect on Satiety Subject Side Effects Effect on Satiety 1 none No effect 23 Bloating Ate less 2 Increased bowel movem ents No effect 24 none Full 3 Bloating Ate less 25 none No effect 4 none No effect 26 Increased bowel movements Ate less 5 Bloating Thirsty Full 27 none No effect 6 Increased bowel movements No effect 28 Increased bowel movements Full 7 none Full 29 n one Full 8 increased bowel movements Ate less 30 Increased bowel movements Ate less 9 Gas Thirsty Full 31 none Full 10 none Ate less 32 none No effect 11 Bloating Stomach Discomfort Ate less 33 none Full 12 none Full 34 Stomach discomfort Ate less 1 3 none Ate less 35 none Ate less 14 Stomach Discomfort Gas Ate less 36 Increased bowel movements No effect 15 Thirsty Ate less 37 WITHDRAWN 16 Bloating Gas Ate less 38 none Ate less 17 none No effect 39 none No effect 18 none No effect 40 none Ate l ess 19 none No effect 41 none Ate less 20 none No effect 42 none Full 21 WITHDRAWN 43 Increased bowel movements Stomach discomfort Full 22 none No effect 44 WITHDRAWN

PAGE 37

37 Table 4 3. Number of subjects with side effects in group A, group B, total and p ercent of the total Figure 4 2. Bar chart c omparing p ercent apoptotic cells (mean SD) on day 21 seen in the groups consuming one (n = 21) or two (n = 20) servings of cereal Statistical comparisons were determined using a t test (p = 0.255 ). Side Effects One Serving Two Servings Total (Percent) Increased Bowel Movements 4 4 8 ( 19.5% ) Bloating 4 1 5 ( 12% ) Gas 3 0 3 ( 7% ) Stomach Discomfort 3 1 4 ( 10% ) Thirsty 3 0 3 ( 7% ) Full/Ate Less 14 13 27 ( 66% ) 1 Serving 2 Servings

PAGE 38

38 Figure 4 3. Box plot comparing the me dian values of the percent of apoptotic cells seen in the baseline (n = 43) and day 21 (n = 41) blood draws. Statistical comparisons were determined using a Mann Whitney Rank Sum test on medians due to unequal variance (p = 0.837 ). Day 0 Day 21

PAGE 39

39 CHAPTER 5 DISCUSSION Die tary antioxidants reduce the risk of many chronic diseases. Previous literature suggests that they are mainly found in fruits and vegetables. Emerging studies confirm that wheat bran contains phenolic acids that can offer extensive antioxidant benefi ts to the consumer. However, the ir binding to fiber ma y impact their bioavailability. The results of the DPPH assay show that there is antioxidant activity in wheat bran cereal. When compared to the amount seen in fresh squeezed orange juice it is quite small. The effect of wheat bran cereal on oxidative stress in vivo has not been investigated. The purpose of this pilot study was to see if the antioxidants in the wheat bran would provide protection to healthy human subjects when consumed as a cereal. In the pr esent study, forty four subjects were randomized to one of two groups with different serving sizes of cereal. Their blood was drawn at day 0 and they were asked to consume their cereal each day for three weeks and come back to give a final blood draw. As s een in Figure 4 3, significant differences were not found between the percent of apoptotic cells when consuming one or two servings of cereal. All results were then pooled together to compare the percent of apoptotic cells in the blood draws for day 0 and day 21. Figure 4 3 shows no significant difference between the baseline blood draw and the final blood draw. While this data does not show increased antioxidant activity after consuming the wheat bran cereal, it cannot be definitively stated that the whea t bran cereal does not contain antioxidants that may provide antioxidant activity. There were some disadvantages to this study. Compliance was measured by having the subjects report how many days they did not eat the cereal as well as having them returned unused

PAGE 40

40 bags of cereal. It is possible that some of the participants were not entirely truthful. They should have had an extra bag of cereal and some did not bring back any bags stating they ate the cereal every day. Also, looking at the reports of fullness after eating the cereal, you would expect to see more reports of being full after the eating the cereal in group B who had two servings of cereal compared to group A. This was not the case which may mean poor compliance of study participants Another limitation related to the purpose of this study is the variability in DNA strand breaks among subjects. To correct for this, further studies could include the use of a control cereal containing low fiber and a low amount of antioxidants. A crossover study design would reduce the influence of confounding covariates because each crossover patient would serve as their own control.

PAGE 41

41 APPENDIX A COMPOSITION OF STUDY MATERIALS

PAGE 42

42 A PPENDIX B FLYER FOR RECRUITING PARTICIPANTS

PAGE 43

43 APPENDIX C IN CLUSION/EXCLUSION EV ALUATION TO DETERMIN E PARTICIPANT ELIGIB ILITY Inclusion/Exclusion Evaluation P.I. Dr. Percival and Suzanna Bonard These q uestions are for evaluating inclusion and exclusion for purposes of enrollme nt in the study. These answer s to these questions are not recorded in association with an individual identification. Names and telephone numbers are kept once their eligibility to enroll is established. The participant is then assigned a number that is used exclusively for identification while their name and phone number (the key to their ID number) remain in a locked file cabinet within a Introductory Statement: [ Thank you for calling] (or) [We are returning your call] abo ut a research study we will be doing at th e University of Florida in the D epartment of Food Science & Human Nutrition. The purpose of the study is to evaluate the effects of a wheat bran cereal on human immu ne function and to determine if bioactives in wh eat bran increase antioxidant activity in humans. Particip a tion in this study would last 21 days and will require 2 visits to our lab. You will be required to consume cereal daily for 21 days and come to the lab for blood draws between 8:00a.m. 9 :00a.m. after fasting overnight. To see if yo u might qualify for this study, I need to ask you some questions about your health history and present condition. [for female participants} If you are pregnant or planning to become pregnant you should not participat e. I will now ask you about your age, height and weight, current health and medication. Questions: 1. Are y ou between the ages of 1 8 and 50 years old? Yes/no: yes is acceptable into the study 2. What is your current weight and height? BMI over 35 is not acc eptable only because we do not have the equipment to take blood pressure 3. Do you have any ongoing or chronic illness or infection ? Yes/no: no is acceptable 4. Are you on any of the following: antihypertensive medication, immunosuppressive drugs, antibiotics, or chronic use of NSAIDS? Yes/no; no is acceptable 5. Do you take any dietary supplements? If yes, are you willing to refrain from taking them during the 21 days of the study? [ Note: Subjects consuming a daily vitamin/mineral supplement with can be recruited into the study]. Yes/no: yes is acceptable 6. During the study, will you consume no more than 2 glasses of alcoholic beverages per day? Yes/no: yes is acceptable Thank you. You qualify for participation in the study. [an appointment locatio n, time and date are set] your interest.

PAGE 44

44 APPENDIX D STUDY EMAIL Dear If you could be so kind, please reply to this email to let me know that you have received i t and read it completely. Thank you again for volunteering to participate in our nutrition study on Wheat Bran Cereal. You are scheduled for your first visit on This first visit should take less than an hour. You will need to fast after midnight the night before (no food, however, you can and should drink water). We will call you to remind you on When you come in on morning, first you will have some paperwork to fill out. After that, we will take your blood pressure and your blood will be drawn by our phlebotomist. We will provide a grab and go breakfast for you. You will also receive cereal supply along with instructions and additional information regarding the study and subsequent visits. After consuming the cereal for 21 days, you will return t o the clinical lab for your second blood draw, complete your final questionnaire and return uneaten bags of cereal. If are not familiar with our location, I have attached a map. When you get to the Food Science & Human Nutrition Building please come to the North end of the building, enter through the double glass doors and take the elevator to the 2 nd floor, Room 227 If you have any problems on the morning of the study, please call 352 392 1991 ext. 287 I am also attaching an Informed Consent form fo r you to read over before you arrive for the study. Reading this in advance should shorten the time you are here during your visit. Note: Both attachments to this email are pdf documents. If you cannot open these files, please email me back and I will sen d them to you in a different format. If you have any questions about any of this information, please call me at 392 1991 x255 or email me at suzanana@ufl.edu Finally, if you know of anyone else who might be inter ested, please have them call us. With Thanks, Suzanna Bonard Graduate Student in University of Florida 449 Food Science & Human Nutrition Gainesville, FL 32611

PAGE 45

45 APPENDIX E INFORMED CONSENT I NFO RMED C ONSENT F ORM to Participate in Research, and A UTHORIZATION to Collect, Use, and Disclose Protected Health Information (PHI) I NTRODUCTION Name of person seeking your consent: Place of employment & position: This is a research study of how wheat bioactives may benefit immune function in humans. Could participating in this study offer any dir ect benefits to you? Yes as described on page 49 Could participating cause you any discomforts or are there any risks to you? Yes as described on page 48 Please read this form which describes the study in some detail. I or one of my co workers will also describe this study to you and answer all of your questions. Your participation is entirely voluntary If you choose to participate you can change your mind at any time and withdraw from the study. Y ou will not be penalized in any way or lose any benefits to which you would otherwise be entitled i f you choose not to participate in this study or to withdraw If you have questions about your rights a s a research subject, please call the University of Florida Institutional Review Board (IRB) office at (352) 846 1494. If you decide to take part in this study, please sign this form on page 54

PAGE 46

46 G ENERAL I NFORMATION ABOUT THIS S TUDY 1. Name of Participant ("Study Subject") ___________________________________________________________________ 2. What is the Title of this research study? Wheat Bioactives and Immune Function 3. Who do you cal l if you have questions about this research study? Dr. Susan S. Percival work: 352 392 1991 x217 cell: 352 562 9670 email: percival@ufl.edu 4. Who is paying for this research stud y? The sponsor of this study is Kelloggs Corporate Citizen's Fund 5. Why is this research study being done ? The purpose of this research study is t o evaluate the effects of wheat bioactives on human immune function and to determine if wheat bioacti ves provide antioxidant protection to DNA. You are being asked to be in this research study because you are a healthy individual between the ages of 21 and 50. W HAT C AN YOU E XPECT IF YOU P ARTICIPATE IN THIS S TUDY ? 6. What w ill be done as part of your normal clinical care (even if you did not participate in this research study )? Nothing will be done as part o f your normal clinical care because you are a healthy volunteer and therefore do not have any normal clinical care.

PAGE 47

47 Because this study is not related to your normal clinical care, your physician will not be informed that you are taking part in this study. 7. What will be done only because you are in this research study ? This is a 3 week long study. At the beginning of the study, you will first com plete the initial paperwork which includes this informed consent form and the form required to pay you, which should take about 10 minutes. A trained phlebotomist will obtain a sample of venous blood ( four teaspoons total i n two tubes) from you for an immune assessment, i f you decide to take part in this study You will be randomly assigned to consume either one serving (50 grams) or two servings (100 grams) of wheat cereal. You are required to fast a minimum of 6 hours pri or to the blood draw and it is preferable if you fast overnight. 1. You will be given a 3 week supply of your assigned cereal serving. For the first 4 days you will consume in the morning and at night. By the 5 th day you will consume each serving in its entirety. Water consumption should be maximized as well. 2. After 3 weeks of consuming the cereal, you will be asked to return to have a trained phlebotomist obtain a sample of venous blood ( four teaspoons total in two tubes) from you for immune assess ment. You are required to fast a minimum of 6 hours prior to the blood draw and it is preferable if you fast overnight. 3. Your blood pressure will be measured at both of your two (2) visits to the lab. If on your first visit, if either one of your blood p ressure values are high, defined as 140/90, you will not be allowed to participate. If you have any questions now or at any time during the study, please contact Dr. Susan S. Percival in question 3 of this form. 8. How long will you be in this research study? The total time commitment for this research is estimated to be 4 hours, over a 3 week period. Each of the two blood draw sessions (first day and at the end of 3 wee ks) is expected to last no more than 1 hour (equivalent to 2 hours total). You will be

PAGE 48

48 required to come to the Food Science and Human Nutrition building for the blood draws. You will consume either one serving (50 grams) or two servings (50) of cereal ev ery day for 3 weeks. The time to eat the cereal daily and to fill in the final questionnaire is expected to total approximately 2 hours. 9 How many people are expected to take part in this research study ? 40 people are needed to part icipate in this research study W HAT ARE THE R ISKS AND B ENEFITS OF THIS S TUDY AND W HAT ARE Y OUR O PTIONS ? 10 What are the possible discomforts and risks from taking part in this research study ? There are no risks associated with consuming one to two servings of cereal a day. The increased fiber may cause some discomfort, at first, in the form of gastrointestinal distress. This may include, but is not limited to bloatin g, cramps, and gas. To minimize the discomfort of high fiber, we ask the participant to gradually increase their intake of the high fiber cereal over 4 days. We also recommend that participants drink a lot of water to help with the digestion of the fiber The risks of drawing blood from a vein include discomfort at the site of puncture; possible bruising and swelling around the puncture site; rarely, an infection; and, uncommonly, faintness from the procedure. An overnight fast is required and may cau se physical discomfort, however we will n Other p ossible risks to you may include: Researchers will take appropriate steps to protect any information they collect about you. However, there is a slight risk that information about you could be revealed inappropriately or accidentally. Depending on the nature of the information, such a release could upset or embarrass you, or possibly affect your insurability or employability. Questions 17 21 in this form discuss what information about you will be collected, used, protected, and shared. This study may include risks that are unknown at this time. Participation in more than one research study or project may further increase the risks to you. If you are already enrolled in another research study, p lease inform Dr. Susan S. Percival (listed in question 3 of this consent form) or the person reviewing this consent with you before enrolling in this or any other research study or project.

PAGE 49

49 Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study. If you wish to discuss the information above or any discomforts you may experienc e, please ask questions now or call the PI or contact person listed on the front page of this form. 1 1 a. What are the potential benefits to you for taking part in this research study? You may or may not personally benefit from participating in this stud y. You may experience better immune health if the wheat bioactive works as we predict. 1 1 b. How could others possibly benefit from this study ? Consuming the wheat bran cereal may result in benefits such as stronger immunity. 11c. How could the researchers benefit from this study? In g eneral, presenting research results helps the career of a scientist. Therefore, Dr. Susan S. Percival may benefit if the results of this study are presented at scientific meetings or in scientific journals. Dr. Percival does not receive any compensation, monetary or otherwise, from the sponsor outside of the funding for this research. 1 2 What other choices do you have if you do not want to be in this study? The option to taking part in this study is doing nothing I f y ou do not want to take part in this study, tell the Principal Investigator or her assistant and do not sign this Informed Consent Form. You have been invited to participate in this research project because you qualify as a member of the generally healthy population The investigators associated with this project may or may not teach in your college or be associated with courses for which you are enrolled or might be expected to register in the future. Your participation in this study is voluntary and any decision to take part or not to participate will in no way affect your grade or class standing. If you believe that your participation in this study or your decision to withdraw from or to not participate in this study has improperly affected your grade( s), you should discuss this with the dean of your college or you may contact the IRB office.

PAGE 50

50 1 3 a. Can you withdraw from this study? You are free to with draw your consent and to stop participating in this study at any time. If you do withdraw your consent, you will not be penalized in any way and you will not lose any benefits to which you are entitled. In addition, you have the right to ref use to answer any specific question that you do not want to answer. If you decide to withdraw your consent to participate in this study for any reason, please contact Dr. Susan S. Percival at 352 392 1991 ext. 217 or study coordinator at 352 293 1991 ext. 255 They will tell you how to stop your participation safely. If you have any questions regarding your rights as a research subject, please call the Institutional Review Board (IRB) office at (352) 846 1494. 1 3 b. If you withdraw, can information about you still be used and/or collected? If you withdraw, no new information will be collected about you. However, information that was already collected may still be used and disclosed to others if the researchers have relied on it to complete and protect the validity of the research. 1 3 c. Can the Principal In vestigator withdraw you from this study? You may be withdrawn from the study without your consent for the following reasons: You are unable to keep appointments, complete a final questionnaire or take the s tudy capsules as directed, o r the study is canc elled by the Food and Drug Administration (FDA), the National Institutes of Health (NIH), the company supplying the study treatment, and/or other administrative reasons. You may also be withdrawn from the study if you have a change in your medical healt h status including blood pressure. W HAT ARE THE F INANCIAL I SSUES IF Y OU P ARTICIPATE ? 14. If you choose to take part in this research study, will it cost you anything? It will not cost you anything to take part in this study. The grain cereal will be provided at no cost to you while you are participating in this study. The Sponsor will pay for all activities provided as part of your participation in this study. There will be no cost to you. If you receive a bill related to this study, please contact Dr. Susan S./ Percival at 352 392 1991 x 217 or the study coordinator at 352 392 1991 x 255

PAGE 51

51 1 5 Will you be paid for taking part in this s tudy? You will receive compensation for taking part in this study. We will pay you $25 for participation in each of the two blood draws and $ 50 for comp leting the final questionnaire, totaling $10 0. You will receive payment after the study is completed Please allow between 4 8 weeks after the completion of the study for payment. If you are paid for taking part in this study, your name and social security number will be reported to the appropriate University administrative personnel for purposes of making and recording the payment. The information will be used for the purpose of payment and will be kept co nfidential. You are responsible for paying income taxes on any payments provided by the study. If you are a University of Florida employee, taxes will be deducted from your payment which will be added to your regular paycheck. If the payments total $600 or more, the University must report the amount you received to the Internal Revenue Service (IRS). 1 6 What if you are injured because of the study? If you are injured as a direct result of your participation in this study, any resulting medical expenses will be billed to you or your insurance provider. You will be responsi ble for any deductible, co insurance, or co payments. Some insurance companies may not cover costs associated with research studies. Please contact your insurance company for additional information. No additional compensation is offered. The Principal In vestigator and others involved in this study may be University of Florida employees. As employees of the University, they are protected under state law, which limits financial recovery for negligence. Please contact the Principal Investigator listed in question 3 of this form if you experience an injury or have questions about any discomforts that you experience while participating in this study. 17. How will your health information be collected, used and shared? If you agree to participate in this study, the Principal Investigator wi ll create, collect, and use private information about you and your health This information is called protected health information or PHI In order to do this, the Principal Investigator needs your authorization The following section describes what PHI wi ll be collected, used and shared, how it will be collected, used, and shared, who will collect, use or share it, who will have access to it, how it will be secured, and what your rights are to revoke this authorization.

PAGE 52

52 Your protected health information may be collected, used, and shared with others to determine if you can participate in the study, and then as part of your participation in the study. This information can be gathered from you or your past, current or future health records, from procedures such as physical examinations, x rays, blood or urine tests or from other procedures or tests. This information will be created by receiving study treatments or participating in study procedures, or from your study visits and telephone calls. More speci fically, the following information may be collected, used, and shared with others: name height weight blood pressure measurements S ocial S ecurity number for compensation purposes address phone number birth date a list of the medications you are ta king and the conditions for which they were prescribed diaries and questionnaires. This information will be stored in locked filing cabinets or on computer servers with secure passwords, or encrypted electronic storage devices. S ome of the information collected could be included in a "limited data set" to be used for other research purposes. If so, the limited data set will only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social security number, photographs, or other codes that link you to the information in the limited data set. If limited data sets are created and used, agreements between the parties creating and receiving the limited dat a set are required in order to protect your identity and confidentiality and privacy. 18. For what study related purposes will your protected health information be collected, used, and shared with others? Your PHI may be collected, used, and shared with others to make sure you can participate in the research, through your participation in the research, and to evaluate the results of the research study. More specifically, your PHI may be collected, used, and shared with others for the following study rela ted purpose(s): To evaluate the effects of wheat bioactives on human immune function and to determine if grain bioactives provide antioxidant protection to DNA. Once this information is collected, it becomes part of the research record for this study.

PAGE 53

53 19. Who will be allowed to collect, use, and share your protected health information? Only certain people h ave the legal right to collect, use and share your research records, and they will protect the privacy and security of these records to the extent the law allows. These people include the: the study Principal Investigator Dr. Susan S. Percival and research staff associated with this project. other professionals at the University of Florida or Shands Hospital that provide study related treatment or procedures the University of Florida Institutional Review Board (IRB; an IRB is a group of people who are responsible for looking after the rights and welfare of people taking part in research). 20. Once collected or used, who may your protected health i nformation be shared with? Your PHI may be shared with: the study sponsor Kellogs Corporate Citizen's Fund United States and foreign governmental agencies who are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections Government agencies who are responsible for overseeing public health concerns such as the Centers for Disease Control and f ederal, s tate and local health department s Malcom Randall VA Medical C enter (Gainesville) Your insurance company for purposes of obtaining payment Otherwise, your research records will not be released without your permission unless required by law or a court order. It is possible that once this information is shared with authorized persons, it could be shared by the persons or agencies who receive it and it would no longer be protected by the federal medical privacy law. 21. If you agree to take part in this research study, how long will your protected health information be used and share d with others? Your PHI will be used and shared with others for up to three (3) years after the study ends. If you withdraw your permission for the use and sharing of your protected health information, then your information will be removed from the databse.

PAGE 54

54 You are not required to sign this consent and authorization or allow researchers to collect, use and share your PHI. Y our refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consen t and authorization. You have the right to review and co py your protected health information. However, we can make this available only after the study is finished. You can revoke your authorization at any time before, during, or after your participation in th is study If you revoke it no new information will be collected about you. However, information that was already collected may still be used and shared with others if the researchers have relied on it to complete the research. You can revoke your authorization by giving a written request with your signa ture on it to the Principal Investigator.

PAGE 55

55 S IGNATURES As a representative I have explained to the participant the purpose, the procedure s, the possible benefits, and the risks of this research study; the alternative information will be collected, used, and shared with others: Signature of Person Obtaining Consent an d Authorization Date risks; the alternatives to being in the study; and how your protected health information will be collected, used and shared with others. You ha ve received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask questions at any time. You voluntarily agree to participate in this study. You hereby authorize the collect ion, use and sharing of your protected health information as described in section s 17 21 above. By signing this form, you are not waiving any of your legal rights. Signature of Person Consenting and Authorizing Date

PAGE 56

56 APPENDIX F FINAL QUESTIONNAIRE Subject # ____ Thank you once again for participating in our Wheat Study. Please answer the following questions as completely as possible. 1. During the study, did you experience any side effect(s) that might be attributed to the cereal? Yes ___ No ___ If Yes, please explain: ___________________________________________________________ _____________________________________________________________________________ 2. During the study, did you take any dietary supplement(s) other than a vitamin/mineral? Yes ___ No ___ If Yes, what type(s) and how often: ________________________________________________ _____________________________________________________________________________ 3. During the study, did you notice any changes in your satiety (feeling of fullness) after consuming the cereal? Yes ___ No ___ If Yes, how did this effect your usual eating habits: __________________________________ ______________________________________________ _______________________________ 4. During the study, did you experience any weight loss or weight gain? Yes ___ No ___ 5. Did you consume the cereal we provided to you daily, for the entire 3 weeks? Yes ___ No ___ 6. If you missed eating the cereal, approximat ely how many days did you miss? _____________________________________________________________________________ 7. During the study, did you find yourself feeling more hungry than usual? Yes ___ No ___ 8. Any additional information you would like us to know, or any comments regarding this study? _____________________________________________________________________________ _____________________________________________________________________________ ________________________________________________________________ _____________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

PAGE 57

57 LIST OF REFERENCES 1. Wells, HF, Buzby JC: Dietary assessment of major trends in U.S. food consumption, 1970 2005. USDA, 27, 2008. 2. AACC: AACC international defines whole grain. AACC Int, 1999. 3. USDA: Dietary Guidelines for Americans, 29 30 USDA, 20 1 0. 4. Miller G, Pr akash A, Decker E: Whole grain micronutrients. Whole Grain Foods in Health and Disease, 243 258, 2002. 5. Jacobs DR, Meyer KA, Kushi LH, Folsom AR: Whole grain intake may reduce the risk of ischemic heart disease death in postmenopausal women: the Iowa women 257, 1998. 6. Truswell AS: Cereal grains and coronary heart disease. Eur Clin Nut r, 56 :1 14, 2002. 7. Meyer KA, Kushi LH, Jacobs DR, Slavin J, Sellers TA, Folsom AR: Carbohydrates, dietary fiber, and incident type 2 diab etes in older women. Am J Clin Nutr, 71:921 930, 2000. 8. Van Dam RM, Grievink L, Ocke MC, Feskens EJM: Patterns of food consumption and risk factors for cardiovascular disease in the general Dutch population. Am J Clin Nutr, 77: 1156 1163, 2003. 9. Murtaugh MA Jacobs DR, Jacob B, Steffen LM, Marquart L: Epidemiological support for the protection of whole grains against diabetes. Proceedings of the Nutr Soc, 62 :143 149, 2003. 10. Kasum CM, Jacobs DR, Nicodemus K, Folsom AR: Dietary risk factors for upper aerodiges tive tract cancers. Int J Cancer, 99:267 272, 2002. 11. Jacobs DR, Marquart L, Slavin JL, Kushi LH: Wholegrain intake and cancer: an expanded review and meta analysis. Nutr Cancer 30: 85 96, 1998. 12. Chatenoud L, Tavani A, La Vecchia C, Jacobs DR, Negri E, Levi F, Franceschi S: Whole grain food intake and cancer risk. Int J Cancer 77 :24 28, 1998. 13. Terry P, Lagergren J, Ye W, Wolk A, Nyren O: Inverse association between intake of cereal fiber and risk of gastric cardia cancer. Gastroenterology, 120 :87 391, 2001.

PAGE 58

58 14. Slavin, J. Whole Grains and Human Health. Nutr Research Rev, 17:1 12, 2004. 15. Miller HE, Rigelhof F, Marquart L, Prakash A, Kanter M: Antioxidant content of whole grain breakfast cereals, fruits, and vegetables. J Amer Coll Nutr, 19:312 319, 2000. 16. Adom KK Liu RH: Antioxidant activity of grains. J Agric Food Chem, 50:6182 6187, 2002. 17. Adom KK, ME Sorrells, RH Liu: Phytochemical profiles and antioxidant activity of wheat varieties. J Agric Food Chem, 51:7825 7834, 2003. 18. McCord, JM: The evolution of free ra dicals and oxidative stress. Am J Med, 108:652 659, 2000. 19. Powers SK, Ji LL, Leeuwenburgh C: Exercise training induced alterations in skeletal muscle antioxidant capacity: a brief review. Med Sci Sports Exerc, 31:987 997, 1999. 20. De Rijk MC, Bretler MM, den Breeijen JH, Launer LJ, Grobbee DE, van der Meche FG, Hofman A: Dietary antioxidants and Parkinson disease. Arch Neurol, 54:762 765, 1997. 21. Engelhart MJ, Geerlings MI, Ruitenberg A, van Sweiten JC, Hofman A, Witteman JC, Breteler M: Dietary intake of anti oxidants and risk of Alzheimer disease. JAMA 287:3223 3229, 2002. 22. Klipstein Grobusch K, Geleijnse JM, den Breeijen JH, Boeing H, Hofman A, Grobbee DE, Witteman JC: Dietary antioxidants and risk of myocardial infarction in the elderly: the Rotterdam study. Am J Clin Nutr, 69:261 266, 1999. 23. Kushi LH, Folsom AR, Prineas RJ, Mink PJ, Wu Y, Bostick RB: dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women. N Engl J Med, 334:1156 1162, 1996. 24. Zhou K, Su L, Yu L: Phytochemica l and antioxidant properties in wheat bran. J Agric Food Chem, 52:6108 6114, 2004.

PAGE 59

59 25. Moore J, Hao Z, Zhou K, Luther M, Costa J, Yu L: Carotenoid, tocopherol, phenolic acid, and antioxidant properties of Maryland grown soft wheat. J Agric Food Chem, 53:6649 6657, 2005. 26. Zhou K, Yin J, Yu L: Phenolic acid, tocopherol and carotenoid compositions, and antioxidant functions of hard red winter wheat bran. J Agric Food Chem, 53:3916 3922, 2005. 27. Moore J, Liu J, Zhou K, Yu L: Effects of genotype and environment on t he antioxidant properties of hard winter wheat bran. J Agric Food Chem, 54:5313 5322, 2006. 28. Kahkonen M P, Hopia AI, Vuorela HJ, Rauha J, Pihlaja K, Kujala TS, Heinonen M: Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food C hem, 47:3954 3962, 1999. 29. Liu RH: Potential synergy of phytochemicals in cancer prevention: Mechanism of action. J Nutr, 134:3479 3485, 2004. 30. Kim K, Tsao R, Yang R, Cui SW: Phenolic aci d profiles and antioxidant activities of wheat bran extracts and the effect of hydrolysis conditions. Food Chem, 95:466 473, 2006. 31. Graf E: Antioxidant potential of ferulic acid. Free Radical Bio and Med, 13:435 448, 1992. 32. Poquet L, Clifford MN, Williamso n C: Transport and metabolism of ferulic acid through the colonic epithelium. Drug Metab Dispos, 37:1749 1758, 2009. 33. Hatcher D, Kruger DW: Simple phenolic acids in flours prepared from Canadian wheat: Relationship to ash content, color, and polyphenol oxi dase activity. Cereal Chem, 74:337 343, 1997. 34. Rondini L, Peyrat Maillard MN, Marsset Baglieri A, Fromentin G, Durand P, Tome D, Prost M, Berset C: Bound ferulic acid from bran is more bioavailable than the free compound in rat. J Agric Food Chem, 52:4338 4343, 2004. 35. Onyeneho SN, Hettiarachchy NS: Antioxidant activity of durum wheat bran. J Agric Food Chem, 40:1496 1500, 1992. 36. Adom KK, Sorrells ME, Liu RH: Phytochemicals and antioxidant activity of milled fractions of different wheat varieties. J Agric Fo od Chem, 53:2297 2306, 2005.

PAGE 60

60 37. Esposito F, Arlotti G, Bonifati AM, Napolitano A, Vitale D, Fogliano V: Antioxidant activity and dietary fiber in durum wheat bran by products. Food Res Int, 38:167 1173, 2005. 38. Liyana Pathirana CM, Shahidi F: Antioxidant acti vity of commercial soft and hard wheat ( Triticum aestivum L.) as affected by gastric pH conditions. J Agric Food Chem, 53:2433 2440, 2005. 39. Soler M, Bosetti, Franceschi S, Negri E, Zambon P, Talamini R, Conti E, Vecchia C: Fiber intake and the risk of oral pharyngeal, and esophageal cancer. Int J Cancer, 91:283 287, 2001. 40. Pietinen P, Rimm EB, Korhonen P, Hartman AM, Willett WC, Albanes D, Virtamo J: Intake of dietary fiber and risk of coronary heart disease in a cohort of Finnish men. Circulation, 94:2720 2727, 1996. 41. Saura Calixto F, Perez Jimenez J, Goni I: Contribution of cereals to dietary fibre and antioxidant intakes: Toward more reliable methodology. J Cer Sci, 50:291 294, 2009. 42. Miller G. Whole grain, fiber and antioxidants. CRC Handbook of Dietary Fiber 453 460, 2001. 43. Kroon PA, Faulds CB, Ryden P, Robertson JA, Williamson G: Release of covalently bound ferulic acid from fiber in the human colon. J Agri Food Chem, 45 :661 667, 1997. 44. Eberhardt MV, Lee CY, Liu RH: Antioxidant activity of fresh apple s. Nature, 405:903 904, 2000. 45. Rapola JM, Virtamo J: Randomised trial of alpha tocopherol and beta carotene supplements on incidence of major coronary events in men with previous myocardial infarction. Lancet, 349:1715, 1997. 46. Virtamo J, Pietinen P, Huttun en JK, Korhonen P, Malila N, Virtanen MK: Incidence of cancer and mortality following alpha tocopherol and beta carotene supplementation. JAMA 290:476 485, 2003. 47. Adam A, Crespy V, Levrat Verny MA, Leenhardt F, Leuillet M, Demigne C, Remesy C. The Bioavai lability of Ferulic Acid is Governed Primarily by the Food Matrix Rather than its Metabolism in Intestine and Liver in Rats. J Nutr, 132:1962 1968, 2002.

PAGE 61

61 48. Kern SM, Bennett RN, Mellon FA, Kroon PA, Garcia Conesa MT. Absorption of hydroxycinnamates in humans after high bran cereal consumption. J Agric Food Chem, 51:6050 6055, 2003. 49. Anson NM, Aura AM, Selinheimo E, Mattila I, poutanen K, van den Berg R, Havenaar R, Bast A, Haenen GR: Bioprocessing of wheat bran in whole wheat bread increases the bioavailabili ty of phenolic acids in men and exerts anti inflammatory effects ex vivo. J Nutr, 141:137 143, 2011.

PAGE 62

62 BIOGRAPHICAL SKETCH Michelle Patricia Smith was born in Bellevue, Washington. Michelle grew up in Clearwater, Florida where she attended Countryside H igh School. After high school, she attended the University of Florida where she e arned a Bachelor of Science in f ood s cience and human n utrition, specializing in nutritional s ciences in May of 2009. In the summer of 20 09, Michelle was accepted as a m aster Human Nutrition Department at UF and began her classes that fall. She began working with Dr. Susan Percival in March of 2010. After graduation, she intends on working toward becoming a r egistered d ietician.