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1 GALACTOOLIGOSACCHARIDE SUPPLEMENTATION REDUCES STRESS INDUCED GASTROINTESTINAL DYSFUNCTION AND DAYS OF COLD OR FLU: A RANDOMIZED, DOUBLE BLIND, CONTROLLED TRIAL IN HEALTHY UNIVERSITY STUDENTS By CHRISTINE HUGHES 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
2 2011 Christine Hughes
3 To my husband, my family and all those who helped me along the way
4 ACKNOWLEDGMENTS I thank my supervisory committee for their mentoring and guidance and would like to give a special thanks to my advisor for all her encouragement and help throughout this resea rch project. I thank all the graduate students and undergraduate students for their help in the logistics of the study none of which could be completed without the fabulous parti support. I thank my parents and my husband for their loving enco uragement during my research
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATI ONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Introductory Notes ................................ ................................ ................................ .. 12 Galactooligosaccharides ................................ ................................ ......................... 12 GOS and Infants ................................ ................................ ............................... 16 EFSA Response to Health Claim ................................ ................................ ..... 17 Microbiota and Immune Function ................................ ................................ ..... 18 Th e GSRS and Bristol Stool Scale ................................ ............................. 19 GOS and microbiota ................................ ................................ .................. 20 Academic Stress Model ................................ ................................ .......................... 22 Cold Assessment in Research ................................ ................................ ................ 25 Supplements Affecting Immunity ................................ ................................ ............ 26 The Common Cold ................................ ................................ ........................... 26 Treatment and patient beliefs ................................ ................................ ..... 28 Mechanism of transmission of rhinovirus & the immune system ................ 28 Ascorbic Acid ................................ ................................ ................................ .... 29 Zinc ................................ ................................ ................................ .................. 30 Selenium ................................ ................................ ................................ .......... 32 Garlic ................................ ................................ ................................ ................ 33 Echinacea ................................ ................................ ................................ ......... 34 Vitamin D ................................ ................................ ................................ .......... 34 Carotenoids ................................ ................................ ................................ ...... 35 Green Tea ................................ ................................ ................................ ........ 36 2 INTRODUCTION ................................ ................................ ................................ .... 38 3 METHODS ................................ ................................ ................................ .............. 40 Subjects ................................ ................................ ................................ .................. 40 Experimental Design ................................ ................................ ............................... 40 Fiber Administration Protocol ................................ ................................ .................. 41 Online Study Questionnaires ................................ ................................ .................. 41
6 Statistical Analyses ................................ ................................ ................................ 43 4 RESULTS ................................ ................................ ................................ ............... 45 5 DISCUSSION AND CONCLUSION ................................ ................................ ........ 53 APPENDIX A IRB APPROVAL LETTER ................................ ................................ ....................... 57 B IRB INFORMED CONSENT ................................ ................................ ................... 59 C RECRUITM ENT MATERIALS AND QUESTIONNAIRES ................................ ....... 69 LIST OF REFERENCES ................................ ................................ ............................... 76 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 86
7 LIST OF TABLES Table page 3 1 Subject characteristics and compliance ................................ .............................. 49 3 2 Weekly gastrointestinal symptoms and daily stool characteristics in control subjects and subjects supplemented. ................................ ................................ 50
8 LIST OF FIGURES Figure page 1 1 (1,4) galactooligosaccharide molecule. ................................ 37 1 2 galactosidase ................... 37 3 1 Flow chart of subject recruitment, allocation and analysis. ................................ 48 3 2 Cold/flu symptom intensity (SI) score by level of stress. ................................ ..... 51 3 3 Average percentage of days with a cold/flu symptom intensity ( SI) score greater than 6 by BMI ................................ ................................ ........................ 52
9 LIST OF ABBREVIATION S BMI Body Mass Index DP Degree of Depolymerization EFSA European Food Safety Authority FOS Fructooligosaccharides GI Gastrointestinal Tract GOS Galactoligosaccharide s GSRS Gastrointestinal Symptom Response Scale IBS Irritable Bowel Syndrome IgA Immunoglobulin A ICAM 1 Intercellular Adhesion Molecule 1 IL Interleukin ILra 1 Interleukin 1 Receptor Antagonist NFKB Nuclear Factor Kappa B NK Natural Killer Cells SCFA Short Chain Fatty Acids SEM Standard Error of the Mean SI Symptom Intensity
10 Abstract of Thesis P resented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science GALACTOOLIGOSACCHARIDE SUPPLEMENTATION REDUCES STRESS INDUCED GASTROINTESTINAL DYSFUNCTION AND DAYS OF COLD OR FLU: A RANDOMIZED, DOUBLE BLIND, CONTROLLED TRIAL IN HEALTHY UNIVERSITY STUDENTS By Christine Hughes May 2011 Chair: Bobbi Langkamp Henken Major: Food Science and Human Nutrition Acute psychological stress induced by academic exams is associated with dysregulated gastro intestinal and immune function. The purpose of the study was to examine whether supplementation with galactooligosaccharides (GOS) reduced gastrointestinal dysfunction and percentage of days with cold/flu in academically stressed undergraduate students. In a randomized, double blind study, subjects (n=427) received 0 g, 2.5 g, or 5.0 g GOS for eight weeks around the time of fall final exams. Level of stress and cold/flu symptoms and inte nsity (SI, 0=not experiencing to 3=severe) were re corded daily. The SI from nine cold/flu symptoms was summed with a day of cold/flu defined as a sum greater than six The Gastrointestinal Symptom Response Scale was completed weekly, and associations betwe en the categories of symptoms and GOS were examined. Stress was positively related to diarrhea, indigestion, and reflux syndromes, abdominal pain, average daily cold/flu SI score, and the percentage of days with cold/flu. Gastrointestinal symptom scores fo r diarrhea (P=0.0298), constipation (P=0.0342), abdominal pain (P=0.0058), and indigestion
1 1 (P=0.0003) syndromes were lower with GOS. The cold/flu SI score was affected by GOS and stress (P<0.0001); 2.5 g was associated with a lower SI score across all leve ls of stress but 5 .0 g was protective only at lower levels of stress. The percentage of days with cold/flu was associated with GOS within different BMI categories (P=0.0002) wherein a 40% reduction in the percentage of days with cold/flu was observed in no rmal weight (BMI=18.5 24.9) individuals with 5 .0 g of GOS. This effect was not observed in overweight/ obese individuals; however 2.5 g was protective in overweight and obese individuals. Acute psychological stress was directly related to symptoms of gastr ointestinal dysfunction and cold/flu. GOS supplementation reduced these symptoms and days with cold/flu.
12 CHAPTER 1 LITERATURE REVIEW Introductory Notes It is known that the common cold and influenza are an econ omic burden, and that they also adversely a ffect student academic performance On e method to study colds and flu is with the academic stress model, in which a percentage of students undergoing stress get sick and exhibit cold or flu symptoms in a defined period of time. Nutritional interventions could be employed to maintain good health by decreasing cold and flu symptoms. For example prebiotics, non digestible carbohydrates fermented in the gastrointestinal tract, have the ability to stimulate the growth of potentially beneficial bacteria which may reduce the immune burden and maintain health Stressors, such as psychological stress, alter the microbiota and im pair health by dysregulating immune function ( 1) Research is lacking regarding the effect of galactooligosaccharide s (GOS), a prebiotic, on the immune outcomes of undergraduate students undergoing stress. This literature review will discuss galactooligosaccharides and the research pertaining to infants, as well as a recent EFSA health c laim. Topics will also include the academic stress model, the Gastrointestinal Symptom Rating Scale ( 2) gastrointestinal microbiota and immune function cold assessment in research, and other nutrients affecting im munity. Galactooligosaccharides The benefits of pre and probiotics arise from the impact they have on the gastrointestinal microflora. Probiotics a re live microorganisms, such as l actobaccili, that act by modifying endogenous bacteria and increasing the proportion of beneficial bacteria. However, the effects of probiotics are difficult to control as the amount of
13 bacteria reaching the gastrointestinal tract is not known ( 3) non digestible food ingr edient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, thus improving host health ( 4) A revised definition states that prebiotic is a in gredient that allows specific changes, both in the composition and/ or activity in the gastrointestinal tract that confers benefits upon host well being and health ( 5) Preb iotics are known to be fully fermented in the gastrointestinal tract, producing short chain fatty acids, fuel for the colonocytes ( 6, 7) By strengthening colonocytes, prebioti cs maintain gastrointestinal integrity and promote general go od health Prebiotics such as GOS facilitate the colonization of gastrointes tinal microbiota in the gastrointestinal tract of newborn infants ( 6) Human breast mi lk is believed to be the a concentration of 1 g/dL ( 8) The oligosaccharide ratio believed to be most similar to breast milk commonly researched in infant studies is 9:1, GOS: fructooligosaccharide (FOS) ( 9) GOS is added to various foods in Japan and Europe as a health promoting ingredient ( 10) Galactoliogosaccharides are comprised of a chain of galactose units arising from tr ansgalactosylation reactions with a terminal glucose unit (Figure 1 1 ) The degree of polymerization can range from 2 to 8 units. In vitro evidence shows they are resistant to salivary and digestive enzymes and are most likely fermented in the gastrointest inal tract ( 6, 11) Commercial p roduction of GOS occurs through a transgalactosylation reaction ( 12) The enz galactosidase produces several oligomers of differing lengths including GOS ( 5, 10) GOS is a food ingredient that has
14 GRAS status in the United States and a fter purification GOS can be added to food products ( 5) The GOS employed in the research study was a white powder de rived from food grade lactose and galactosidase obtained from B. circulans LOB 377 which is a non toxogenic, non pathogenic organism (Figure 1 2) Synthesis of GOS [Purimmune, GTC Nutrition, Golden, Co lorad o] starts with a lactose powder which is dissolved in hot water (90 C) ( 13) Adjustments are made for pH to make the environment slightly acidic ( hydrochloric acid and sodium hydroxide ) ( 13) The solution is stirred unti l oligosaccharide concentration is greater than 50% w/v then the temperature is ra ised to inactivate the enzyme. The enzyme is removed; the product is then processed to remove impurities, concentrated, and further purified. The supplement contains 86% GOS, with 21% DP2 and 63% DP3 and above. The influence of GOS on allergy and eczema development in infants, cholesterol l evels in young adults, and calcium absorption in post menopausal women has been studied ( 14 18) However r esearch is lacking is regarding the effect of GOS on immune outcomes in a student population. It is thought that GOS aid s gastrointestinal immunity by directly affecting the gastrointestinal epithelium ( 10) GOS has been shown to prevent the adhesion and invasion of pathogens in the small intestine such as Escherichia coli ( 19) The authors propose that GOS may act as a rece ptor mimic and may competitively inhibit adhesion and invasion of pathogens into the gastrointestinal epithelium ( 19) Another proposed mechanism by which GOS interacts with the immune system involves changing the gastrointestinal microflora by selectivel y stimulating the growth of
15 lactobacilli and bifidobacteria ( 6) These bac teria are considered beneficial. Breastfed infants are believed to have a more beneficial gastro intestinal microflora which produces short chain fatty acids (SCFA), acetate and lacta te that lower the p H. These inhibit the growth of potentially detrimental bacteria such as certain Escherichia coli ( 20) Thus, researchers examined the fecal pH of breastfed infants in comparison to infants on a placebo consisting of maltodextrin ( 21 24) Ben et al. (n=371) and Bakker Zierikzee et al. (n=57) ( 21, 22) found that fecal pH decrease d with an increase of lactate, suggesting that conditions are unfavorable for e nterobacteria. Moro et al. ( 2002 ) also found a decrease in fecal pH with oligosaccharide supplementation versus the placebo ( 24) The authors also reported a dose dependent effect of oligosaccharides ( 0.4 g/ dL and 0. 8 g/dL) on the stimulation of bifidobacteria and l actobacilli ( 24) In contrast Bouhnik et al. examining GOS in adults (n=8) found no significant difference in fecal pH and no difference in fecal concentrations of enterobacteria ( 23) These findings did show an increase in bifidobacteria and over time a decrease in breath hydrogen suggesting that the fermentation of trans galactooligosaccharide has changed over time possibly more effectively metabolized in the colon ( 23) A study by Tiihonen et al. examining GOS and a probiotic mixture in young Dutch men showed no effect on fecal bifidobacteria. However the authors suggest this could potentially be due to the already high counts of bifidobacteria due to the high fiber content of the typical Dutch diet ( 3) Another study showed that the bifidobacteria increase was more significant in populations with a low baseline number of bifidobacteria ( 25) Nonetheless, in part b ecause of the complexity of the gastrointestinal tract discrepanci es exist in the literature ( 21)
16 GOS and Infants Various studies examine the effect of GOS on infants. GOS supplemented in infant formula ha s been shown to b e well tolerated ( 21) Infants supplemented with GOS have shown similar gastrointestinal microflora as breastfed infants ( 21) GOS relieves constipated infants by contributing to softer stoo l formation whilst not increasing stool freq uency ( 26) Infants fed with a formula containing GOS have the same growth and stool characteristics as breast fed infants ( 27) A study by Ben and colleagues (n=371) found that a low dose of GOS ( 2 .4 g/ L) was shown to be bif idogenic ( 22) Oligosaccharides in human breast milk are found in a concentration of 8 12 g/ L in mature milk and 25 g/ L i n colostrum ( 28) Researchers preferred the low dose intervention to avoid potentially irritating ef fects associated with hi gher dose s in infants ( 22) Scholtens found that infants supplemented with GOS and long chain FO S (n=215) had an increase in fecal sIgA with an increase in bifidobacteria, suggesting a benefit to gastrointestinal mucosa ( 29) Oligosaccharides (GOS/FOS) has been shown to decrease stool transit time and increase stool viscosity in premature infants ( 30) Alternatively, p remature formula fed infants tend to have hard stools and delayed gastrointestinal transport ( 30) As human milk is rich in oligosaccharides breast fed infants are shown to have incre ased bifidobacteria and l actoba cilli in their intes tines ( 24) It is this bifidogenic effect that researchers are interested in replicating by performing intervention trials with prebiotics, such as GOS Moro et al. found that supplementing formula fed infants with GOS and FOS (either 0.4 g/ dL or 0. 8 g/ d L) resulted in a dose dependent effect on bidifobacteria and l actobacilli over the 28 days study period, versus the placebo ( 24)
17 Scholtens found that during weaning infants supplemented with GOS (4.5 g/d) had bifidobacteria counts similar to exclusively breast fed infants ( 31) Early mode of feeding, breastfeeding or formula feeding, and gastrointestinal microbiota a re of interest Kalliomaki et al. hypothesized that early microbiota could be associated with children to becoming obese later in life ( 32) EFSA Response to Health Claim Recently a health claim regarding GOS and its effects on immunity has been investigated for its validity. In response to a health claim application, the European Food Safety Authority (EFSA) examined various studies regarding the effects of GOS/FOS (9:1) (brand name Immunofortis Danone Baby Nutrition, The Netherlands ) on im mune function and the incidence of a topic eczema in infants ( 33) EFSA stated that the immune system was not adequate ( 33) One such study sought to examine immune function in infants, and showed a decrease in infection rate and allergic manifestations ( 15, 16) A ccording to t he EFSA, the researchers did not adequately investigate the basis of each diagnosis whether it would be determined from clinical symptoms or even microbiological testing. Mothers reporting all ergy symptoms for their infants is not reliable ; this is interes ting to note because evidence shows that adults self reporting cold and flu data is reliable The EFSA also stipulated that a healthy immune system includes an adequate response from the adaptive immune system and that a decrease in infection rate does not show a dir ect effect on immune function. The EFSA stated that there is lack of evidence showing that an incre ase in bifidogenic bacteria and a decrease in enterobacteria would be beneficial ( 33) Also, the board did not find adequate evid allergic infections or eczema based on
18 the literature supplied by the researchers ( 15 17, 34 37) With better design outcome measures the mechanism by which oligosacchar ide supplementation impacts immune outcomes may be demonstrated Microbiota and Immune Function The gastrointestinal t ract is the largest organ of the immune system and changes in gastrointestinal microbiota could lead to changes in immune health. T hi s section will briefly discuss gastrointestinal microbiota and immune health the role of stress in gastr ointestinal function, the Gastrointestinal Symptom Response Scale, the Bristol Stool Scale, microbiota. The number of bacteria found in the colon and in the feces is 10 12 cells per gram ( 38) The gastrointestinal tract is home to a diverse microflora providing beneficial effects to the host. The gastrointestinal microflora has been shown to facilitate excretion of toxins and occupy attachment sites for potentially pathogenic microorganis ms ( 38) It a lso interacts with immune tissue associated lymphoid tissues ( 38) The most prevalent organisms in the gastrointestinal tract are the Bacteriodes Prevotella group (gram negative anaerobe) and Clodistrium species (gram positive anaerobes). Aerobic bacteria are much less prevalent ( 38) Th e gastrointestinal microflora differs among people This begs the question as to the effect of differing microflora on hosts ( 38) Future research aims to determine which species of bacteria have most significant effects on human health and how nutritional interventions can alter this microbiota. New e vidence suggests that changing m icrobiota affects human health. Ley has shown that gastrointestinal microbiota differs in le an and obese people and that weight
19 loss can change the microbiota ( 39) ata shows less Bacterioidetes in obese individuals and a high er proportion of Firmicutes compared to lean individuals ( 39) Weight loss of the obese groups can result in an inc reased proportion of Bact eriodetes, more closely resembling that of lean individuals ( 39) A weight loss of at least 6% in the fat restricted diet and at least 2% in the carbohydrate restricted diet was necessary to see this effect. This suggests that weig ht loss, or possibly a change in diet can alter gastrointestinal microflora. O besity is a disorder characterized by systemic inflammation ( 40) Neyrick theorizes that prebiotics could be beneficial in the treatment of the inflammation associated with obesity through alter ing the gastrointestinal microbiota ( 41) Stress is known to cause gastrointestinal dysfunction Sixteen medical and premedical students reported increased abdominal pain during examinations ( 1) Acute stress has been shown to inhibi t gastric emptying and decrease colonic transit time ( 42) Research is lacking regarding the effect of academic stress on gastrointestinal symptoms. The GSRS and Bristol Stool Scale The Gastrointestinal Symptom Rating Scale (GSRS) was developed for irrita ble bowel syndrome ( 43) The GSRS has been validated for its ease of use and excellent construct ( 44 46) The GSRS consists of five syndromes with approximately three symptoms each including: diarrhea syndrome (diarrhea, loose stools, urgent need for defecation), constipation syndrome (constipation, hard stools, and feeling of incomplete evacuation), abdominal pain (abdominal pain, hunger pains, and nausea), indigestio n syndrome (rumbling, bloating, burping, and gas) and reflux syndrome (heartburn and acid regurgitation).
20 The Bristol Stool Form Scale is a seven point stool consistency scale devised by as an estimate of stool transit time ( 47) Stools are rat ed as such: 1 (separate hard lumps like nuts), 2 (sausage shaped but lumpy), 3 (like a sausage or snake with cracks on its surface), 4 (like a sausage or snake, smooth and soft), 5 (soft blobs with clear cut edges), 6 (fluffy pieces with ragged edges, a mu shy stool) and 7 (watery, no solid pieces) ( 47) T he scale has been used in research and in clinical practice ( 48, 49) The Bristol Stool Form Scale helps to clarify whether self reported stress levels have an impact on gastrointestinal transit time GOS and m icrobiota There ha ve been few studies describing how GOS changes the gut microbiota and affects immune function and health outcomes. Davis et al. found that doses of 5 .0 g/d and 10 g/d produced an increase in bifidobacteria counts and 2.5 g/d did not ( 50) It is interesting to note that even when a high dose of GOS (10 .0 g/d) was administered for three weeks some participants did not show the bifidogenic effect ( 50) One study by Bouhnik and colleagues in healthy adul ts found that GOS was bifidogen ic at 2.5 g/d to 10 g/d (n=64) ( 25) Vulevic et al. is the only study to date examining immune function and GOS in healthy elderly volun teers ( 51) Participants on GOS (2.64 g /d ) had a significant increase in bifidobacteria counts a nd a decrease in other less desirable bacteria such as some strains of clodis tria and enterobacteria They also found an increase in anti inflammatory cytokines (IL 10) and a reduction in pro inflammatory c ytokines (IL 6) after peripheral blood mononuclear cell stimulation by lipopolysaccharide This study shows that immune function can be modulated with the addition of GOS.
21 GOS, or more specific ally trans galactooligosaccharide has been examined for its p otential effects on irritable bowel syndrome patients (IBS) Silk et al. employed an in tervention with either 1.7 g /d 3.3 g /d or a placebo and asked participants to rate gastrointestinal sympt oms on a 7 point Likerd scale. Results indicate that 1.7 g of tr ans galactooligosaccharide enhanced fecal bif idobacteria numbers and lessened flatulence and bloating ( 52) The researchers sugg est that these results may provide insight into the mechanism of GO S in regards to improving IBS. The researchers state that anti inflammatory markers in the peripheral blood were normalized with increased bifidobacteria ( 52) A study by Marcos examined whether taking a probiotic could alter gastrointestinal microbiota and impact immune response in students ( 53) One hundred and fifty five undergraduate students undergoing academic stress consumed a fermented yogurt with Lactobacillus casei D N 114001 ( 53) Marcos and colleagues found that stress c auses a dysregulation of the immune system, particularly a decrease in lymphocytes and decreased natural killer cell a ctivity ( 53) The addition of the fermented yogurt containing Lactobacillus casei to the diet of stre ssed students improved immune function as seen with an increase lymphocyte number as well as CD56 cell activity ( 53) Pregliasco et al. found that long term administration (90 days) of a syn biotic improved bowel functions, and also reduced the incidence of the common cold ( 54) A syn biotic is a prebiotic and a probiotic and act of live microbial dietary supplements in the gastrointestinal tract of the host ( 10) Participants were asked to record sym ptom severity (on a scale of 1 5 with 1= no symptoms and 5=severe symptoms) and whether there were any changes in bowel movements
22 (increase, decrease or no change) ( 54) The symbiotic ( Lactobacillus planterum, Lactobac illus rhamnosus and Bifidobacterium lactis, lactoferrin and FOS or GOS) increased bowel movements and decreased incide nce, severity and average duration of colds ( 54) Immune outcomes have been measured for probiotics, and synbiotics. O ne study examined th is relationship with GOS in aged adults; however n o studies have examined whether GOS affects immune outcomes in students undergoing an academic stress. Academic Stress Model This section will explore the research regarding the academic stress model and h ow it relates to the presented research. For students passing or failing examinations influences a ( 55) Researchers examining health benefits such as decreasing cold and flu symptoms with a particul ar nutritional intervention have various methods at their disposal to see whether the intervention produces an effect. One way to examine health and colds would be to use a viral challenge study design by injecting a rhinovirus into participants under stress consuming either the placebo or the compound of interest and to measure what percentage of participants on each treatmen t exhibit cold symptoms ( 56) Cohen inoculated 394 healthy participants with viruses (rhinovirus 2, 9 or 14 or respiratory syncytial virus or coronavirus 229E) and examined whet her psychological stress impaired immunity ( 57) The researchers linked higher psychological stress and increased susceptibility to illness ( 57) Another way to assess colds and flu would be to monitor study pa rticipants for an extended time period in which participants will almost inevitably get sick ( 58) The idea behind this meth od is that the compound of interest could prevent colds symptoms.
23 Another way of measuring cold symptoms would be by employing a model which shows that a certain percentage of participants would get sick during the time period (i.e., the academic stress mo del ) This model is a method which looks at sickness in undergraduate students undergoing academic stress and the occurrence of cold and flu symptoms in a short time frame ( 59, 60) Th e academic stress model can be employed when injection of a virus c annot be justified, or the long time frame of other study designs would deter students from participating. This model has been validated by other studies ( 59, 60) Many studies have used the academic stress model to evaluate its effects on immunity. In a naturalistic model, Cobb et al examined whether psychological style, coping style and family environment had an impact on susceptibility to upper respiratory tract infections ( 61) The r esearchers followed 107 participants for one year prior to the study and 15 weeks of the study and found that the risk of illness was higher in those with a higher perceived stress and high life event stress ( 61) Interestingly, the avoidant coping style w as protective against colds in high life stressors. The researchers suggest that avoidance style of coping may prevent people from dwelling on the negative aspects of their lives and thereby shortening their time of vulnerability ( 61) Sleep quality and du ration are believed to be important predictor s of immunity. Cohen at al. performed a study regarding sleep duration and immunity. The researcher found that those with poorer sleep efficiency and sleep duration in the weeks prior to infection exposure had a higher probability to develop a cold ( 62) The researchers explain that sleep disturbance can be influenced by the regulation of pro inflammatory cytokines ( 62)
24 Physical or physiological stressors can alter immune function thereby potentially increasing susceptibility to infections. Uchakin and colleagues monitored immune responsiveness of first year medical students during first examinations ( 63) Blood was collected at either 24 hours or 48 hours after examinations. Peripheral blood mononuclear cells we re stimulated with phorbol myristate acetate and lipopolysaccharide for four hours ( 63) Results indicate that stress can alter immune cells in vitro as seen by a decrease in natural killer cell numbers and changes in other immune cells ( 63) A major limitation of this study is that for some parameters there was no adequate power to detect significant changes. Acute stress increases the activity of the hypothalamus pituitary adrenal (HPA) axis and raise salivary immunoglobulin A (IgA). A study by Takat suji et al. examined the effect of examination stres s on female nursing students (n=15) by salivary IgA and cortisol levels prior to and immediately after an examination. The researchers found and increase in salivary IgA with no significant changes in cor tisol levels ( 55) In contrast Ng et al. studied salivary IgA and cortisol levels in dental undergraduate students prior to and immediately after examination ( 64) The researchers wanted to link these data with perceived stress and found pre test stress correlated with an increase in corti sol but no change in IgA ( 64) An explanation for these diverge nt results could be accounted for by the fact that cortisol is highest after waking and decreases throughout the day ( 65) As the researchers did not specify the time of day that salivary cortisol was measured this could account for the differing results.
25 Kiecolt Glaser et al. took blood samples from medical students (n=75) one month before examination and on the first day of final exams ( 66) They found that stress led to a decrease in natural killer cell activity with no changes in salivary IgA ( 66) Stress can negatively impact the gastrointestinal tract. Knowles et al. examined whether academic stress impacted salivary cortisol and lactic acid b acteria in the stools of students ( 65) The results indicate that stress led to a decrease in lactic aci d bacteria counts during time of high stress (final examination) ( 65) The authors suggest that stress causes alterations in the microbiota leading to increased susceptibility to illness ( 65) Cold Assessment in Research Colds and flu have been studied but criteria is lacking regarding what actually constit utes a cold. A wea kness in cold research is the lack of well developed outcome measures. The Jackson scale is typically employed to determine cold symptoms and severity. The index developed in the 1950 s assess 8 symptoms (sneezing, nasal obstruction, nasal discharge, sore t hroat, cough, headache, chilliness and malaise) rating these symptoms on a three or four point scale ( 67) The scale was shown to have ( 68) Another method to assess colds is the Wisconsin Upper Respir atory Symptom Survey (WURSS), developed to assess the quality of life during colds. The WURSS which was found to be reliable, is a 44 item index that consists of impairments to daily living, breathing, sleeping, working and interpersonal relations hips ( 69) Barrett et al. investigated whether there was a relationship between questionnaires (mainly the Jackson scale and the WURSS). The researchers looked at an Echinacea study in which participants were inoculated with the rhinovirus and laboratory
26 biomarkers including IL 8, nasal neutrophil cou nt, mucus weight and viral titer were examined ( 70) These biological markers were used to assess whether the participants developed a cold. Modest associations were found and neither questionnaire was a good predictor of infection but both correlate with laboratory assessed measures ( 69) The addition of quality of life questions on the WURSS did not strengthen the association with laboratory biomarkers ( 69) This study suggests that the WURSS while providin g information regarding quality of life did not provide any additional information pertaining to the actual cold. Supplements Affecting Immunity The purpose of this secti on is to explore supplements affecting immunity. First, a background of the common co ld will be discussed including the mechanism of transmission. A literature review pertaining to ascorbic acid, zinc, selenium, garlic, Echinacea, vitamin D, carotenoids, green tea and how these supplements affect immunity will be discussed. The C ommon C old The economic cost of the common cold is staggering. It is estimated that a working adult loses on average 8.7 work hours when inflicted with a cold and 1.2 work hours when attending to children under the age of 13 inflicted with a cold or flu episode ( 71) This amounts to 25 billion dollars lost in productivity annually ( 71) The cost for students is also large. A 6 month study by Nichol et al. revealed that 91% (n=3,249) had a cold ( 72) Cold s and flu accounted for bed days, missed school days and work da ys, resulting in doing poorly on exams or assignments ( 72) A reliable, simple, cost effective way to reduce the incidence and duration of the common cold is desired P rograms aimed to reduce sickness absence days by
27 providing physical activity and stress management have been examined One such study examined whether an intervention on nursing assistants would have a lasting impact, after 3 years follow up no lasting impact was found ( 73) It would be beneficial to find nutritional interven tions to decrease colds and flu The incidence of upper respiratory tract illnesses in adults is from one to six episodes a year ( 74) The most common cause s of upper respiratory tract infections (colds and flu) include the rhinovirus, influenza, parainfluenza, adenovirus, and syncytial virus ( 74) The peak incidence of colds in temperate regions occurs in the autumn months, for reasons unknown ( 74) A study performed with young adults (n= 346) found that re verse transcription polymerase chain reaction for picornavirus and virus isolation for human rh inovirus confirmed 83% of self reported colds during the autumn months (September thru October) ( 75) Tyrrell et al. found that after inoculation of picanovirus self diagnosis ag reed with the ( 76) The study also found that the only difference between the viral strands was the incubation time ( 76) The onset of symptoms of upper respiratory tract infection is usually one to two days after the viral infection an d the peak of symptoms being two to four days after exposure ( 76) The illness includes complaints of scratchy throat, sneezing, nasal discharge, sore throat, hoarseness, cough, heada che, myalgia, malaise, feverish ness, an d chilliness ( 77) In the beginning of a cold, symptoms include nasal obstruction, rhinorhhea, and sneezing ( 76) Cough is associated with 30% of colds ( 78) The cold can last about a week and up to 25% can last 2 weeks ( 78)
28 Treatment and p atient b eliefs The goals in the management of the common cold include resolving of symptoms, decreasing the person to person spread and preventing complications ( 79) The common cold is ge nerally associated with low mo rbidity but complications can include otitis media and sinusitis ( 79) A survey of pediatricians in 2009 confirmed that 96% ( 80) Treatment for colds typically focuses on symptom relie f such as cough suppressants, antihistamines and, decongestants ( 81) Good quality evidence exists that antibiotics should not be used to treat colds as they do not reduce symptom duration or severity and cause gastrointestinal side effects ( 81) A telepho ne survey by Braun (2000) (n= 249 respondents) regarding beliefs related to colds showed that there are erroneous beliefs about the cause and treatment of colds ( 82) Respondents were called after visit to a primary care physician in which they were seekin g care for their children or themselves. Of those respondents 44% believed that a ntibiotics could help colds, and 42% believed that colds could result from bacteria and viruses. This is astoundingly high as it has been reported that only 5% of colds can be attributed to bacteria, it is therefore a rare occurrence ( 83) Respondents believed that v itamin C (67%) and inhalation of steam (70%) could reduce cold symptoms. The respondents were well educated, employed, fully insured and in good health. These resul ts cannot be extrapolated to a population which is less affluent and/or less educated. Mechanism of t ransmission of r hinovirus & the i mmune s ystem Rhinoviru s is delivered to the eye (usually by a finger and travels down the lachrymal duct) or to the nose as an aerosolized particle where it goes to the back of the
29 throat and the adenoid area ( 84) This area contains crypts which have lymphoid follicles. These follicles contain intracellular adhesion molecule 1 (ICAM 1) ( 84) ICAM 1 is the cellul ar receptor for 90% of rhinovirus serotypes ( 84) The peripheral white blood cell count increases in the first 2 3 days of the virus challenge ( 77) The increase in white blood cells is a result of an increase in circulating polymorphonuclear leukocytes ( 7 7) In human studies there is an association between IL 8 and the common cold ( 77) Increased levels of IL 8 are present in the nasal secretions of participants with the rhinovirus. In experimental rhinovirus infections there is a dose dependent relationsh ip between IL 8 concentrations and the severity of symptoms ( 77) Virus induced oxidative stress in virus induced NFKB which in turn activates IL 8 production ( 77) Studies also show that antioxidants inhibit IL 8 production ( 77) Ascorbic Acid Ascorbic acid, commonly referred to as v itamin C is a water soluble vitamin found in fruits and vegetables. The most notable source of vitamin C is citrus. Vitamin C is important for iron absorption, wound healing, and collagen formation. Deficiency in vitamin C causes scurvy. The Institute of M edicine recommends that adult males consume 75 mg/d (females 90 mg) and those who smoke, which is an oxidative stress, should consume an additional 35 mg/d. Research shows that vitamin C cannot prevent a cold but can decrea se symptom severity. Baird et a l. supplemented (n=362) with orange juice containing 80 mg/d ascorbic acid or a placebo ( 85) A significant reduction in total cold symptoms was s een with the ascorbic acid ( 85)
30 Zinc Zinc lozenges have an unpalatable taste may cause mouth irritation and even taste distortion ( 86) The effectiveness of zinc lozenges may be related to the amount of charged zinc ions and not total dosage ( 86) The literature regarding the effectiveness of zinc on cold and flu prevention is i nconclusive. Some literature shows a positive effect of zinc on reduction of cold symptoms but these studies have not been adequately blinded due to adv erse effects of the zinc ( 87) S tudies with zi nc supplementatio n showing no significant results on cold and flu and may be attributed to inadequate sample size or inadequate doses ( 87) Zinc was sh own to inhibit viral growth and in a randomi zed placebo controlled trial to decrease the length of a cold by seven days ( 88) Subjects were diagnosed to have a cold by a doctor and given either 23 mg lozenges or a calcium lactate placebo Subjects were asked to report presence and severity o f cold symptoms on a scale of 0 to 3 (with 0 being no symptoms and 3 being severe). S ide effects included unpalatable taste and mouth irritation. A double blind, randomized, placebo controlled trial consisting of 50 participants recruited within the first 24 hours of developing a cold, showed that consuming zinc lozenges (13.3 mg zinc ac etate) every two to three hours had shorter overall duration of colds, cough, and nasal discharge ( 89) The authors concluded that the improvement in cold symptoms by the zinc was due to its antioxidant and anti inflammatory properties, as evidenced by sig nificant differe nces in soluble interleukin 1 receptor antagonist (IL 1ra) and ICAM 1 Interleukin 1 receptor antagonist is anti inflammator y and inhibits IL oluble intercellular adhesion molecule 1 is a major cellular receptor for rhinoviru s ( 89) The authors suggest ed that zinc acts as a n antiviral by decreasing
31 ICAM 1 or that zinc binds with ICAM 1 thereby preventing the bi nding of rhinovirus to the cell In a study conducted by Eby et al. zinc gluconate, which was administered in a nasal spray (i.e., avoid ing unpleasant taste of zinc lozenges ), showed no effectiveness in reduction of cold symptoms ( 90) In another study, z inc orotate lozenges were used due to their slightly sweet if not bland taste ( 90) In this double blind placebo controlled trial participants (n=77) rated symptoms on a scale of 0 to 3 (0 being absent and 3 being severe). Participants were told to use t o nasal spray two to three times every 15 to 30 minutes while awake to keep the nose tissue moist and to take a lozenge every two to three hours while awake. No significant differences were found (i.e., cold duration was the same for those taking the zinc as those on the placebo ) The main side effect of the nasal spray in some p articipants was long lasting nasal pain Th e nasal pain suggests t hat 10 mmol of zinc gluconate may be the maximum tolerable amount. The researchers concluded that it is unethical to use zinc or any other metal into the interior of the nose to treat colds (90) A study conducted by Turner and Cetnarowski with 273 participants with an experimental rhinovirus and 281 participants with naturally occurring colds took either zinc gluconate zinc acetate, or a placebo ( 91) Subjects were asked to rate seven symp toms related to cold and flu on a scale of 0 to 4 (with 0 being absent and 4 being severe). After speaking with study staff daily the total daily symptom score was recorde d. For a given participant t he onset of the cold was defined as the start of the stu dy and the end of the cold was defined when the participant had two consecutive symptom scores of less than or equal to one ( 91) Evaluation of blinding revealed that
32 taste alone was not sufficient to determine the identity of the supplement ( 91) N o sign ificant differences i n duration or severity of colds were reported Selenium Selenium incorporates into selenoproteins and has a n effect on oxidative stress, redox and critical cellular processes ( 92) Selenium has been shown to be important for both innate and adaptive immune responses ( 93) It plays a role in redox reactions, antioxidant function, and membrane integrity and protects against DNA damage ( 94) A good source of selenium is the Brazil nut and moder ate levels of selenium are found in fish and shellfish ( 95) In the US wheat is a good source because of the availability of s e lenium in the soil, while in Europe wheat is not an adequate source because of low levels found in soil ( 95) a cardiomyopathy, affects regions of China with selenium deficient soil. ted to the coxsackievirus. Selenium elevate s antiviral immunity, and prevent s genetic adaptations in the viral genomic RNA that lead to virulence and cardiac pathology ( 96) Se lenium deficient mice infected with influenza have higher lavage total cell volumes, suggesting inflammation when compared with selenium adequate mice ( 96) th suboptimal selenium levels supplemented with either 50 g, 100 g or a placebo for 15 weeks experienced changes in immune function and rates of clearance of a live attenuated polio virus ( 97) Participants were va ccinated and re vaccinated against the polio virus in order to elicit an immune response characterized by memory B cells and T cells. The results show ed selenium supplementation increased plasma selenium c oncentrations and cytosolic glutathione peroxidase activity ( 97) Selenium also
33 increase d interferon gamma production, resulted in an earlier p eak T cell proliferation, and increase d helper T cell production. Additionally p articipants showed an increased clearance of poliovirus These factors suggest improved immune function ( 97) Though sele nium has been shown to improve immune function no evidence exists regarding its effects on cold and flu severity or duration. Garlic Published literature regarding the effect of garlic on immune function in clinical studies is sparse. A Cochrane review of the garlic and the common cold revealed just one double blind, placebo controlled article ( 98) The reviewer concluded that there is insufficient evidence regarding the effects of garlic on the common cold ( 98) Josling et al. examined the effect of a garlic supplement on preventing the common cold in 146 participants, matched for age, sex and garlic consumption for 12 weeks ( 99) The researcher instructed participants to record symptoms daily in a diary (5= well, no pro blems, 4= quite well with occasional sneeze, not disruptive to normal routine, 3=can feel a cold coming on, 2=feeling low and beginning to exhibit symptoms, and 1=full cold symptoms). A cold was determined by a score of 3 that preceded a score of 1 or 2. C old duration was determined by the number of days the participant had a score of 1 or 2 ( 99) The placebo group reported more days w ith a cold and longer symptom duration. Ishikawa et al. examined the effect of an aged garlic supplement on quality of life and natural killer (NK) cell a ctivity in participants with inoperable live r cancer, pancreatic cancer or colon cancer ( 100) Participants rand omized to the aged garlic experienced increase s in the number NK and NK ce ll activity a fter 6 months; however, they saw no
34 changes in quality of life (assessed by the Functional Assessment of Cancer Therapy questionnaire) ( 100) Echinacea Echinacea is a flowering plant in the same family as the daisy. Barrett et al. examined the effects of dried E chinacea root (10 g on the first day, then 5 g daily or a placebo) on the common cold ( 70) Results suggest that Echinacea had no effect on cold duration or symptom severity and there was inadequate power to detect significant results ( 70) The same group also examined the effects of an unrefined Echinacea at treating colds in a college age population and no sig nificant differences were found (101). Echinacea purpurea extract did help in cold prevention with athletes ( 102) Echinacea has been known to interact with commonly prescribed drugs and caution must be taken when supplementing. Vitamin D When skin is exposed to sun light more specifically ultraviolet light, a large amount of cholecalciferol is made from 7 dehydrocholesterol Cholecalciferol is then transport ed to the liver where it is hydroxylated to calcidiol (25 hydroxy vitamin D) From there calcitriol is ma de when calcidiol is hydroxylated in the kidneys to 1 25 hydroxyvitamin D Ergocalciferol (vitamin D2 ) can be found in cold water fish, nuts and egg yo lks. Cholecalciferol or D3 is typically found as the form in supplement s Vitamin D deficiency results in rickets or osteomalacia and is characterized by insufficient bone mineralization. Ginde employing the National Health and Nutrition Examination Surv ery (NHANES) found an inverse relationship between serum 25 hyd roxyvitaminD and the incidence of upper respiratory tract infections ( 103)
35 Kriesel et al. found that administering calcitriol (1, 25 dihydroxyl vitamin D) to hea lthy young participants who rec eived the flu vaccine resulted in no significant differences in viral titers when compared with the placebo ( 104) Carotenoids Carotenoids are colored pigments found mainly in plants. The main carotenoids found in the diet are: lycopene (found in tomatoes and processed tomatoes), lutein (peas, dark leafy cryptoxanthin (mandarins, apric ots, orange peppers), and beta carotene (carrots, broccoli, spinach and, apricots) ( 105) Carotenoids act as antioxidant agents and quench free radicals. Epidemiologic al studies have shown a link between a decreased development of cancer and a diet containing carotenoids ( 105) carotene (30 mg/d) would protect against long wave ultraviolet light (UV) exposure in young men as measured by delayed type hypersensitivity ( 106) The researchers hypothesized that because UV light decreases immune function carotenoids can provide photoprotection S upplementing participants with the antioxidant would protect them against an immune dysfunction ( 106) The researchers fo und their hypothesis to be correct protects against photosuppression of immune function ( 106) carotene on lymphocyte pro liferation but resul ts are conflicting Some r eports show increased T helper a nd natural killer cells carotene supplementation and others do not replicate those positive results ( 107) Possible reasons for discrepancies include d ifferent carotene and diffe rent a dministration lengths used in study protocols ( 105)
36 Research is limited regarding other carotenoids effect on immunity. A study by Corridan et al. provided participants with either 13.5 mg lycopene, carotene or a placebo for 12 weeks to s ee whether cell mediated immunity would be changed in free living adults. The amounts in intervention capsules were based on normal amounts of lycopene and carotene obtained from a diet rich in fruits and vegetables. The researchers saw no change in T cell subsets or lymphocyte proliferation, suggesting that healthy aged adults may not benefit from low doses of carotenoids ( 108). Green Tea Black, green and oolong tea are manufactured similarly except for the oxidative state of catechins ( 109) G reen tea is manufactured from fresh tea leaves that have been dried at elevated temperatures to avoid destroying antioxidant potential ( 109) Catechins and polyphenols are the antioxidants found in tea s; the latter also contributes to the flavor of the tea The polyphenol with the most antioxidant activity is epigallocatechin 3 gallate (EGCG). Green tea has been hypothesized to improve immunity, decrease cancer and, prevent inflammation ( 109) Human randomized clinical trials are lacking regarding the effec t s of green tea on colds and flu
37 Figure 1 1. T (1,4) galactooligosaccharide molecule Figure 1 2. galactosidase
38 CHAPTER 2 I NTRODUCTION Acute upper respiratory illnesses are common in college students. Over a six month period including the winter months, 80% to 90% of students recorded at least one d ay of cold/flu symptoms, 22% reported at least one healthcare visit, 41% missed at least one day of class, and 28% self reported that they did poorly on an exam due to upper respiratory illnesses ( 58, 72) Acute psychological stress, such as that induced by academic exams, is associated with dysregulated immune and gastrointestinal function ( 1, 65, 1 10) Classic studies showed a reduction in cellular immunity in first year medical students undergoing academic exams ( 66, 111) Activity of natural killer cells, one of the front line defenses against viral infections, was reduced during exams, and those students who scored higher for stressful life events had lower natural killer cell activity ( 66) Cytokine production was also associated with acute psychological str ess, i.e., increased (IL) 6 and IL 10 and decreased interferon with increased stress ( 110) In addition to the changes in immune function, academic stress is associated with increased cortisol production, abdominal pain, and a disruption of the intestinal microbiota resulting in lower viable lactic acid bacteria counts ( 1, 65) Psychological stress can modulate the gastrointestinal and immune systems through the hypothalamus pituitary adrenal (HPA) axis. Glucocorti coids (e.g., cortisol), which play a key role in the HPA, are likely responsible for the observed changes in natural killer cell function, dysfunction of the gastrointestinal tract, and altered microbiota ( 65, 112) Recently, the concept of the brain gut e nteric microbiota axis was introduced suggesting bidirectional communication between the gastrointestinal tract
39 and brain with input from the enteric microbiota ( 113) Another model where cortisol may be driving changes in immune function and enteric micro biota is that of immunosenescence ( 114) Vulevic et al. supplemented the diets of older adults with the preb iotic, GOS and observed increased numbers of beneficial bacteria (i.e., bifidobacteria), decreased production of IL 6, and increased natural killer cell activity ( 51) With these concepts in mind, it was of interest to determine whether GOS supplementation would reduce gastrointestinal dysfunction and the percentage of days of cold/flu in university students undergoing academic stress. If successful, such an intervention may help reduce personal, academic, and financial costs associated with cold or flu viruses.
40 CHAPTER 3 METHODS Subjects Participants, from the University of Florida, were recruited via listservs, flyers, posters, and announcements i n early fall of 2009. Participants were healthy full time students aged 18 years or older who suffered from at least one cold in the past year. Potential subjects were excluded if they 1 ) had chronic allergies involving the upper respiratory tract (chronic was defined as taking allergy medicine daily), milk allergy or immunosuppressive illnesses or treatments in the past year; 2 ) would not have Internet access for the duration of the protocol; 3 ) did not have at least one scheduled final exam; iv) were a cu rrent smoker; 4 ) received antibiotic therapy during the two months prior to the start of the study; 5 ) were unwilling to discontinue any fiber or potentially immune enhancing dietary supplements (e.g., prebiotics, probiotics, Echinacea, fish oil, vitamin E [>100% of the RDA or >15 mg/day]); or 6 ) had a cold on the day of enrollment. Subjects gave written informed consent and all study procedures followed were in accordance with the ethical standards of the University of Florida Institutional Review Board (Appendix A ) Experimental D esign Subjects (n=427) were randomly assigned to a supplement group during the first week in November of 2009 and followed for eight weeks including the time of fall final exams. Exams were held over the span of one week during the sixth week of the academic course schedule. Once students completed their last exam, subjects were on semester break through the remainder of the study.
41 The study was a pr ospective, randomized, parallel, double blind, placebo controlled trial. Subjects were proportionally stratified based on gender (50/50) and randomized via sealed envelopes to receive 0 g, 2.5 g, or 5.0 g GOS (Purimmune TM GTC Nutrition, Golden, CO). The st ratification and randomization schemes were generated by the study statistician who did not have direct contact with any subjects. Fiber Administration Protocol The GOS supplements were provided in coded packets that were similar in size and shape to comm ercially (sucrose) was added to the 0 g and 2.5 g packets so that all packets were the same weight and looked similar. A flow agent (silicon dioxide) was added to all packets to improve emptying of packag e contents. The supplement contain s 86% GOS with 21% DP2 and 65% DP3 and above. The final weights of the packets provided 0 g, 2.5 g, and 5.0 g GOS. The subjects were instructed to pour the contents of the packet into any beverage, mix well, and consume th e beverage in its entirety each day for eight weeks. Both the GOS and sucrose had a slight sweet taste. Subjects were unable to distinguish the GOS packets from the placebo. The proportion of subjects who thought they were receiving 0 g, 2.5 g, and 5.0 g o f GOS was 44%, 42%, and 14%, respectively with no differences among groups. Online Study Questionnaires On the day of randomization subjects met with investigators to have their height and weight measured, be instructed on study procedures, and obtain the supplement packets. Each subject was assigned a study number to use as their login user name for online questionnaires. Subjects provided their own password. User names were not linked to University records; however, paper records retained by the study co ordinators
42 were instructed on how to complete online baseline, daily, weekly, and final questionnaires. The questionnaires used throughout the eight week protocol were administered through the University of Florida hosted E Learning System (Blackboard Learning System, Washington D.C.). Questions regarding demographic information were contained on a short questionnaire at baseline. Daily online questionnaires asked about level of stress on a scale from 0 (no stress) to 10 (extremely stressed), consumption of the supplement to the nearest quarter packet, cold and flu symptoms, number and consistency of stools, hours of sleep, and antibiotic use. Subjects were asked to rate symptom intensity (0=none, 1=mild, 2=moderate, 3=severe) for running/congested nose, stiffness or chills, headache, cough, fatigue, fever, sore throat, achiness, and ear discomfort. The cold/flu symptom intensity (SI) score was calculated as the sum of the individual symptom intensities ( 115) Subjects were asked to score their stool consistency using the Bristol stool score ( 116) The stool scale, which ranged from 1 (hard to pass stools) to 7 (entirely liquid stools), was shown graphically on the question naire. The baseline and weekly questionnaires inquired about gastrointestinal symptoms using the Gastrointestinal Symptom Scale Score ( 2) On the final questionnaire, subjects were asked whether they received an annual influenza vaccination (2008 2009 and 2009 2010 flu seasons) or the 2009 H1N1 vaccination and to guess the amount of GOS (0 g, 2.5 g, 5.0 g) they received in their daily packets. Each daily questionnaire was to be completed by noon the following day and weekly and final questionnaires were to be completed within three days of online release. Study coordinators monitored subject compliance online on a daily basis and contacted
43 subjects via email or phone if they missed these time limits. Subjects were discouraged from completing questionnaires t hat were three days overdue. Questionnaires were no longer available to subjects once completed. Statistical A nalyses Compliance was assessed by summing the daily reported percentage of the supplement packet consumed and dividing by 56 days. Missing daily questionnaires were considered as an intake of 0% of the supplement packet. We assumed that there would be a lag time between the start of the supplement and effects on study outcomes; therefore, data from the first seven days were not used in analyses of supplement modulated outcomes in the cold/flu symptoms. Differences in demographic data and the average number of days with cold/flu symptoms among groups were analyzed using a one way ANOVA or the Kruskal Wallis one way analysis of variance on ranks. Tests of comparisons Categorical data wer e compared using the chi square statistic. Variables included in all models for daily cold/flu symptoms and SI were stress; lag stress with a one, two, and three day lag; gender; day of randomization; ethnicity; antibiotic use; vaccination against season al influenza for the 2009 2010 and 2008 2009 flu seasons; vaccination against H1N1; number of final exams; BMI; age; and hours of sleep. The two way interactions were also included. Variables and variable interactions that were not significant were elimina ted from the model. To adjust for personal differences in scoring cold/flu SI, subjects were categorized based on the average sum of their SI score from the first week of supplementation. Symptom intensity score categories were an average sum of less tha n 1 (60% of subjects), 1 to 3 (29% of subjects), and greater than 3 (11% of subjects) over
44 the first week of supplementation. The average SI sum for the remaining weeks of supplementation was significantly different among categories (<1, 1.2 + 0.01; 1 to 3 2.0 + 0.1; and >3, 3.4 + 0.1; P <0.0001) suggesting that the personal differences in scoring SI continued throughout the study. The daily average SI score from study weeks two through eight was compared among the three groups using a general linear mixed model with an autoregressive correlation structure to account for correlation of daily observation. Before analyzing data and while still blinded to treatment, the daily cold/flu SI scores for individuals were reviewed to identify a cut point above which a subject was likely to have a cold/flu (i.e., be sick). The cut point was to differentiate cold/flu symptoms from symptoms associated with stress. An SI score above six, which would represent a minimum of three symptoms with at least two of moderate and one of severe intensities, was selected as the SI that reflected a sick day. An indicator variable the response variable with an autoregressive correlation structure to acc ount for the distribution was used. Fifteen gastrointestinal symptoms divided among five categories (diarrhea syndrome, constipation syndrome, abdominal pain, indigestion syndrome, and reflux syndrome) were each rated from 1 = no discomfort to 7 = very severe discomfort. Scores within each category were summed and weekly gastrointestinal symp toms were
45 for the week prior to randomization. A generalized linear mixed model was then used to model gastrointestinal symptoms categories. Because the data were not norma lly distributed an exponential distribution was specified and an autoregressive correlation structure was used to correct for the correction over time that was caused by individuals being measured repeatedly over the course of the study. The degrees of fre edom were corrected using the Kenward Roger method to control the family wise error rate. For the weekly gastrointestinal symptoms model, the daily covariates were replaced with their average value for the week. For example, daily stress levels were replac ed with average weekly stress. The same process was repeated for hours of sleep. The lag daily stress variables were not used. Unless stated otherwise, data represent least squares means + SEM. Analyses were completed using SigmaPlot (version 11.0, 2008, S ystat Software, Inc., San Jose, CA) and the generalized linear mixed models were fitted using SAS v9.2 (SAS Institute, Cary, NC). CHAPTER 4 RESULTS Four hundred and eighty one subjects were consented and assessed for eligibility (Figure 3 1 ). Of this group 24 did not meet inclusion criteria, eight declined to participate, and 22 did not return for the randomization visit. Of the 427 remaining subjects, 141, 142, and 144 subjects were randomized to 0 g, 2.5 g, and 5.0 g of GOS, respectively. Two subjects dr opped out before beginning the supplement. Five subjects completed seven days of records or fewer and were therefore not included in any of the analyses. Data were analyzed on an intent to treat basis from 419 subjects who completed on average 55 out of 56 daily records and consumed an average of more than 93% of the supplement packets each day (Table 3 1).
46 There were no differences in the number of final exams, vaccination against H1N1 in 2009 or seasonal influenza in 2008 2009, antibiotic use, or subject characteristics among the groups with the exception of age and the number vaccinated against the 2009 2010 influenza (Table 3 1). Subjects consuming 5 .0 g of GOS each day were on average six months younger than those subjects who received the placebo. On average, subjects reported one cold/flu symptom per day. Subjects reported fatigue an average of 12 days over the eight weeks, followed by 10 days of running/congested nose, seven days of headache, seven days of cough, six days of sore throat, five days of achiness, stiffness or chills, two days of ear discomfort, and one day of fever. There were no differences among groups in the average number of cold/flu symptoms reported by subjects (data not shown). Average daily stress was not different among groups ( Table 3 1). Peak average stress occurred the week of final exams (5.0 + 0.2), and the lowest level of stress (1.3 + 0.2) was reported during semester break (i.e., the final weeks of the study). The average cold/flu SI score was significantly associated with s tress and GOS supplementation (P<0.0001). Stress was positively related to SI score and a lower SI score was associated with 2.5 g GOS across all levels of stress (Figure 3 2 ). At lower levels of daily stress, 5.0 g of GOS was also associated with a lower average SI score. The protective effect of 5.0 g of GOS was not detected at higher levels of stress. The probability of having a sick day was positively related to level of stress (P<0.001) and significantly associated with GOS supplementation for differe nt categories of BMI ( interaction of BMI and GOS, P =0.0002, Figure 3 3 ). Among individuals with a healthy weight (64% of the study population), those receiving 5 .0 g of
47 GOS reported a 40% reduction in the probability of having a sick day as compared with t hose receiving 0 g and 2.5 g GOS. This protective effect was not seen in overweight and obese individuals (30% of the study population); however, among these individuals, a lower percentage of sick days was reported in those receiving 2.5 g GOS compared wi th 0 g and 5.0 g. Across all treatment groups, s tress was positively related to the five gastrointestinal symptom score categories and was highly significant (P<0.01) for all but constipation syndrome ( P =0.1017, data not shown) Across all levels of stress, g astrointestinal symptom scores were significantly lower with GOS supplementation for all symptom categories (e.g., diarrhea syndrome [treatment group effect, P=0.0298], constipation syndrome [P=0.0342], abdominal pain [P=0.0058], and indigestion s yndrome [P=0.0003]) except reflux syndrome (P=0.2073, Table 2). There were no differences in the number of stools per day; however, daily stool consistency was softer with 2.5 g of GOS (Table 3 2).
48 Figure 3 1 Flow chart of subject recruitment, allocat ion and analysis.
49 Table 3 1 Subject characteristics and compliance 0 g GOS 1 (n=140) 2.5 g GOS (n=140) 5.0 g GOS (n=139) Gender (M/F) 69/71 70/70 68/71 Race/Ethnicity [n,(%)] 2 White Hispanic Asian Black 83 (59%) 21 (15%) 18 (13%) 18 (13%) 72 (51%) 28 (20%) 23 (16%) 17 (12%) 76 (55%) 25 (18%) 21 (15%) 16 (12%) Age (y) 20.1 + 0.1 19.9 + 0.1 19.6 + 0.1 3 BMI (kg/m 2 ) 23.8 + 0.3 23.9 + 0.3 23.4 + 0.3 Final exams (n) 2.9 + 0.1 2.8 + 0.1 2.9 + 0.1 Daily stress level (0=no stress, 10=extreme stress) 3.1 + 0.1 3.1 + 0.1 3.1 + 0.1 Hours of sleep each night 7.2 + 0.1 7.2 + 0.1 7.2 + 0.1 Average d on antibiotic over study period 0.018 + 0.0 06 0.015 + 0.00 5 0.016 + 0.00 4 Vaccinated against the 2008 2009 seasonal influenza [n,(%)] 27 (20%) 23 (17%) 26 (24%) Vaccinated against the 2009 2010 seasonal influenza [n,(%)] 4 13 (9%) 23 (16%) 31 (22%) Vaccinated against 2009 H1N1 [n,(%)] 24 (17%) 12 (9%) 16 (12%) Daily questionnaires completed (out of 56 d) 54.7 + 0.4 54.8 + 0.4 54.8 + 0.3 Percentage of supplement packet consumed per d 95% + 1% 93% + 1% 94% + 1% 1 GOS, galactooligosaccharides. Differences among groups were calculated using a one way ANOVA or Kruskal Wallis one way analysis of variance on ranks. Tests of treatment effects wer comparisons. Categorical data were compared using the chi square statistic. Data are reported as mean + SEM unless stated otherwise. 2 One subject in the 5.0 g GOS group did not report his race/ethnicity. 3 P=0.002 vs. 0 g GOS. 4 P=0.013. The observed proportions among groups were significantly different than expected (chi square test).
50 Table 3 2 Weekly gastrointestinal symptoms and daily stool characteristics in control subjects and subjec ts supplemented with galactooligosaccharides. 0 g GOS 1 (n=140) 2.5 g GOS (n=140) 5.0 g GOS (n=139) GSRS 2 Diarrhea syndrome 3 4.2 4 + 0.2 0 a 4.06 + 0.17 a,b 3.97 + 0.15 b Constipation syndrome 4 4.5 1 + 0.1 0 a 4.4 1 + 0.09 a,b 4.2 0 + 0.08 b Abdominal pain 5 4.6 5 + 0.08 a 4.3 2 + 0.07 b 4.5 4 + 0.08 a Indigestion syndrome 6 6.88 + 0.13 a 6.2 3 + 0.1 0 b 6.5 3 + 0.1 1 c Reflux syndrome 7 2.27 + 0.0 3 a 2.2 0 + 0.0 3 a 2.2 5 + 0.0 3 a Number of stools/d 1.5 0 + 0.0 3 a 1.47 + 0.0 3 a 1.5 1 + 0.0 3 a Daily stool consistency 8 3.29 + 0.0 3 a 3.4 2 + 0.0 3 b 3.25 + 0.0 3 a 1 GOS, galactooligosaccharides. 2 Gastrointestinal Symptom Response Scale (GSRS). Gastrointestinal symptoms were scored at baseline and weekly during the eight weeks of supplementation with 1=no discomfort to 7=severe discomfort. Individual scores were summed within each category. Symptom scores from baseline questionnaires were used to control for personal variation in responses to the subsequent eight treatment questionnaires. A generalized linear mixed model was used to model gastrointestinal symptom categories. Data for symptoms are bac k transformed least squares means and approximate SEMs for the back transformed least squares means. Data represent the least squares means + SEM unless stated otherwise. Means with different superscript letters within each row are significantly different at P < 0.05. 3 Diarrhea syndrome included questions regarding diarrhea, loose stools, urgent need for defecation. 4 Constipation syndrome included questions regarding constipation, hard stools, and feeling of incomplete evacuation. 5 Abdominal pain included questions regarding abdominal pain, hunger pains, and nausea. 6 Indigestion syndrome included questions regarding rumbling, bloating, burping, and gas. 7 Reflux syndrome included questions regarding heartburn and acid regurgitation. 8 Stool consistency repres ents the average daily Bristol stool score with a score of 1 denoting hard to pass stools and a score of 7 denoting an entirely liquid stool.
51 Figure 3 2 Cold/flu symptom intensity (SI) score by level of stress in individuals receiving 0 g, 2.5 g, 5.0 g galactooligosaccharides (GOS) The SI for nine cold/flu symptoms was scored for intensity from 0 (not experiencing) to 3 (severe) and summed to reflect the SI score. Level of stress was scored from 0 (no stress) to 10 (extremely stressed). The average S I score from the first week of supplementation was used to control for personal differences in scoring symptoms. Few subjects (n=17) had an average daily level of stress above 6. The daily average SI score was compared among groups using a general linear m ixed model. At each level of stress, bars with different letters are significantly different at P<0.05. The number on each bar denotes the number of subjects within each supplement group at each level of stress. Data represent the least squares means + SEM
52 Figure 3 3 Average percentage of days with a cold/flu symptom intensity (SI) score greater than 6 by BMI in individuals receiving 0 g, 2.5 g, 5.0 g galactooligosaccharides (GOS; interaction of BMI and GOS, P =0.0002) An SI score above six would repr esent a minimum of three symptoms with at least two of moderate and one of severe intensities. The average percentage of days from study weeks two through eight was compared among supplement groups using a generalized linear mixed model. Within each BMI ca tegory, bars with different letters are significantly different at P<0.05. Data represent least squares means + SEM.
53 CHAPTER 5 DISCUSSION AND CONCLUSION This study provides new information on the benefit of GOS on gastrointestinal and immune health outcomes in apparently healthy young adults undergoing an academic stress. As anticipated, in the eight weeks before, during, and after fall final exams, university undergraduates self reported daily stress that was positively related to gastrointestinal s ymptoms, cold/flu symptom intensity, and the percentage of days with cold/flu. G alactooligosaccharide supplementation modulated all of these symptoms. The dose of 2.5 g GOS was more effective than the 5.0 g dose at reducing symptoms associated with abdomin al pain (i.e., abdominal pain, hunger pains, and nausea) and indigestion syndrome (i.e., rumbling, bloating, burping and gas). This is of interest to note considering that GOS is fermented by the intestinal microbiota, which results in gas production ( 16 ) The higher dose of GOS (5.0 g) was better than the placebo in regards to indigestion syndrome (Table 2). Although 2.5 g of GOS was associated with slightly improved stool consistency, 5.0 g of GOS was associated with the decreased likelihood of constipation syndrome. To our knowledge this is the first study to demonstrate a benefit of GOS, a prebiotic, on modulating stress induced gastrointestinal dysfunction in adults. Previously, Diop a nd colleagues demonstrated a protective effect of a probiotic mix containing Lactobacillus acidophilus and Bifodobacterium longum on stress induced gastrointestinal symptoms ( 116) In that three month study, adults aged 18 to 60 years who were affected dai ly by symptoms of stress reported significantly less nausea and vomiting and abdominal pain with the probiotic compared with the placebo. The authors suggested that effects on the residual intestinal microbiota, intestinal barrier, and
54 immune system may ha ve modulated these symptoms ( 116) In our study, GOS may have worked through similar mechanisms or through the HPA axis ( 65, 112, 113) GOS intake has previously been shown to increase numbers of bifidobacteria and lactobacilli ( 25, 51) as well as fecal short chain fatty acids, which have been associated with decreased epithelial permeability ( 117, 118) It is possible that gastrointestinal symptoms actually contributed to th e reported level of stress. If this were the case, it may help to explain why 5.0 g of GOS lost its effectiveness at incr eased levels of stress (Figure 3 2 ). At lower levels of stress 5.0 g of GOS was associated with lower average cold/flu symptom intensit y, but this protective effect was lost at higher levels of stress. The 5 .0 g dose of GOS was not as effective as 2.5 g at reducing abdominal pain (Table 3 2). It could be that abdominal pain contributed to overall stress and ultimately risk of cold/flu sym ptoms. In healthy weight individuals, supplementation with 5 g of GOS reduced the average percenta ge of days of cold/flu (Figure 3 3 ). This protective effect was not seen in underweight, overweight, and obese individuals. It should be noted that because 6 4% of the subjects fell within the healthy weight category, we are most certain about these data. Additionally, emerging evidence points to differing microbiota including a reduction in bifidobacteria in obese versus lean individuals ( 39, 119, 120) If GOS improved gastrointestinal and immune function by changing the microbiota, then it is possible that we would observe different effects in individuals within different BMI categories. This study is one of the first to demonstrate a protective effect of GOS in relation to the percentage of days with cold/flu; however, there are a number of limitations that need to be addressed. Academic stress was used as a model of acute psychological
55 stress; though students only reported an average daily stress of three on a 10 point scale with a high of five during exam week. Populations with higher levels of stress may experience more severe gastrointestinal and immune dysregulation. With a greater degree of daily stress, the GOS associated health benefits may have been o bserved with a smaller sample size or alternately, not observed to the same degree. Another limitation is in how a day of cold/flu was defined as an SI su m of greater than six which would represent a minimum of three different symptoms of more than minima l intensity. Although this is a fairly conservative definition in comparison to that used in other studies ( 72, 121 123) the presence of cold/flu associated pathogens was not confirmed It was advantageous to report the percentage of days with cold/flu rather than incidence and duration because it is difficult to determine when one cold ends and a new cold begins. Another potential limitation is the use of Internet based questionnaires in place of traditional paper questionnaires. Although student responses to the Internet version of the questionnaires were not compared to that of the traditional paper version, these comparisons have been done with other questionnaires. Ritter and collea gues tested the reliability of 16 different health related instruments administered online compared to the traditional mailed paper questionnaires ( 124) In every case, the Internet based instrument appeared to be reliable and the Internet participation in the study was as good if not better than that of the traditional method ( 124) For the undergraduate students in our study, the convenience of completing questionnaires online and the ability of the investigators to follow subject compliance in real time likely contributed to the high compliance rate.
56 In summary, acute psychological stress, such as that associated with academic exams, was directly related to symptoms of gastrointestinal dysfunction and cold/flu. GOS supplementation attenuated these sympto ms. Future studies should determine the mechanisms by which GOS improves health outcomes within the brain gut enteric microbiota axis as these findings may have wide applicability beyond ac ademic s tress
57 APPENDIX A IRB APPROVAL LETTER
59 APPENDIX B IRB INFORMED CONSENT
69 APPENDIX C RECRUITMENT MATERIALS AND QUESTIONNAIRES
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86 BIOGRAPHICAL SKETCH Christi food science and human nutrition with an emphasis in d ietetics from the University of Florida in 2009 and a n nutritional sciences in 2011. She has worked for Dr. Bobbi Langkamp Henken for 4 years on 6 clinical nutrition studies. In the fall of 2011 she plans to become a registered dietitian by completing her dietetic internship. Christine is married to a wonderf ul man and has a fabulous Yorkshire terrier named Sophie. Christine h as hi gh hopes that one day galactooligosaccharides will be taken routinely by academically stressed undergraduate students.