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Mutagens in feces of vegetarians and non-vegetarians Bergstrom, Danielle Cantin 1982

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MUTAGENS IN FECES OF VEGETARIANS AND NON-VEGETARIANS by -DANIELLE CANTIN BERGSTROM B'.H.Sc, Laval U n i v e r s i t y , 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DIVISION OF HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 (c) Danielle Cantin Bergstrom, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of H u m n/U fV utfi it i <\*J SCHOOL, or Uo/Ti/S £CO/UOSY)/C$ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Mutagens in feces have been suggested to be an indica t o r for r i s k of colon cancer. Groups consuming vegetarian d i e t s are known to have lower mortality from colon cancer. The purpose of t h i s study was to assess muta-genic a c t i v i t y i n feces of persons h a b i t u a l l y consuming vegetarian or non-vegetarian diets and to try to i d e n t i f y dietary factors or other health habits which contributed to f e c a l mutagenicity. Eleven s t r i c t vegetarians, six ovo-lacto vegetarians and twelve non-vegetarians, a l l from the Greater Vancouver area, p a r t i c i p a t e d i n t h i s study. Data on c e r t a i n demographic va r i a b l e s and health habits, as well as dietary intake (food frequency and food records), were taken. One f e c a l sample was c o l l e c t e d from each subject for the study. Aqueous extracts of the feces were prepared and analyzed for mutagens using the f l u c t u a t i o n t e s t with Salmonella typhimurium TA100 and TA98. Levels of mutagenicity on each organism were then s t a t i s t i c a l l y c orrelated with frequency of consumption of food groups, nutrient intake, demographic data and health habits. Ovo-lacto vegetarians and s t r i c t vegetarians, as groups, had s i g n i f i -cantly lower l e v e l s of f e c a l mutagens than non-vegetarians i n the TA100 assay. With TA98, only the s t r i c t vegetarians had lower l e v e l s of mutagens compared to the non-vegetarians. The presence of several d i f f e r e n t mutagenic compounds was indicated. S i g n i f i c a n t negative c o r r e l a t i o n s were found with mutagenicity on TA98 for a l l subjects with the following d i e t a r y v a r i a b l e s : f r u i t s and j u i c e s , f i b e r and i r o n . Similar negative c o r r e l a t i o n s were found for t o t a l carbo-hydrate and Southgate f i b e r intakes and mutagenicity on TA100. Within the i i i group of non-vegetarians, there were negative c o r r e l a t i o n s with mutagenicity on TA98 and t o t a l p rotein and with mutagenicity on TA100 and calcium. With the demographic va r i a b l e s and health habits, no clear pattern emerged to indicate factors which would predict lowered mutagenicity for a l l subjects. It i s concluded that vegetarians have lower l e v e l s of f e c a l mutagenicity and that several dietary factors are l i k e l y to contribute to t h i s phenomenon. 0 i v TABLE OF CONTENTS Abstract i i L i s t of Tables v i i L i s t of Figures ix Acknowledgements x I Introduction 1 II Review of the L i t e r a t u r e 3 A. Epidemiology of Colon Cancer 3 1. Incidence and M o r t a l i t y 3 2. Correlations with Other Cancers and Diseases 4 3. Role of Environmental Factors i n Colon Carcinogenesis 4 4. Diet as an E t i o l o g i c a l Factor i n Colon Carcinogenesis 7 B. Dietary Hypotheses 8 1. Fat and Animal Protein 8 a. C o r r e l a t i o n Studies Between Human Intake of Fat and Animal Protein and Colon Cancer Incidence 8 b. Case-Control Studies i n Relation to Human Intake of Fat and Animal Protein and Colon -Carcinogenesis .' 10 c. Proposed Mechanism of Action of Fat i n Colon Carcinogenesis 11 d. Human Studies of Fecal F l o r a and Steroids 12 e. Animal Studies Implicating Fat and Fecal Steroids i n Colon Carcinogenesis 13 2. Fiber 14 a. D e f i n i t i o n of Fiber 14 b. Protective Role of Fiber i n Colon Carcinogenesis 16 c. E f f e c t s of Fiber on Transit Time 16 d. E f f e c t s of Fiber on Fecal Bulk and D i l u t i o n of Possible Carcinogens 17 e. Correlation Studies Between Human Intake of Fiber and Colon Cancer Incidence 19 f. Animal Studies on the E f f e c t s of Fiber on Colon Carcinogenesis 20 g. Other Possible E f f e c t s of Fiber on Colon Carcinogenesis 21 V . 3. Vegetables 21 C. Fecal Mutagens and Colon Cancer 22 1. Presence of Mutagenic Compounds i n Feces of Individuals and Populations 22 2. Methods for Evaluating Fecal Mutagenicity 25 III Materials and Methods 27 A. Groups Sampled 27 B. C o l l e c t i o n of Dietary Data and Other Information 28 1. Questionnaire 28 2. Four Day Food Records 28 C. C o l l e c t i o n of Fecal Samples 29 D. Fecal Analyses 29 1. Preparation of Extracts 29 2. Mutagen Analyses 29 E. A d d i t i o n a l Measurements 31 F. Data Analyses 31 1. Data from Fecal Samples 32 a. Fecal Extracts from Groups Sampled 32 b. Other Experimental Measurements 33 2. Dietary Data and Other Information 33 a. Questionnaire 33 b. Four Day Food Records 38 c. Additional Dietary Analyses 39 3. Level of Significance 39 IV Results 40 A. Fecal Mutagenicity Data 40 1. Comparison of Mutagenicity in Extracts from Vegetarians and Non-Vegetarians 40 2. C o r r e l a t i o n of Mutagenicity with Other Parameters of Fecal Samples 47 B. Dietary Data and Other Information 51 1. Questionnaire, Part I 51 a. Demographic Variables, Dietary Practices and Health Status and Habits of Groups Sampled 51 v i b. C o r r e l a t i o n Between Mutagenicity and Dietary P r a c t i c e s , Health Status and Habits of Groups Sampled 59 2. Questionnaire, Part II 63 a. Comparison of Food Frequencies Between Diet Groups 63 b. Correlation Between Food Frequencies and Mutagenicity with TA100 and TA98 68 3. Four Day Food Records 74 a. Comparison of Nutrient Intake with the Dietary Standard for Canada 74 b. Comparison of Nutrient Intake Among Diet Groups 76 c. Correlation of Nutrient Intake with Mutagenicity 80 4. V a l i d a t i o n of Food Frequency Questionnaire 84 V Discussion 87 A. Fecal Mutagenicity 87 B. Relationship Between Fecal Mutagenicity and Age, Sex and Miscellaneous Dietary and Health Data 91 C. Dietary Data and Fecal Mutagenicity 93 VI Conclusion 97 Bibliography 99 Appendices 110 A. Consent Form 110 B. Explanation of the Project and Instructions to Follow .. I l l C. Sample of Food Record Sheet 112 D. Questionnaire 113 E. Dietary Standard for Canada, Revised 1975 132 F. Raw Data: Food Frequency Intake by Food Items for Each Subject 133 G. Raw Data: D a i l y Nutrient Intakes for Each Subject Derived from Four Day Food Records 135 v i i LIST OF TABLES 1. Individuals at Increased Risk for Colon Cancer 5 2. Dietary Fiber '. 15 3. L i s t of Foods Included i n Each Food Group from Food Frequency 34 4. Number of Revertant Tubes out of 50 i n Dependence of the Fecal Extract Concentration for TA100 41 5. Number of Revertant Tubes out of 50 i n Dependence of the Fecal Extract Concentration for TA98 42 6. Comparison of Diet Groups with the Mann-Whitney U Test (One-Tailed) 46 7. Spearman Rank Co r r e l a t i o n Between Mutagenicity with TA100 and TA98 48 8. Wet Weight, Percent Dry Weight and pH of Fecal Samples from Three Diet Groups ^9 9. Spearman Rank Co r r e l a t i o n Between Mutagenicity and pH of the Fecal Homogenates 50 10. Frequency D i s t r i b u t i o n of Demographic Variables by Diet Groups 52 11. Frequency D i s t r i b u t i o n of Dietary Habits Variables by Diet Groups 53 12. Frequency D i s t r i b u t i o n of Health Status and Habits Variables by Diet Groups 56 13. Spearman Rank Correlations Between Questionnaire Variables and Mutagenicity with TA100 61 14. Spearman Rank Correlations Between Questionnaire Variables and Mutagenicity with TA98 62 15. Comparison of Food Frequencies Between Diet Groups by the Mann-Whitney U Test (Two-Tailed) 64 16. Spearman Rank Cor r e l a t i o n Between Food Frequencies and ' Mutagenicity with TA100 (Two-Tailed) 69 17. Spearman Rank Cor r e l a t i o n Between Food Frequencies and Mutagenicity with TA98 (Two-Tailed) 7 2 18. Mean Nutrient Intake Per Day for Each Subject Derived from Four Day Food Records 75 v i i i 19. Comparison of Nutrient Intake Among Diet Groups by the Mann-Whitney U Test (Two-Tailed) 77 20. Spearman Rank Cor r e l a t i o n Between Nutrient Intake by Diet Groups and Mutagenicity with TA100 (Two-Tailed) 82 21. Spearman Rank Co r r e l a t i o n Between Nutrient Intake by Diet Groups and Mutagenicity with TA98 (Two-Tailed) 83 22. Pearson C o r r e l a t i o n Between Frequency of Intake from Questionnaire and Four Day Food Record (One-Tailed) 85 IX LIST OF FIGURES 1. Average Number of Revertant Tubes out of 50 i n Dependence of the Extract Concentration Using Salmonella typhimurium TA100 .... 44 2. Average Number of Revertant Tubes out of 50 in Dependence of the Extract Concentration Using Salmonella typhimurium TA98 45 2 3. Nomograph for Body Mass Index (kg/m ) 58 ACKNOWLEDGEMENTS Several people have contributed t h e i r valuable time and energy to help make t h i s thesis a r e a l i t y . I would l i k e to thank my senior advisor, Dr Harriet Kuhnlein, for her guidance and support throughout the course of th i s study; Dr Urs Kuhnlein for h i s assistance and advice i n regard to the mutagen analysis; Dr Melvin Lee and Dr Joseph Leichter f or t h e i r advice i n the thesis preparation. Special thanks are expressed to Dr F.P. Glick from the Department of Health Care and Epidemiology for h i s recommendations on s t a t i s t i c a l pro-cedures; Lewis James and Frank Flynn for t h e i r assistance with computer programs; the s t a f f of the B.C. Cancer Research Centre f o r t h e i r encouragement; Rosemary Johnson for typing this thesis; and to the par-t i c i p a n t s , whose cooperation has made the study possible. The f i n a n c i a l support of the Summer Graduate Student Bursary (1978) and the B.C. Cancer Foundation Summer Studentship (1979) i s g r a t e f u l l y acknowledged. I am e s p e c i a l l y grateful to my husband, Craig Bergstrom, for his support, patience and love always, but e s p e c i a l l y during the duration of t h i s study. 1 CHAPTER I INTRODUCTION It i s c u r r e n t l y believed that 90% of human cancers are caused by environmental factors ( D o l l , 1977; Wynder and Gori, 1977). The environ-mental factors which most reasonably apply to colon cancer, accounting for 2-4% of a l l deaths i n the Western countries, are of dietary o r i g i n . Several studies support the involvement of dietary components i n the etiology of t h i s disease (Wynder et a l . , 1966; Correa and Haenszel, 1978; Wynder, 1975). Diets low i n f i b e r and/or high i n animal fats and meat have been associated with a high r i s k for colon cancer ( D o l l , 1979). Vegetarianism, on the other hand, seems to be protective against colon cancer ( D o l l , 1979; P h i l l i p s , 1975; Walker and B u r k i t t , 1976). To reduce the incidence of colon cancer, i t i s of great importance to obtain more d e t a i l e d information about the food items and nutrients which may a f f e c t colon carcinogenesis. However, the long time span between the i n i t i a t i o n of the cancer l e s i o n and the diagnosis of colon cancer i s not conducive to c o n t r o l l e d human studies. Research would be greatly f a c i l i -tated i f the food metabolites promoting cancer could be i d e n t i f i e d and measured. It has been suggested that colon cancer i s caused by carcinogens which are formed during the digestive process. Diet would, i n large part, deter-mine the formation of these carcinogens, e i t h e r by acting as substrate for t h e i r production i n the gut or by modifying t h e i r e f f e c t s ( D o l l , 1979). Bruce et a l . (1977) and Varghese et a l . (1977) f i r s t demonstrated the presence of mutagens i n human feces and found that the mutagens were lowered by dietary f a c t o r s , such as ascorbic a c i d , vitamin E and f i b e r (Bruce et a l . , 2 1979). Ehrich et a l . (1979) have also observed that a greater percentage of white South A f r i c a n s , at high r i s k for colon cancer, excreted f e c a l mutagens compared to urban and r u r a l blacks, at low r i s k for t h i s disease. S i m i l a r l y , f e c a l mutagens were found more frequently i n high r i s k New York i n d i v i d u a l s consuming a Western di e t than i n F i n n i s h residents (low r i s k ) whose die t was high i n f i b e r (Reddy et a l . , 1980). It i s possible that f e c a l mutagens play a major ro l e i n colon cancer e t i o l o g y , since 90% of mutagenic substan-ces exhibit carcinogenic a c t i v i t y (McCann and Ames, 1975; P o i r i e r and Simmon, 1976). The purpose of t h i s study was to measure the mutagenic a c t i v i t y present i n f e c a l samples of vegetarian subjects and of subjects on "normal" non-vegetarian d i e t s . Detailed analyses of the d i e t and health habits were also investigated to i d e n t i f y factors i n h i b i t i n g or enhancing f e c a l muta-gen i c i t y . 3 CHAPTER II REVIEW OF THE LITERATURE A. EPIDEMIOLOGY OF COLON CANCER 1_. Incidence and M o r t a l i t y Bowel cancer represents a major health concern i n most Western count-r i e s . It causes an estimated 45,000 deaths each year i n the United States alone (Berg et a l . , 1973; Devesa and Silverman, 1978). In Canada, the age-standardized incidence rates per 100,000 of the population are 24.2 for males and 26.9 for females. Deaths caused by colon cancer are i n the range of 16.2 and 16.3 per 100,000 of the population f o r males and females, res-p e c t i v e l y (Ministry of Supply and Services, 1981). Colon cancer incidence i s c u r r e n t l y showing a s l i g h t increase i n white males, more pronounced i n non-white males, whereas i t i s decreasing i n white females but r i s i n g i n non-white females. Colonic cancer has a sex r a t i o that favors women before menopause but which approaches unity a f t e r meno-pause. For r e c t a l cancer, a decrease i s noted for a l l females and white males, while the incidence i s increasing f o r non-white males. It i s more prevalent i n males than i n females (Devesa and Silverman, 1978). In general, countries with high rates of colon cancer also exhibit high rates of r e c t a l cancer (Wynder et a l . , 1967). It has been suggested, how-ever, that colon and r e c t a l cancer may have a d i f f e r e n t e t i o l o g y for the following reasons: a. The low incidence of colon cancer i n Japan does not correspond with a low rate of r e c t a l cancer (Wynder and Shigematsu, 1967). 4 b. Male and female rates for colon cancer are generally s i m i l a r , whereas those for the rectum show considerable differences (MacLennan, 1979). 2^ . C o r r e l a t i o n with Other Cancers and Diseases Colon cancer has been p o s i t i v e l y c o r r e l a t e d with breast cancer, sugges-t i n g a common etio l o g y (Drasar and I r v i n g , 1973). A p o s i t i v e c o r r e l a t i o n between cancer of the colon and cancer of the pancreas, kidney and prostate has also been established (Wynder and Reddy, 1974). Gastric cancer i n c i -dence and death rates are strongly negatively co r r e l a t e d with those of colon cancer i n several countries (Drasar and I r v i n g , 1973; Wynder and Shigematsu, 1967). Among other diseases c o r r e l a t e d with colon cancer i s myocardial i n f a r c t i o n , which has been associated with factors such as smoking, eleva-ted blood c h o l e s t e r o l l e v e l s , obesity and hypertension. However, no l i n k has been established between these factors and colon cancer (Wynder and Shigematsu, 1967). Patients s u f f e r i n g from the diseases l i s t e d i n Table 1 are also at high r i s k f o r colon cancer ( L i p k i n , 1978). 3. Role of Environmental Factors i n Colon Carcinogenesis The r e s u l t s of several epidemiological studies of colon cancer suggest that environmental factors are strongly involved i n the development of t h i s disease ( B u r k i t t , 1971; Drasar and I r v i n g , 1973; Haenszel and Correa, 1971; Wynder and Shigematsu, 1967; Wynder and Reddy, 1974). These studies have shown that the incidence of colon cancer i s higher i n i n d u s t r i a l i z e d countries such as the United Kingdom, Northern and Western Europe, New Zealand and North America. Japan and Finland are exceptions. It i s com-Table 1 Individuals at Increased Risk for Colon Cancer F a m i l i a l Polyposis Syndromes: - Inherited adenomatosis of colon and rectum - Gardner's syndrome ( O l d f i e l d ) - Turcots syndrome - Diffuse g a s t r o i n t e s t i n a l polyposis Colonic adenomas Previous colon, breast, endometrial or bladder cancer S i t e - s p e c i f i c colon cancer Cancer family syndrome Inflammatory bowel disease Common va r i a b l e immune deficiency Residence i n geographic areas having high frequencies of colon cancer From L i p k i n (1978). 6 p a r a t i v e l y low i n A f r i c a , South America (except i n Argentina and Uruguay), Asia and Puerto Rico, which are countries of r e l a t i v e l y lower economic status (Wynder and Shigematsu, 1967). Bibra (1976) points out that "the differences i n incidence are so great that the findings cannot be a t t r i b u t e d to differences i n diagnostic procedures or the e f f i c i e n c y of medical care". Thus, i n the United States, which i s a country with good diagnostic f a c i l i -t i e s , colon cancer incidence i s higher" i n the north than i n the south and more prevalent i n urban than i n r u r a l areas (National Cancer I n s t i t u t e , 1971). Socioeconomic f a c t o r s do not seem to have an e f f e c t on incidence within the high r i s k areas. However, i n a low r i s k area such as Japan, populations i n good socioeconomic p o s i t i o n s exhibit a higher incidence of cancer of the bowel (Reddy and Wynder, 1973). This i s also seen i n C a l i (Colombia), where a f o u r f o l d excess r i s k e x i s t s i n upper classes for both sexes (Haenszel and Correa, 1971). Most of the evidence supporting the involvement of environmental fac-tors i n the etiology of colon cancer stems from migrant studies. Groups moving to an area of d i f f e r e n t r i s k for t h i s disease seem to acquire the r i s k of the new place of residence i n a r e l a t i v e l y short period of time. This i s well i l l u s t r a t e d by the studies of Japanese migrating to Hawaii (Issei) and those of Japanese descent born i n Hawaii ( N i s e i ) . A higher i n -cidence of colon cancer was observed in subjects who no longer followed the pra c t i c e of eating at least one Japanese meal per day (Haenszel and Kurihara, 1968; Correa and Haenszel, 1978). A s i m i l a r trend was seen i n Japanese i n C a l i f o r n i a (Buell and Dunn, 1965) and i n Norwegian, P o l i s h , Puerto Rican and Chinese immigrants to the United States (Sherlock et a l . , 1975). Migration within the United States a l t e r s colon cancer incidence i n a manner s i m i l a r to that of foreign migrants, mortality again being r e f l e c -t i v e of the place of residence rather than of that of b i r t h (Haenszel and 7 Dawson, 1965). 4. Diet as an E t i o l o g i c a l Factor i n Colon Carcinogenesis Several investigators consider d i e t the most l i k e l y environmental fac-tor to be involved i n colon cancer et i o l o g y (Wynder et a l . , 1966; Correa and Haenszel, 1978; Wynder, 1975). As pointed out by D o l l (1979), there i s not as yet a s t r i c t proof that an element of n u t r i t i o n i s carcinogenic or anticarcinogenic. However, the evidence on which the r e l a t i o n s h i p of diet and cancer i s based comes from: a. Retrospective studies of a f f e c t e d patients and c o n t r o l s . b. Prospective studies of groups of i n d i v i d u a l s with known dietary status. c. C o r r e l a t i o n studies between d i f f e r e n t countries in time with the consumption of food and drink and incidence of cancer. d. Laboratory demonstrations that a dietary substance i s mutagenic or carcinogenic. From these studies, several constituents of the diet have been linked to colon cancer, notably diets high i n f a t and meat (Armstrong and D o l l , 1975; Wynder et a l . , 1969), high i n beef (Correa and Haenszel, 1978), low i n f i b e r ( B u r k i t t , 1978), high i n c h o l e s t e r o l (Liu et a l . , 1979), low i n c e r t a i n vegetables (Bjelke, 1973), and high i n animal protein (Gregor et a l . , 1969). The major dietary factors implicated i n cancer etiology w i l l be reviewed i n the following section of t h i s chapter. 8 B. DIETARY HYPOTHESES 1_. Fat and Animal Protein a. C o r r e l a t i o n Studies Between Human Intake of Fat and Animal Protein and Colon Cancer Incidence Wynder and associates f i r s t emphasized the worldwide c o r r e l a t i o n of colon cancer incidence and f a t consumption (Wynder, 1975; Wynder and Shigematsu, 1967). Colon cancer was noted to be lower i n Japan. An analysis of the intake of nutrients i n the United States and i n Japan i n 1962 revealed that the Japanese di e t was lower i n saturated fats than the American d i e t . However, second generation of Japanese i n Hawaii, whose diet content i n fat was f a i r l y s i m i l a r to the American d i e t , were at an increased r i s k for colon cancer. The authors suggested that a high dietary fat intake may contribute to the r e l a t i v e l y high r i s k among the Jewish population of New York. Sup-porting t h e i r theory was the fact that Seventh Day Adventists i n C a l i f o r n i a had r e l a t i v e l y low rates of colon cancer, and t h e i r diet was low i n saturated fats and meat. They also had low serum c h o l e s t e r o l l e v e l s . Gregor and associates (1969) cor r e l a t e d food intake with death rates i n 28 countries for two periods, 1947-1948 and 1962-1963. They found a high c o r r e l a t i o n between death from colon cancer and animal protein consumption. The intake of f i b e r and f a t was not analyzed. Gregor et a l . (1969) hypothe-sized that diet could play a r o l e i n promoting rather than i n i t i a t i n g colon cancer. Dunn and B u e l l (1966) made the same hypothesis while studying time trends i n Japanese immigrants to C a l i f o r n i a . Drasar and Irving (1973), while comparing mortality and incidence of 9 colon cancer i n 37 countries with FAO dietary data, found a s i g n i f i c a n t c o r r e l a t i o n with intake of animal protein and t o t a l f a t . A p o s i t i v e corre-l a t i o n between intake of t o t a l fat and vegetable f a t with the incidence of colon cancer i n the United States was also established by Enig et a l . (1978). According to Reddy (1980), these data indicate that t o t a l dietary f a t , rather than the type of f a t , i s a determinant of colon cancer i n c i -dence. Armstrong and D o l l (1975) reviewed the data of cancer incidence and FAO food disappearance s t a t i s t i c s . They observed that meat and animal pro-t e i n consumption were the v a r i a b l e s most highly correlated with colon can-cer. Total fat was also c o r r e l a t e d but to a lesser degree. S i m i l a r l y , strong epidemiological c o r r e l a t i o n s with meat, i n p a r t i c u l a r beef, have been reported (Berg and Howel, 1974). Enig et a l . (1978) have c r i t i c i z e d the animal fat hypothesis, b e l i e v -ing that i t i s an increase i n vegetable f a t , mostly containing trans-double bonds, which has contributed to increased f a t consumption i n the United States. They have suggested that processed vegetable f a t s should be inves-tigated as possible e t i o l o g i c a l factors i n colon cancer. Also against the animal fat hypothesis i s the f a c t that Finland has a r e l a t i v e l y low i n c i -dence of colon cancer despite a high animal fat intake (Enig et a l . , 1978). ' Challenging these arguments, Correa (1981) has pointed out that the marked increase i n vegetable f a t intake was not accompanied by a reduction i n other f a t consumption. It i s therefore d i f f i c u l t to assess which type ( i f any one i n p a r t i c u l a r ) i s c o n t r i b u t i n g to colon cancer incidence, since the o v e r a l l fat intake i s high. However, no c o r r e l a t i o n with t o t a l f a t intake and colon cancer incidence was found i n an epidemiological study including 4,137 subjects of f i v e ethnic groups i n Hawaii (Kolonel et a l . , 10 1981). Enstrom (1975) has also taken strong exception to the hypothesis that beef and fat consumption i s involved i n colon cancer etiology. His c r i t i -cisms are based on h i s review of incidence and m o r t a l i t y trends re l a t e d to differences i n socioeconomic status, education, geographic locations and consumption of these two dietary components. He also r e f e r s to the rather large increase i n beef intake over the l a s t 40 years, as compared to only a s l i g h t increase in colon cancer for the same period. While Seventh Day Adventists, who eat very l i t t l e meat, have a low incidence of bowel cancer, Mormons with s i m i l a r rates for t h i s disease are among the highest consumers of beef i n the United States (Lyon et a l . , 1976). b. Case-Control Studies i n Relation to Intake of Fat and Animal Protein and Colon Carcinogenesis Very few epidemiological studies comparing cancer patients with controls have demonstrated a c l e a r a s s o c i a t i o n between colon cancer and meat or fat consumption (Armstrong and D o l l , 1975; Enig et a l . , 1978). Only the studies of Haenszel et a l . (1973), i n a series of 179 cases and 357 c o n t r o l s from Hawaii, and a study of 41 subjects and 123 c o n t r o l s by P h i l l i p s (1975), have confirmed t h i s hypothesis. Studies by Wynder and Shigematsu (1967) i n New York, by Higginson (1966) i n the Midwest, and by Graham et a l . (1978) i n Buffalo have found no differences i n meat intake between cases and controls. A recent study among Japanese i n Japan f a i l e d to show a higher r i s k of colon cancer for meat consumers (Haenszel et a l . , 1980). 11 c. Proposed Mechanisms of Action of Fat i n Colon Carcinogenesis If d i e t a r y f a t i s indeed associated with colon cancer, by which mechanisms does i t t r a n s l a t e to higher cancer r i s k ? It has been hypothe-sized by Aries et a l . (1969) and H i l l et a l . (1971) that: 1. The amount of fat determines both the concentration of acid and neutral s t e r o l substitutes in the large bowel and the composition of the microflora on such substrates. 2. The gut m i c r o f l o r a metabolize acid and neutral s t e r o l s to carcino-gens active i n the large bowel. Reddy (1981) has summarized the reasons why the p o t e n t i a l carcinogenic a c t i v i t y of c e r t a i n b i l e acids and neutral s t e r o l s has generated so much i n t e r e s t : 1. They are s i m i l a r to carcinogenic p o l y c y c l i c aromatic hydrocarbons. 2. They may be converted chemically to 3-methylcholanthrene. 3. The f u l l aromatization of the b i l e acid nucleus would y i e l d a carcinogenic metabolite based on cyclopentaphenanthrene. 4. The human gut f l o r a , e s p e c i a l l y a group of Clostridia, has been shown to achieve p a r t i a l aromatization of the s t e r o l r i n g . 5. Several b i l e acids have induced carcinomas at the s i t e of i n j e c t i o n i n experimental animals. 6. Microflora-mediated reactions are u n l i k e l y to y i e l d p o l y c y c l i c hydrocarbons but are more l i k e l y to give products that act as colon tumor promoters and accelerators rather than complete carcinogens (Reddy, Weisburger and Wynder, 1978). d. Human Studies of Fecal F l o r a and Steroids 12 Several studies have been c a r r i e d out to determine whether the composition of mic r o f l o r a i n the colon may produce carcinogenic or c o c a r c i -nogenic substances from food, g a s t r o i n t e s t i n a l secretions or metabolites (Sherlock et a l . , 1980). S i g n i f i c a n t differences have been reported i n f e c a l f l o r a and metabolic constituents from people i n d i f f e r e n t parts of the world (Reddy and Wynder, 1973). Fecal samples from people l i v i n g i n areas with high m o r t a l i t y rates such as England, Scotland and the United States, contained higher amounts of anaerobic b a c t e r i a than feces from people of low r i s k areas such as India, Japan and Uganda. The same observations were noted i n people on a Western di e t versus people on vegetarian d i e t s (Aries et a l . , 1969; H i l l et a l . , 1971; Reddy and Wynder, 1973). Acid and neu-t r a l s t e r o l concentrations, as well as f e c a l secondary b i l e a c i d s , were higher i n meat consumers (Reddy and Wynder, 1973; H i l l and A r i e s , 1971). Similar changes were observed i n volunteers a l t e r n a t i n g from high meat to low meat di e t s (Reddy et a l . , 1975). Populations at high r i s k for colon cancer have been shown to have greater f e c a l content of anaerobic Clostridia and Baotevioides with fewer Lactobacilli (Reddy et a l . , 1975). H i l l (1975) has observed that these two organisms could contribute to the development of compounds s t r u c t u r a l l y s i m i l a r to some known carcinogens, since they are the only ones known to dehydrogenate the st e r o i d nucleus. When colon cancer patients were compared to control p a t i e n t s , a higher amount of f e c a l b i l e acids was found i n the former group ( H i l l et a l . , 1975; Reddy and Wynder, 1977). An increase i n dehydrogenating Clostridia ( H i l l et a l . , 1975) and f e c a l b a c t e r i a l 7a-dehydroxylation of primary b i l e acids was also observed (Reddy and Wynder, 1977). 13 Other studies, however, have reported very few or no s t a t i s t i c a l l y s i g n i f i c a n t differences i n f e c a l f l o r a of i n d i v i d u a l s on Japanese and Wes-tern diets (Finegold, 1974), between s t r i c t vegetarian Seventh Day Adven-t i s t s and i n d i v i d u a l s on an "American" d i e t (Goldberg et a l . , 1974), or between vegetarian and non-vegetarian Seventh Day Adventists (Finegold et a l . , 1977). Maier et a l . (1974) have found minor changes in the f e c a l f l o r a i n volunteers r o t a t i n g between normal, low and high meat d i e t s . Many short-term studies with volunteers whose die t s were manipulated have shown no changes in the f e c a l f l o r a , with the exception of normal adults fed a chem-i c a l l y defined, residue-free diet ( H i l l , 1981). It was suggested by the same author that periods up to 12 months may be needed to bring about chan-ges i n the f e c a l f l o r a . e. Animal Studies Implicating Fat and Fecal Steroids i n Colon Carcinogenesis Animal studies support the hypothesis that b i l e acids may be pro-moters of carcinogenesis rather than carcinogens and these studies have been reviewed by Reddy (1981). Acid s t e r o l s have been shown to act as cocarcino-gens, whether ingested (Raicht et a l . , 1978), r e c t a l l y i n s t i l l e d (Narisawa et a l . , 1974), s u r g i c a l l y diverted through the colon (Chomchai et a l . , 1974) or adsorbed to cholestyramine (Nigro et a l . , 1973). However, the mechanisms by which b i l e acids promote colon cancer are not yet well under-stood (Kay, 1981). Several animal model studies indicate that t o t a l dietary f a t rather than the type of fat demonstrates a promoting role i n colon carcinogenesis (Reddy, 1981). Nigro et a l . (1975) have shown that when rats were treated with azoxymethane, they exhibited more tumors and metastases when fed a 14 high beef fat diet than those on a low f a t d i e t . In another study by Reddy'et a l . (1976), animals were given the same dietary regimen for two generations before dimethylhydrazine treatment. More tumors were induced by DMH i n animals fed either 20% l a r d or 20% corn o i l compared to those fed 5% f a t . F344 rat s fed combination di e t s of e i t h e r high p r o t e i n , high fat of animal o r i g i n or from vegetable sources had more tumors when treated with DMH than the controls fed lower amounts of p r o t e i n and f a t of either o r i g i n (Reddy, Narisawa and Weisburger, 1976). The studies of Reddy, Watanabe and Weisburger (1977), Bansal et a l . (1978) and Rogers and Newberne (1973) also point to the r o l e of t o t a l fat rather than the type of f a t i n colon cancer. High intake of dietary fat has also been shown to enhance b i l i a r y se-c r e t i o n and f e c a l excretion of b i l e acids (Reddy et a l . , 1977; Cummings, Wiggins et a l . , 1978). Although the mechanisms by which high dietary f a t promotes colon tumors are not yet c l e a r , i t i s suggested that promotion takes place through a mechanism in v o l v i n g increased colonic b i l e a c i d con-tent (Reddy, 1981). 2. Fiber a. D e f i n i t i o n of Fiber Dietary f i b e r i s cu r r e n t l y defined as "the remnant of plant c e l l s r e s i s t a n t to hydrolysis by the alimentary enzymes of man" (Trowel, 1972). It i s not a chemical e n t i t y but a mixture of d i f f e r e n t types of polysaccha-rides and l i g n i n (Southgate et a l . , 1976). A d e s c r i p t i o n of the d i f f e r e n t components of dietary f i b e r and t h e i r c h a r a c t e r i s t i c s i s given i n Table 2. The composition and physical properties of t o t a l dietary f i b e r are also i n -fluenced by the foods present i n the d i e t and may also vary within the same Table 2 Dietary Fiber Type Chemical C h a r a c t e r i s t i c s Representative Molecular Size (Daltons) Plant C e l l Function Special Physical Properties C e l l u l o s e Unbranched 1-4 B-D-glucose polymers 6 x 10 5 C e l l wall structure Hydrophyllic Hemicellulose Pentose or hexose polymers often branched; soluble i n cold a l k a l i 3 x 10 4 Wall s t a b i l i t y Ion binding Pectins 8-1,4-D-galacturonic acid polymers usually a s s o c i -ated with other poly-saccharides 6-9 x 10 4 Wall s t a b i l i t y Gel-forming a b i l i t y Ion binding A l g a l poly-saccharides Some sim i l a r to plants, others have sulfated polymers such as agar and carrageenan 2 x 10 5 A l g a l c e l l wall structure Gel-forming a b i l i t y L ignin Non-carbohydrate s u b s t i -tuted phenylpropanes 1-5 x 10 3 C e l l wall strength Possible b i l e s a l t binding a b i l i t y From Gray and Fogel (1979). 16 p l a n t , depending upon i t s state of maturity and the condition under which i t was grown (Southgate et a l . , 1976). Worth mentioning i s the f i n d i n g by Robertson (1972) that, although the sources of f i b e r i n the die t have changed i n the l a s t century, with a s h i f t towards more f r u i t s and vegetables and less cereal products, there has been l i t t l e change i n the t o t a l dietary f i b e r intake. Glober et a l . (1974) have also reported that Japanese migrants i n Hawaii who adopt the Western diet a c t u a l l y increase t h e i r t o t a l dietary f i b e r intake. b. Protective Role of Fiber i n Colon Carcinogenesis Another popular theory suggests an inverse r e l a t i o n s h i p between f i b e r intake and colon cancer ( B u r k i t t , 1971; Bur k i t t et a l . , 1972). It i s postulated that a high f i b e r diet r e s u l t s i n a shorter t r a n s i t time of foods and other substances through the i n t e s t i n e . This could reduce the opportu-n i t y for the gut b a c t e r i a to produce carcinogens and minimize the action of a carcinogen on the mucosal wall. By increasing f e c a l bulk, f i b e r could also d i l u t e any carcinogens ingested or formed i n the bowel. As pointed out by Bibra (1976), i t i s d i f f i c u l t to assess the evidence for and against t h i s hypothesis, since chemically i l l - d e f i n e d f i b e r has been used i n several experimental studies. S p i l l e r and Freeman (1981) have also suggested that when the e f f e c t s of f i b e r are studied, one must recognize the differences between high f i b e r d i e t , such as i n A f r i c a , and Western d i e t s , to which some f i b e r has been added. c. E f f e c t s of Fiber on Transit Time Variable r e s u l t s have been reported when the e f f e c t s of high f i b e r 17 diet on t r a n s i t time were studied. Some researchers have found no e f f e c t on t r a n s i t time (Eastwood et a l . , 1973) or decreased t r a n s i t time of sub-jec t s who were act i n g as t h e i r own controls (Cummings, Branch et a l . , 1978; Walters et a l . , 1975; Payler, 1973). Harvey et a l . (1973) have shown that with added dietary f i b e r , t r a n s i t time was decreased i n those with o r i g i -n a l l y long t r a n s i t time, increased i n those with o r i g i n a l l y short t r a n s i t time, and unchanged i n those with moderate t r a n s i t times. Glober et a l . (1974) have reported shorter t r a n s i t time i n Japanese Hawaiians compared to Caucasians from the same area, despite a s i m i l a r r i s k of colon cancer i n both ethnic groups. In another study, Hawaiian Japanese who have a high incidence of colon cancer, were found to have a s i m i l a r t r a n s i t time to Japanese from Japan who are at low r i s k for t h i s disease (Glober et a l . , 1977). Both groups had shorter t r a n s i t time than Hawaiian Caucasians. How-ever, the stool, weights were smaller i n the two Hawaiian groups compared to the Japanese from Japan. A study comparing low r i s k populations i n Kuopio, Finland, with those at high r i s k i n Copenhagen showed no diffe r e n c e i n t r a n s i t time between the two groups despite a higher f i b e r intake i n the former (IARC, 1977). The hypothesis that t r a n s i t time may play an e t i o l o g i c a l r o l e i n colon cancer i s challenged by the fa c t that constipation, use of lax a t i v e s and frequency of bowel movements did not seem to be associated with t h i s disease (Wynder and Shigematsu, 1967). d. E f f e c t s of Fiber on Fecal Bulk and D i l u t i o n of Possible Carcinogens There seems to be a considerable amount of evidence supporting the r o l e of high f i b e r diets on the d i l u t i o n of possible carcinogens such as 18 f e c a l s t e r o i d s . Eastwood et a l . (1973) have observed an increased stool weight and reduced f e c a l b i l e a c i d concentrations when wheat bran or c e l l u -lose was added to the normal English d i e t . In another study by Walters et a l . (1975), dietary supplements of wheat bran or bagasse had no e f f e c t on the t o t a l loss of neutral s t e r o i d s , although a d i l u t i o n e f f e c t was observed because of increased f e c a l bulk. Wheat bran reduced the f e c a l concentra-tions of acid steroids but not bagasse, which increased the excretion of b i l e a cids. Degradation of acid or neutral steroids by b a c t e r i a was not af-fected. These r e s u l t s were d i f f e r e n t from those of Pomare and Heaton (1973) who showed a reduction i n the metabolism of b i l e s a l t s by i n t e s t i n a l bac-t e r i a , with increased excretion of b i l e acids when-wheat bran was added to the d i e t . These r e s u l t s suggest that the d i f f e r e n t types of f i b e r consumed may a f f e c t f e c a l b i l e excretion d i f f e r e n t l y . This i s further substantiated by the work of Cummings, Branch et a l . (1978). They reported that f i b e r from c a r r o t , cabbage, apple, bran and guar germ had a d i f f e r e n t e f f e c t on f e c a l weight i n humans, possibly r e l a t e d to the content of pentose-contain-ing polysaccharide i n the f i b e r . Wheat bran caused the greatest increase followed by cabbage, c a r r o t , apple and guar, r e s p e c t i v e l y . A shorter tran-s i t time and a d i l u t i o n of the f e c a l marker were observed with the addition of the f i b e r . In another study, a d i l u t i o n of f e c a l b i l e acids and i n -creased f e c a l weights were observed by the addition of cereal f i b e r to the diet (Cummings et a l . , 1968). The same r e s u l t s were obtained by Kay and Truswell (1977) when wheat f i b e r was added to the d i e t . However, when wheat f i b e r was substituted f or p e c t i n , an increase i n f e c a l steroids and b i l e acid output was reported. Epidemiological studies have also confirmed that low r i s k populations for colon cancer tend to have b u l k i e r stools than high r i s k populations. High f e c a l weights were reported i n Japanese from Japan compared to Hawaiian 19 Japanese (Glober et a l . , 1977), i n residents of Kuopio compared to those from Copenhagen (IARC, 1977) and from the United States (Reddy et a l . , 1978). In the l a t t e r study by Reddy et a l . (1978), the intake of fat was si m i l a r i n both groups, although the main sources d i f f e r e d , being milk and dairy products for the residents of Kuopio and meat for the subjects from the • United States. The intake of f i b e r was higher i n the Finn i s h residents. No difference i n the t o t a l excretion of secondary b i l e acids was observed, due to the large f e c a l output by the Finnish. However, the concentrations of these b i l e acids, as well as the a c t i v i t y of f e c a l ^-glucuronidase, were lower i n the Kuopio residents. e. C o r r e l a t i o n Studies Between Human Intake of Fiber and Colon Cancer Incidence Very few epidemiological studies have correlated the intake of f i b e r to colon cancer incidence. In the study of Armstrong and D o l l (1975), the negative c o r r e l a t i o n with c e r e a l intake was n a t u r a l l y concurrent with a po s i t i v e c o r r e l a t i o n with meat intake. When f i b e r sources were separated, a small but s i g n i f i c a n t c o r r e l a t i o n was observed with colon cancer but not for crude f i b e r . In a case-control study i n Tel Aviv (Modan et a l . , 1975), a s i g n i f i c a n t negative c o r r e l a t i o n was found between colon cancer and inges-t i o n of fi b e r - c o n t a i n i n g foods, although the evidence i s challenged by Cruse et a l . (1978). One small case-control interview study by Dales et a l . (1979) found that low f i b e r - h i g h f a t diets were associated with higher r i s k for colon cancer, compared to high fiber-low f a t d i e t s . As pointed out by Graham and M e t t l i n (1979), fibrous foods contain properties other than f i b e r , which possibly account for the e f f e c t a t t r i b u t e d to the f i b e r . 20 f. Animal Studies on the E f f e c t s of Fiber i n Colon Carcinogenesis Animal studies show that the e f f e c t of f i b e r on cancer et i o l o g y depends on the type of f i b e r . While some f i b e r components may be protective or without e f f e c t , others may enhance or possibly cause cancer of the colon ( S p i l l e r and Freeman, 1981). Wheat bran at high l e v e l s was found to decrease the dimethylhydrazine-induced colon tumors i n several studies (Wilson et a l , , 1977; Barbolt and Abraham, 1978). This e f f e c t was not reproduced for duodenal tumors (Barbolt and Abraham, 1978). Chen et a l . (1978) reported a s i m i l a r e f f e c t on mice. The addition of dietary bran at 4.8% and 20% of the d i e t f a i l e d to a l t e r colon cancer incidence or mortality of rats treated with DMH f o r one year, although r a t s fed the bran diet survived longer than rats fed a f i b e r - f r e e d i e t . Rats fed high l e v e l s of r e f i n e d c e l l u l o s e and treated with azoxymethane had a reduced incidence of cancer of the small i n t e s t i n e but not of the colon, compared to the controls r e c e i v i n g a semi-synthetic d i e t only (Ward et a l . , 1973). However, food intake differences were observed, possibly due to the bulking e f f e c t of the c e l l u l o s e . This may have an e f f e c t on tumor formation (Alcantera et a l . , 1976). Freeman et a l . (1978), i n a dou-b l e - b l i n d study, have reported a lower incidence of colon tumors i n rats treated with dimethylhydrazine when fed a diet supplemented with 4.5% p u r i -f i e d c e l l u l o s e . Bauer and associates (1979) observed an increased number of colonic tumors i n rats fed a 6.5% pe c t i n d i e t and treated with dimethylhydrazine, compared to the rats fed a pectin-free d i e t . Freeman et a l . (1980) studied the e f f e c t of d i f f e r e n t amounts of p u r i f i e d c e l l u l o s e and pectin fed to 21 rats treated with dimethylhydrazine. The incidence of colon tumors was decreased i n the rats fed added c e l l u l o s e , regardless of the dose. Pectin did not seem to have an e f f e c t on colon carcinogenesis. Watanabe and colleagues (1979) have also shown that semi-purified diets with v a r i a b l e content of a l f a l f a , pectin and wheat bran produced d i f f e r e n t e f f e c t s on the number of tumors caused by azoxymethane or methylnitrosourea i n the colon. These substances also a f f e c t e d d i f f e r e n t l y the excretion and concentration of f e c a l b i l e acids. g. Other Possible E f f e c t s of Fiber in Colon Carcinogenesis Another possible mechanism by which dietary f i b e r may protect the colon against the action of p o t e n t i a l carcinogens i s the adsorption of these substances onto the f i b e r and increased excretion. Several types of f i b e r can bind b i l e acids (Kritchevsky and Story, 1974; Nigro and Campbell, 1976) and carcinogens (Smith-Barbaro et a l . , 1981). Fiber has also been shown to lower the t o x i c i t y of many compounds i n experimental animals (Ershoff, 1960). Reduced mutagenic a c t i v i t y of c e r t a i n carcinogens was also reported a f t e r in vitro incubation with f i b e r polymers (Freeman et a l . , 1980). Fiber could also protect against colon cancer by lowering the colonic pH. This would r e s u l t from the formation of various v o l a t i l e f a t t y acids produced by the f e c a l b a c t e r i a l digestion of f i b e r polymers ( S p i l l e r et a l . , 1980). A lowering e f f e c t on the colonic pH i n rats by supplementing the diet with wheat bran has recently been reported (Jacobs and Lupton, 1982). 3^ . Vegetables It has been postulated by Wattenberg (1971) that a vegetarian diet may 22 protect against colon cancer because of a b e n e f i c i a l e f f e c t of vegetables. He reported that the dietary addition of a v a r i e t y of vegetables such as Brussel sprouts, cabbage, turnips, c a u l i f l o w e r and b r o c c o l i , s u b s t a n t i a l l y reduced chemically-induced tumorigenesis in rodents. This tumor i n h i b i t i o n appears to r e s u l t from increased a c t i v i t y of a r y l hydrocarbon hydroxylase induced by indoles present in these plants. A r y l hydrocarbon hydroxylase might detoxify p o t e n t i a l carcinogens (Wattenberg and Loub, 1978). A protective e f f e c t of these vegetables i n man has also been reported. Bjelke (1973), i n a case-control study i n Norway and Minnesota, has observed a lower r i s k of colon cancer than expected i n t h i s country f o r i n d i v i d u a l s consuming large amounts of cruciferous vegetables, although no c o r r e l a t i o n was established with f i b e r . Hirayama (1979) reported the same findi n g s i n a ten-year prospective study in Japan, including 265,000 subjects. Graham et a l . (1978) also found an inverse r e l a t i o n s h i p between the intake of cer-t a i n vegetables, p a r t i c u l a r l y cabbage, b r o c c o l i and Brussel sprouts, and colon cancer. While Modan's r e s u l t s (1975) indicated a protective e f f e c t f o r f i b e r , a negative c o r r e l a t i o n with cabbage and colon cancer was also established. The same findings were reported i n a recent study of Japanese subjects by Haenszel et a l . (1980). C. FECAL MUTAGENS AND COLON CANCER 1. Presence of Mutagenic Compounds i n Feces of Individuals and  Populations A recent theory postulates that mutagenic substances formed i n the bowel i n response to dietary factors may be involved i n cancer etiology. Using the Ames test (1975), Bruce and associates (1977) f i r s t reported the 23 presence of mutagens i n stools of 20% of i n d i v i d u a l s screened, who consumed a mixed Western d i e t . These substances were i d e n t i f i e d as N-nitroso com-pounds and were believed to be produced in the body rather than ingested (Varghese et a l . , 1977). The a d d i t i o n of ascorbic a c i d , a-tocopherol or f i b e r reduced the f e c a l mutagenicity, while a high meat diet increased the concentration of mutagens (Bruce et a l . , 1979). Mandel et a l . (1977) f u r -ther demonstrated that the stools of some people contained a b a c t e r i a l en-zyme capable of t r a n s f e r r i n g a n i t r o s o group from a nitrosamine to an amide, therefore producing nitrosamide (a carcinogenic substance). However, N-nit r o s o compound formation in the human colon i s now thought to be n e g l i g i - . ble (Lee et a l . , 1981). Population differences i n mutagen production have been demonstrated by Ehrich et a l . (1979). They used the Ames test (1975) and Salmonella typhi-muvium TA100 and TA98 to test freeze-dried feces from three South A f r i c a n populations at d i f f e r e n t r i s k for colon cancer. With TA100, 20% of the 42 samples from urban whites (at high r i s k for colon cancer) were p o s i t i v e . Meanwhile, only 2% of 82 extracts from urban blacks and less than 1% of 108 samples from r u r a l blacks were found to be p o s i t i v e . Mutagenicity with TA98 was lower, being 10%, 5% and 2% i n whites, urban blacks and r u r a l blacks, r e s p e c t i v e l y . The urban and r u r a l blacks had a diet high i n f i b e r and low i n f a t compared to the white population. S i m i l a r l y , f e c a l mutagens were found more frequently i n New York i n d i -viduals with t y p i c a l Western diet than i n Kuopio residents who consumed a high f a t , high f i b e r d i e t (Reddy et a l . , 1980). With TA100 and/or TA98, i n the Ames plate t e s t , 22% of f e c a l samples from New York volunteers were p o s i t i v e . Fecal extracts from Kuopio residents were found p o s i t i v e only i n 13% of the samples with TA98 activated by S9. No f e c a l mutagenicity was detected i n Seventh Day Adventists' f e c a l samples by these researchers. 24 The mutagenic a c t i v i t y of 2-acetylaminofluorene tested with TA98 and TA100 i n the same test system was found to be enhanced by f e c a l extracts of Seventh Day Adventists and non-Seventh Day Adventists. However, f e c a l ex-t r a c t s of non-Seventh Day Adventists showed a s i g n i f i c a n t l y higher co-mutagenic a c t i v i t y with TA98 than did those of Seventh Day Adventists (Reddy, Sharma and Wynder, 1980). Compounds with greater i n t e r a c t i o n with DNA have also been i d e n t i f i e d i n f e c a l extracts of omnivores compared to those of s t r i c t vegetarians (Nader et a l . , 1981). The factors responsible for the presence of mutagens i n feces have not yet been elucidated. Several i n v e s t i g a t o r s have reported the presence of mutagens i n foods such as f r i e d f i s h and meat, f r i e d hamburger and b r o i l e d foods (Nagao et a l . , 1977; Commoner et a l . , 1978; Spingarn and Weisburger, 1979), as well as i n cooked, high starch foods (Spingarn et a l . , 1980). These mutagens seem to d i f f e r from those found i n feces, since they require a c t i v a t i o n with S9, and the f e c a l mutagens have been active i n most cases without S9 (Bruce et a l . , 1977; Ehrich et a l . , 1979; Reddy et a l . , 1980). Wilkins et a l . (1980) bel i e v e that f e c a l f l o r a are responsible for the f o r -mation of mutagenic compounds. Ehrich et a l . (1981) have shown that hepatic enzymes can i n h i b i t f e c a l mutagens, suggesting that v a r i a b i l i t y among i n d i -viduals may r e s u l t from differences i n hepatic microsomal enzymes. In an attempt to define i n d i v i d u a l v a r i a b i l i t y i n f e c a l mutagenic a c t i v i t y , Kuhnlein and Kuhnlein (1980) studied the e f f e c t of r i g i d l y consistent f o r -mula diet on f e c a l mutagenicity using the f l u c t u a t i o n test of Green and Muriel (1976). Fecal mutagenicity with TA100 var i e d s i g n i f i c a n t l y between the subjects up to 72 days, but no s i g n i f i c a n t change in f e c a l mutagenicity was observed within a subject throughout the diet period. They concluded that f e c a l mutagenicity may be influenced by long-term habits (dietary or otherwise) or perhaps by genetic t r a i t s . 25 At the present time, the influence of di e t on f e c a l excretion of muta-gens, as well as the im p l i c a t i o n of the l a t t e r i n colon cancer e t i o l o g y , i s under i n v e s t i g a t i o n i n several laboratories (Sherlock et a l . , 1980). 2. Methods for Evaluating Fecal Mutagenicity Bruce et a l . (1977) f i r s t elaborated extraction procedures for f e c a l specimens. Fecal samples were freeze-dried and extracted with peroxide-free d i e t h y l ether. The solvent was evaporated and the residue redissolved i n dimethyl sulfoxide and tested for mutagenicity i n a standard Ames test (1975). Similar extraction procedures were used by other researchers i n t h e i r studies of f e c a l mutagenicity (Ehrich et a l . , 1979; Reddy et a l . , 1980; Wilkins et a l . , 1980; Ehrich et a l . , 1981; Lederman et a l . , 1981). Others have used d i f f e r e n t solvents for e x t r a c t i o n , such as dichloromethane (Reddy, Sharma and Wynder, 1980), dimethyl sulfoxide or ethanol (Nader et a l . , 1981). In another study, f e c a l homogenate rather than freeze-dried feces was extracted with ether (Hayatsu et a l . , 1981). Kuhnlein and Kuhnlein (1980) omitted the use of solvents and prepared water extracts from f e c a l homogenates. While the Ames test (1975) has often been used to assay f e c a l mutageni-c i t y (Bruce et a l . , 1977; Ehrich et a l . , 1979; Reddy et a l . , 1980; Ehrich et a l . , 1981; Lederman et a l . , 1981), i t i s not without problems. Nader et a l . (1981) claim that the a p p l i c a t i o n of the Ames test to detect mutagens i n f e c a l extracts i s ser i o u s l y hampered by problems of t o x i c i t y . Furthermore, Hayatsu et a l . (1981) have demonstrated that ether extracts of normal human feces showed i n h i b i t o r y e f f e c t s on the a c t i v i t i e s of several mutagens i n the Ames t e s t . These i n h i b i t o r y factors i n the ether extract from f e c a l samples were i d e n t i f i e d as o l e i c and l i n o l e i c acids. The authors 26 concluded that " i n the case of screening mutagens by the Ames t e s t , e s p e c i a l l y i n cases where natural materials are examined, appropriate care must be taken to remove f a t t y acids from the samples to avoid f a l s e nega-t i v e s " . In a study of subjects on c o n t r o l l e d formula d i e t s by Kuhnlein and Kuhnlein (1980) , water extracts from f e c a l samples were tested with the f l u c t u a t i o n test for weak mutagens (Green and Muri e l , 1976). This t e s t i s reported to detect mutagens at one-hundredth of the concentrations required i n the standard Ames tes t ( H o l l s t e i n et a l . , 1979). Since only a small quantity of f e c a l extracts i s needed for t h i s t e s t , i t i s less l i k e l y that toxic or i n h i b i t o r y substances w i l l i n t e r f e r e with the assay. Whether the mutagens detected i n d i f f e r e n t populations and by d i f f e r e n t research techniques are si m i l a r or not remains to be determined. 27 CHAPTER II MATERIALS AND METHODS A. GROUPS SAMPLED Twenty-nine healthy subjects p a r t i c i p a t e d i n t h i s study. They were divided into three basic groups depending upon t h e i r normal dietary habits: Group I: Ovo-lacto vegetarians (n=6): avoiding meat but ingesting dairy products d a i l y . Group II: S t r i c t vegetarians (n=ll): avoiding meat and dairy products from t h e i r diet unless dictated by s o c i a l circumstances. Group I I I : Non-vegetarians (n=12): consuming meat and dairy products on a regular b a s i s . A l l subjects resided i n the Lower Mainland of B r i t i s h Columbia. They were r e c r u i t e d through a preventive medicine c l i n i c i n Vancouver, .the Seventh Day Adventist Church and from personal contact. P a r t i c i p a t i o n i n the study was voluntary, and no incentives were o f f -ered to the subjects other than an evaluation of t h e i r dietary intake com-pared to the Dietary Standard for Canada (see Appendix E). Prio r to the study, each p a r t i c i p a n t received an explanation of the project and a d e t a i l e d l i s t of i n s t r u c t i o n s to ensure t h e i r comprehension of the study. Each subject then signed an informed consent form for t h e i r p a r t i c i p a t i o n i n the study, i n accordance with regulations of the U.B.C. Committee on Research Involving Human Subjects (Appendix A). 28 B. COLLECTION OF DIETARY DATA AND OTHER INFORMATION 1. Questionnaire P a r t i c i p a n t s were requested to complete a two-part questionnaire. The f i r s t part i d e n t i f i e d : a. the age, sex, place of b i r t h and n a t i o n a l i t y of the subjects; b. t h e i r dietary p r a c t i c e s , including f a s t i n g , d i e t i n g , number of years of being vegetarians, trimming f a t o f f meat, etc.; c. t h e i r health status and hab i t s , such as weight changes, bowel habits, medication and supplements taken, family h i s t o r y of cancer, known g a s t r o i n t e s t i n a l disorders, etc. The second part established the frequency of consumption of c e r t a i n foods and food groups which could be c o r r e l a t e d with high and low l e v e l s of f e c a l mutagenicity. The questionnaire i s presented i n Appendix D. Graduate students and Cancer Research Centre co-workers (n=9) pretested the questionnaire f o r i t s ease of comprehension and c l a r i t y , and i t was sub-sequently modified before being d i s t r i b u t e d to the subjects. 2. Four Day Food Records Pa r t i c i p a n t s were instructed to keep a record of four day food intake i n order to assess the nutrient intake of the groups sampled. A 24-hour r e c a l l was f i r s t taken by the researcher to teach the subjects how to keep a food record. Information regarding quantity of intake was recorded a f t e r comparison with measuring cups and spoons. Methods of cooking meals, medi-cations and supplements ingested were included for t h e i r possible influence 29 on nutrient intake. Written i n s t r u c t i o n s accompanied the forms on which subjects would record t h e i r intake (see Appendix B arid C). C. COLLECTION OF FECAL SAMPLES Each subject was requested to c o l l e c t the f e c a l matter of an entire bowel movement on the fourth day of food recording. The samples were de-posited i n preweighed containers and placed immediately on dry ice i n styro-foam boxes provided for t h i s purpose. The samples were c o l l e c t e d from the subjects' houses within four hours and stored at -15°C i n the laboratory. D. FECAL ANALYSES 1. Preparation of Extracts In the laboratory, the samples were thawed and homogenized i n a blender with an equal amount of deionized d i s t i l l e d water and centrifuged at 4°C for 50 minutes at 150,000 g i n a Beckman u l t r a c e n t r i f u g e . The supernatant was s t e r i l i z e d by successive passage through two f i l t e r s : .4 -urn pore size and .2 um pore siz e (Acrodisc, Gelman Sciences Inc.), and then divided into 5 ml aliquots and frozen at -15°C. 2. Mutagen Analyses The aqueous f e c a l extracts were assayed for mutagenicity with the f l u c -tuation test of Green and Muriel (1976). This method of t e s t i n g mutagens was chosen because of i t s high s e n s i t i v i t y . I t detects mutagens at concen-t r a t i o n s less than one-hundredth of the concentrations required i n the stan-30 dard Ames tes t ( H o l l s t e i n and McCann, 1979). It measures b a c t e r i a l mutagens or back mutations i n a sp e c i a l set of tester s t r a i n s requiring the amino a c i d , h i s t i d i n e . The composition of media used i n the f l u c t u a t i o n test was: a. Davis M i n g i o l i s a l t s : 7 g of K^HPO^, 2 g of KH2PC>4, 1 g of (NH 4) 2S0 4, 0.25 g of sodium c i t r a t e , 0.1 g of MgS04-7H20, 5 mg of bromocresol purple, and 1 l i t e r of d i s t i l l e d deionized water. b. H i s t i d i n e : 1 mg/ml. c. Glucose: 20%. d. B i o t i n : 0.1 mg/ml. e. Ba c t e r i a : 10"*/ml. In these experiments, Salmonella typhimurium TA100 and TA98 were used as tester s t r a i n s and stocks of these b a c t e r i a were prepared as follows: an overnight culture grown i n nutrient broth (8 g Difco broth, 5 g NaCl i n 1 l i t e r H 20) was d i l u t e d 10^-fold into nutrient broth and again grown over-night with aeration. Dimethyl sulfoxide was added to a concentration of 10% (v/v) and 1 ml portions were frozen i n l i q u i d nitrogen. A l l experiments were performed with the same batch of subcultures. For the f l u c t u a t i o n t e s t , a 1 ml aliq u o t of b a c t e r i a was thawed and d i l u t e d with 10 ml of nutrient broth and incubated overnight with aeration. One ml of a s o l u t i o n of 10~Vml b a c t e r i a was added to 120 ml of Davis Mingio-l i s a l t s mixed with 2.4 ml of D-glucose, 0.48 ml of b i o t i n , 30 ul of L-h i s t i d i n e and 1 ml or less of f e c a l extract. This mixture was then d i s t r i -buted i n portions of 2 ml into 50 tubes and incubated at 37°C for 5 days. The amount of h i s t i d i n e present i n the medium allowed the b a c t e r i a to 8 grow to about 10 per ml, unless the tube contained a revertant and there-fore permitted a higher concentration of b a c t e r i a . The presence of bromo-31 cresol purple, a pH i n d i c a t o r , changed the purple c o l o r of the medium to yellow when a high concentration of b a c t e r i a was present in the tubes, per-mitting easy scoring. The presence of mutagens was then confirmed when there was an increase in the number of revertant tubes over those due to spontaneous mutations (blank). Green and Muriel (1976) used 3 days of incubation at 37°C before scor-ing i n t h e i r o r i g i n a l procedure with a tryptophan-requiring E. coli tester s t r a i n . In the experiment reported here, i t was found that there was s t i l l an increase i n the number of revertants between the t h i r d and fourth day of incubation with TA98 and TA100. The scoring was therefore c a r r i e d out a f t e r a 5-day incubation period. E. ADDITIONAL MEASUREMENTS Wet weight, dry weight and pH of the f e c a l samples were also determined. A small aliquot of f e c a l homogenate was poured into an aluminium weighing tray and placed in a small oven at 45°C for 36 hours. The weight loss was measured to determine percent dry weight. The pH of the homogenates was determined with an Orion pH meter model 601A and an Orion combination e l e c -trode no. 91-05. F. DATA ANALYSIS Programs from the S t a t i s t i c a l Package for the Social Sciences (SPSS; Nie et a l . , 1975) were used i n a l l the s t a t i s t i c a l procedures, unless other-wise stated. The dose-response curve for f e c a l mutagenicity was not l i n e a r and i t 32 was uncertain whether f e c a l mutagenicity among the subjects followed a nor-mal d i s t r i b u t i o n . Non-parametric s t a t i s t i c a l tests were therefore c a r r i e d out, i n most cases, to analyze the data. These recommendations were made by Dr F.P. Gl i c k of the Department of Health Care and Epidemiology, U.B.C. 1. Data from Fecal Samples a. Fecal Extracts from Groups Sampled As mentioned previously, the presence of mutagens, as measured by the f l u c t u a t i o n t e s t , i s confirmed when there i s an increase of revertant tubes compared to the blank (spontaneous mutations). The s i g n i f i c a n c e of t h i s increase was tested by the Chi-square test for two independent samples, as recommended by Green and Muriel (1976). The test was considered p o s i t i v e i f PO.01. To evaluate the difference i n the amount of mutagens between vegetarians and non-vegetarians, the number of revertant tubes at each concentration was corrected f or the blank (which do not contain f e c a l extracts) and summed for every subject. The ranks of these sums were then analyzed with the Mann-Whitney U t e s t . Since vegetarians were expected to have lower l e v e l s of f e c a l mutagens than the non-vegetarians, the one-tailed test was used. Spearman rank c o r r e l a t i o n was applied to determine i f high l e v e l s of mutagenicity with TA100 corr e l a t e d with high l e v e l s of mutagenicity using TA98. The same test was used to r u l e out any c o r r e l a t i o n between high muta-geni c i t y and high blank values, which would be suggestive of some experi-mental e r r o r . 33 b. Other Experimental Measurements The difference in the pH, dry weight and wet weight of f e c a l homogenates between diet subgroups was analyzed by the Mann-Whitney U test (two-tailed). Associations between these parameters were determined by Spearman rank c o r r e l a t i o n analyses. 2. Dietary Data and Other Information a. Questionnaire The questionnaire was coded a f t e r completion by the respondants. The f i r s t part of the questionnaire was analyzed to describe and character-ize the groups sampled and to define t h e i r health status and c e r t a i n d i etary hab i t s . The Spearman c o r r e l a t i o n test was applied to the data to determine which of these c h a r a c t e r i s t i c s were associated with mutagenicity. Variables tested included age, sex, trimming of fa t from meat, use of spices and f r e -quency of use, f a s t i n g , number of years as vegetarians, use of o r a l contra-ceptives, bowel frequency, bowel i r r e g u l a r i t y , use of l a x a t i v e s , smoking, family h i s t o r y of cancer, use of vitamins or other nutrient supplements. The second part of the questionnaire (food frequency) was coded using SPSS procedures. Consumption of food groups and food items were ca l c u l a t e d on a monthly basis and tabulated for each subject. Foods included in each food group are l i s t e d i n Table 3. The Mann-Whitney U test was used to test the data for differences i n the intake of c e r t a i n foods or food groups between vegetarians and non-34 Table 3 L i s t of Foods Included i n Each Food Group from Food Frequency Group Number Name of Group Foods i n Each Group I. Dairy products 1A Cheeses Cream cheese Skim milk cheese Cheddar, Swiss Cottage cheese IB Cream Sweet cream Sour cream IC Ice cream Ice cream Sherbet ID Milk Whole milk Chocolate milk 2% milk Skim milk Evaporated milk Buttermilk P l a i n yoghurt Flavored yoghurt Goat milk I I . Meat, poultry, f i s h and alternates 2A Beef Regular hamburger Lean hamburger Commercial hamburger Stewing meat Roast Steak Corned beef Organ meats L i v e r Veal 2B Lamb Roast Chops 2C Pork Bacon s t r i p s Sausage Ham Back bacon Roast 35 Table 3 (cont'd) Group Name of Foods i n • Number Group Each Group 2D 2E 2F 2G Variety meat Game meat Poultry F i s h and seafoods 2H 21 Eggs Nuts and legumes III. Vegetables Salami Luncheon meats Game Chicken Fresh f i s h Canned f i s h S h e l l f i s h Other f i s h F r i e d egg Boiled egg Other egg Beans (baked) Dried peas Nuts Peanut butter L e n t i l s Soy milk Tofu Mi so Sunflower seeds Sesame seeds Texturized vegetable protein Green and yellow snap beans Corn Onions Cabbage Sauerkraut Greens (lettuce) Green peas Snow peas Root vegetables Tomatoes Bean sprouts A l f a l f a sprouts Pickles Celery B r o c c o l i Cauliflower French f r i e s Other potatoes 36 Table 3 (cont'd) Group Name of Foods i n Number Group Each Group IV. 4A F r u i t s and ju i c e s F r u i t s C i t r u s f r u i t s Apples or sauce Bananas Melon Dried f r u i t s Berries Avocado Other f r u i t s 4B F r u i t j u i c e s Unsweetened j u i c e s Sweentened j u i c e s F r u i t flavored drinks V. 5A Breads and alternates Bread and others White bread Brown bread Corn bread Bran bread Pancakes, crepes, b l i n t z e s 5B Cereals, ready to serve Bran Part bran Granola Other cereals 5C Cooked grains Oatmeal Cream of wheat Brown r i c e White r i c e M i l l e t 5D Other cereal products Macaroni Noodles 5E Other Wheat germ Yeast 5F Baked goods Doughnuts Granola bars Pie Cake 37 Table 3 (cont'd) Group Name of Foods i n Number Group Each Group VI. Sugars and sweets Sugar Honey Syrups Gel a t i n desserts Chocolate bars Others VII. 7A Fats Animal f a t s Butter Meat drippings 7B 7C VIII. Vegetable fats Comb inat ion Miscellaneous Margarine Vegetable o i l Non-dairy creamer Gravy Shortening Prepared salad dressing (mayonnaise type) Beer Wine Liquor or c o c k t a i l s Liqueur Coffee Tea Herbal teas Soft drinks 38 vegetarians. Spearman rank c o r r e l a t i o n tested for a r e l a t i o n s h i p between consumption of c e r t a i n foods or food groups and mutagenicity. b. Four Day Food Records Although a 24-hour r e c a l l was c o l l e c t e d with the four day food records, only the l a t t e r were included i n the analysis since they immediate-ly preceded the f e c a l c o l l e c t i o n . The four day food records were analyzed with the U.B.C. die t program c a l l e d General Nutrient Analysis (GNA). This program was created by Dr M. Lee and Robert Meldrum from the U.B.C. Art Computer Centre i n 1967 and re-vised by Lewis James i n 1979. Data for protei n , fat and f i b e r categories were programmed by Dr Harriet Kuhnlein, Danielle Bergstrom and Penny B e l l from the D i v i s i o n of Human N u t r i t i o n . The program yielded the nutrient intake of the subjects studied by matching a f i l e of t h e i r food intake data with a f i l e of food composition information. The mean nutrient intake for each subject was tabulated and compared to the Dietary Standard for Canada. The Mann-Whitney U test aimed at estab-l i s h i n g a difference i n the intake of c e r t a i n nutrients between the die t groups. The Spearman rank c o r r e l a t i o n test was applied to determine i f an.asso-c i a t i o n , .existed.between c e r t a i n nutrients and high or low l e v e l s of muta-gens. Nutrients which were tested i n t h i s study were: c a l o r i e s , t o t a l pro-t e i n , vegetable p r o t e i n , animal p r o t e i n , t o t a l f a t , animal f a t , carbohydrate, crude f i b e r , Southgate f i b e r , vitamin A, thiamin, r i b o f l a v i n , n i a c i n , v i t a -39 min C, calcium and i r o n . Values f o r vegetable protein and f a t and animal protein and f a t were estimated by reorganizing the food groups i n the GNA program. Foods containing animal protein and animal fat were separated from those with vegetable protein and f a t . For combination foods containing both types of protein and/or f a t , values were obtained by analyzing recipes provided by the subjects with food composition tables. c. A d d i t i o n a l Dietary Analyses The Pearson c o r r e l a t i o n procedure was c a r r i e d out between data from the food frequency questionnaire and data from a food frequency intake derived from the four day food records. This test was performed to v a l i d a t e the use of the food frequency questionnaire i n t h i s experiment. This was achieved by determining i f the frequency of intake of foods and food groups from the four day food intake records could match the frequency of intake from the questionnaire. The four day food frequency intake was derived by c a l c u l a t i n g the num-ber of servings of each food and food group ingested by each subject. The consumption of foods and food groups was converted on a monthly basis by d i v i d i n g the number of servings by four and m u l t i p l y i n g by 30 f o r a month of 30 days. o 3. Level of Significance Unless otherwise stated, the l e v e l of s i g n i f i c a n c e used to judge most s t a t i s t i c a l r e s u l t s was P<?0.05. 40 CHAPTER IV RESULTS A. FECAL MUTAGENICITY DATA 1. Comparison of Mutagenicity i n Extracts from Vegetarians and Non-Vegetarians The number of revertant tubes obtained at d i f f e r e n t extract concentra-tions from 6 ovo-lacto vegetarians, 11 s t r i c t vegetarians and 12 non-vege-tarians are l i s t e d i n Table 4 and Table 5. The highest mutagenic a c t i v i t y with TA100 was detected at a concentra-t i o n of 4.2 pl/ml, where 27 of the 29 subjects were p o s i t i v e . In the case of TA98, the highest mutagenic a c t i v i t y was observed at 8.3 ul/ml, where 24 subjects were p o s i t i v e . The number of revertant tubes at each extract concentration were aver-aged for further analysis. Figure 1 shows that when extracts were assayed with TA100, the non-vegetarians had a higher average number of revertant tubes than both the s t r i c t vegetarians and the ovo-lacto vegetarians. How-ever, no difference i n mutagenicity was seen between s t r i c t vegetarians and ovo-lacto vegetarians. With TA98, a d i f f e r e n t pattern was observed, as seen i n Figure 2. The average number of revertant tubes was almost i d e n t i c a l for a l l three groups at low concentration of extract. However, the non-vegetarians showed a s i g n i f i c a n t increase at higher concentrations of extract compared to the ovo-lacto vegetarians and s t r i c t vegetarians who were s i m i l a r . 41 Table 4 Number of Revertant Tubes out of 50 i n Dependence of the Fecal Extract Concentration f o r TA100 Extract Concentration l [yl/ml) .uieu J U D J C L L. Group Number 0 a 0.5 1.0 2.1 4.2 8.3 Rank 1 22 21 28 35 39 38 5 2 20 15 22 31 37 31 .4 Ovo-lacto 3 22 39 49 49 48 50 29 vegetarians 4 22 21 32 46 45 41 15 5 17 11 18 31 33 23 2 6 16 17 24 34 35 27 6 7 16 17 19 28 39 42 8 8 16 23 25 37 37 24 9 9 24 26 40 47 40 34 10 10 21 24 36 47 40 36 16 S t r i c t 11 16 17 28 41 42 31 17.5 vegetarians 12 17 18 28 33 30 50 13.5 13 17 10 10 15 25 36 1 14 21 25 23 49 49 33 13.5 15 16 24 24 41 46 49 25 16 24 36 41 46 34 34 11 17 17 11 26 20 34 29 3 18 22 26 41 45 47 46 24 19 20 23 26 43 44 50 22 20 21 27 36 46 36 33 12 21 16 20 33 43 44 22 21 Non- 22 20 24 38 50 49 48 28 vegetarians 23 17 18 22 38 43 43 17.5 24 24 29 47 49 50 50 26 25 21 32 37 47 46 32 23 26 24 27 31 45 50 47 19.5 27 20 21 28 39 46 46 19.5 28 20 26 23 31 45 39 7 29 20 26 41 46 47 48 27 For each sub j ec t tested, the number of revertant tubes when no extract was added was also determined. The blanks from the same experiment were averaged. The number of revertant tubes were corrected f o r the blanks and summed for each subject. The sums were then ranked. 42 Table 5 Number of Revertant Tubes out of 50 in Dependence of the Fecal Extract Concentration f o r TA98 Diet Group Subject Numb er Extract Concentration (yl/ml) 0 0.5 1.0 2.1 4.2 8.3 Rank 1 15 15 18 19 17 33 2 2 15 18 24 30 23 29 12.5 Ovo-lacto 3 15 27 32 32 34 50 29 vegetarians 4 16 21 26 25 26 26 6.5 5 15 26 27 22 28 30 15 6 15 21 32 29 30 32 18.5 7 15 17 20 27 31 28 11 8 17 13 20 30 22 25 1 9 17 18 25 29 27 26 3 10 16 23 23 24 25 30 8 S t r i c t 11 16 25 26 24 20 32 10 vegetarians 12 16 28 33 25 31 35 22 13 15 19 28 28 25 33 15 14 17 18 27 31 31 20 5 15 15 22 32 31 30 29 18.5 16 16 21 30 24 25 38 15 17 15 33 25 31 26 36 25 18 15 26 29 26 32 50 27 19 15 27 30 31 29 50 28 20 16 29 22 23 20 35 12.5 21 15 17 23 24 27 25 4 Non- 22 16 24 28 21 33 50 25 vegetarians 23 16 24 26 27 38 36 20.5 24 17 16 34 33 32 41 20.5 25 • . 15 23 18 19 30 29 6.5 26 17 21 31 29 33 34 17 27 16 25 29 36 31 33 23 28 16 21 24 22 29 30 9 29 16 27 29 31 39 30 25 The blank values were averaged and the ranks determined as i n Table 4. 43 The shape of the dose-response curve was almost i d e n t i c a l f or the d i f f e r e n t d i e t groups and also f o r most of the i n d i v i d u a l e x t r acts. With TA100, the number of revertant tubes increased with the amount of extract, l e v e l l e d o f f at 2 ul/ml of extract and decreased at higher concentrations (Figure 1). With TA98, a sharp increase i n the number of revertant tubes was seen at low concentration of e x t r a c t , l e v e l l i n g o f f between 1 and 2 ml of extract and again increasing at higher concentrations (Figure 2). It i s not possible to define a mutation rate because of the complex dose-res-ponse curve. The Mann-Whitney U test (one-tailed) was performed to analyze the differences i n mutagenicity between vegetarians and non-vegetarians. For t h i s t e s t , the number.of revertants at each extract l e v e l was corrected for the blank (number of revertant tubes without e x t r a c t ) , summed for each subject and ranked. Table 6 i s a summary of the r e s u l t s . It can be seen that with both TA98 and TA100 a l l vegetarians (ovo-lacto and s t r i c t vegetarians combined) ranked s i g n i f i c a n t l y lower than non-vegetarians (P<0.05). The same f i n d i n g was observed when s t r i c t vegetarians only were compared to the non-vege-t a r i a n s . The extracts of ovo-lacto vegetarians d i f f e r e d s i g n i f i c a n t l y from non-vegetarians only when tested with TA100. To r u l e out the p o s s i b i l i t y that experimental errors may have influenced the r e s u l t s , Spearman c o r r e l a t i o n analysis was used to test i f there was a r e l a t i o n s h i p between low ranks of mutagenicity and low blank values, using values from Tables 4 and 5. No s i g n i f i c a n t c o r r e l a t i o n was found between these two parameters (r =0.236, P>0.2 for TA100; r =0.230, P>0.2 for TA98). s s Since the storage time between sample c o l l e c t i o n and assay period var-ied from subject to subject, i t was of i n t e r e s t to determine i f storage time t o LU CO 44 Figure 1 Average number of revertant tubes out of 50 in dependence of the extract concentration using Salmonella typhimurium TA 100 50 h UJ > UJ CO UJ o < oc UJ > < 10 • Ovo-lacto vegetarians A Strict vegetarians o Non-vegetarians x ± 8 EXTRACT CONCENTRATION ()Ul/ml) 45 Figure 2 Average number of revertant tubes out of 50 in dependence of the extract concentration using Salmonella typhimurium TA 98 CO UJ CO Z < i — oc UJ > oc UJ CO 3 UJ o < oc UJ > < 50 h 40 TA 98 10k • Ovo-lacto vegetarians A Strict vegetarians o Non-vegetarians 8 EXTRACT CONCENTRATION (fXl/ml) 46 Table 6 Comparison of Diet Groups with the Mann-Whitney U Test (One-Tailed) Diet Group Number of Subjects Average Rank i n the Mann-Whitney U Test Significance TA100 TA98 TA100 TA98 Vegetarians vs 17 11.1 12.8 a b <0.010 <0.050 Non-vegetarians 12 20.5 18.2 S t r i c t vegetarians vs 11 8.0 9.5 <0.010 <0.050 Non-vegetarians 12 15.7 14.3 Ovo-lacto vegetarians vs 6 5.8 7.8 <0.050 n.s.° Non-vegetarians 12 11.3 10.3 Ovo-lacto vegetarians 6 7.8 9.6 vs n.s. n.s. S t r i c t vegetarians 11 9.6 8.7 The ranks of Tables 4 and 5 were analyzed with the Mann-Whitney U tes t as described by Siegel (1956). The s i g n i f i c a n c e i s the p r o b a b i l i t y that the observed differences are due to chance. a<0.010 means >0.001 <0.010. b<0.050 means >0.010 <0.050. n.s. denotes not s i g n i f i c a n t (P>0.050). 47 influenced mutagenicity. The Spearman rank c o r r e l a t i o n s were r g = -0.019 for TA100 and r = -0.1577 for TA98, with P>0.2 i n both cases and therefore s ' not s i g n i f i c a n t . It was also important to determine i f high l e v e l s of mutagenicity with TA100 corresponded to high l e v e l s with TA98, since vegetarians d i f f e r e d s i g n i f i c a n t l y from non-vegetarians i n mutagenicity (Table 7). A s t a t i s t i -c a l l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was found for a l l subjects (P<0.05). When separate d i e t groups were analyzed, the Spearman c o r r e l a t i o n c o e f f i -cient indicated a s i m i l a r c o r r e l a t i o n among the non-vegetarians although not as s i g n i f i c a n t (P<0.1), due to the small number of subjects. No s i g n i f i c a n t c o r r e l a t i o n was found with the s t r i c t vegetarians, the ovo-lacto vegetarians or when both groups were combined. 2. Co r r e l a t i o n of Mutagenicity with Other Parameters of Fecal Samples Table 8 shows the wet weight, percent dry weight and pH of f e c a l sam-ples and the average for each d i e t group. When analyzed with the Mann-Whitney test (two-tailed), none, of these parameters varied s i g n i f i c a n t l y between the vegetarians and the non-vegetarians. No s i g n i f i c a n t c o r r e l a t i o n was found between mutagenicity with ei t h e r TA98 or TA100 and wet weight or dry weight. A negative s i g n i f i c a n t c o r r e l a t i o n (Table 9) was found between the pH and mutagenicity with TA98 when r e s u l t s from a l l subjects were ana-lyzed. This c o r r e l a t i o n was confirmed for the non-vegetarians (P<0.05). Although a negative c o r r e l a t i o n i s seen with TA100 and the pH for a l l sub-j e c t s , i t i s not s i g n i f i c a n t at the P<0.05 l e v e l . No s i g n i f i c a n t c o r r e l a -t i o n was seen between pH and mutagenicity for the s t r i c t vegetarians, the ovo-lacto vegetarians or when the r e s u l t s from these were combined. 48 Table 7 Spearman Rank Cor r e l a t i o n Between Mutagenicity with TA100 and TA98 Spearman Rank Cor r e l a t i o n C o e f f i c i e n t Significance A l l subjects 0.3595 <0.050 • 3. Vegetarians -0.0726 n.s. Non-vegetarians 0.4960 n.s. The c o r r e l a t i o n c o e f f i c i e n t s were determined from the ranks l i s t e d i n Tables 4 and 5, as described by Siegel (1956). Si t n.s. denotes not s i g n i f i c a n t (P>0.050). 49 Table 8 Wet Weight, Percent Dry Weight and pH of Fecal Samples from Three Diet Groups Diet Subject Wet Weight % Dry Group Number (gms) Weight pH 1 175.0 25.0 6.7 2 171.3 27.2 6.8 Ovo-lacto 3 136.0 26.1 6.2 vegetarians 4 85.1 15.3 5.7 5 78.6 18.9 6.5 6 73.6 18.8 7.2 Ma= 119.9 M = 21.9 M = 6.5 S.D. b± 46.9 S.D. ± 4.8 S.D. ± 0.5 7 207.4 16.5 6.4 8 232.0 33.6 6.7 9 110.0 29.0 6.3 10 85.3 32.0 6.1 S t r i c t 11 68.9 27.0 6.0 vegetarians 12 116.0 29.9 6.0 13 21.3 25.1 6.5 14 106.2 30.0 6.3 . 15 324.2 23.2 6.3 16 136.4 16.4 6.5 17 156.8 18.3 6.2 M = 142.2 M = 25.2 M = 6.4 S.D. ± 84.8 S.D. ± 6.2 S.D. ± 0.2 18 50.1 27.2 6.2 19 117.0 28.9 5.9 20 73.6 31.3 6.6 21 38.6 26.6 6.5 Non- 22 116.3 26.7 6.3 vegetarians 23 62.3 33.8 6.7 24 45.5 15.4 6.1 25 127.5 35.2 6.8 26 208.6 11.8 6.8 27 75.9 25.4 5.4 28 265-0 24.1 6.7 29 88.4 31.1 6.3 M = 105.7 M = 26.5 M = 6.4 S.D. ± 68.7 S.D. ± 6.9 S.D. ± 0.4 = mean. ^S.D. = standard deviation. Table 9 Spearman Rank Co r r e l a t i o n Between Mutagenicity and pH of the Fecal Homogenates pH of Homogenate Rank Cor r e l a t i o n C o e f f i c i e n t Significance TA100 TA98 TA100 TA98 A l l subjects -0.3314 -0.4056 n.s. <0.050 Vegetarians -0.2774 -0.1533 n.s. n.s. Non-vegetarians -0.3960 -0.6860 n.s. <0.050 Si.s. denotes not s i g n i f i c a n t (P>0.050). 51 B. DIETARY DATA AND OTHER INFORMATION 1. Questionnaire, Part I a. Demographic Variables, Dietary Practices and Health Status and Habits of Groups Sampled Information about the groups sampled i s provided by frequency d i s t r i b u t i o n of the questionnaire v a r i a b l e s broken down by diet groups and for the t o t a l sample (Tables 10, 11 and 12). Table 10 presents the demographic data of the sample studied. Of the t o t a l samples, 24.1% were between the ages of 19-24, with 37.9% i n the 25-29 year-old category and 27.6% aged between 30-34 years. Only 10.2% of the sample were older than 35 years. Chi-square analysis showed that the age d i s t r i b u t i o n varied among the groups (x 2=15.96, P<0.05), with a greater percentage of non-vegetarians in the 25-29 year-old category and more s t r i c t vegetarians between the ages of 19-24 than i n the other groups. The subjects studied were f a i r l y evenly d i s t r i b u t e d by sex among the groups with 41% males and.58.6% females i n the t o t a l sample. The ovo-lacto vegetarian group comprised 33.3% males and 66.7% females for 36.4% males and 63.6% females i n the s t r i c t vegetarian category and 50% of each sex among non-vegetarians. Of the p a r t i c i p a n t s , 72.4% were born i n North America. A l l subjects had resided i n Vancouver for at l e a s t a year p r i o r to the study. Table 11 gives information about the dietary practices of the p a r t i c i -pants. Among the non-vegetarians, 33.3% always trimmed fat from meat, while 41.7% reported doing so oc c a s i o n a l l y and 25% never removed f a t from meat. Regarding the consumption of hot spices ( c h i l l i e s , curry, e t c . ) , 48.3% 52 Table 10 Frequency D i s t r i b u t i o n of Demographic Variables by Diet Groups Variable Ovo-lacto S t r i c t Non- A l l Vegetarians Vegetarians Vegetarians Subjects (n=6) (n=ll) (n=12) (n=29) Age: 19-24 0 5 2 7 25-29 2 2 7 11 30-34 3 4 1 8 35-39 0 0 2 2 40-45 1 0 0 1 Sex: Male 2 4 6 12 Female 4 7 6 17 Birth p l a c e : Canada 2 5 9 16 United States 1 4 0 5 Europe 3 1 3 7 Other 0 1 0 1 53 Table 11 Frequency D i s t r i b u t i o n of Dietary Habits by Diet Groups Variable Ovo-lacto Vegetarians (n-6) S t r i c t Vegetarians (n=ll) Non-Vegetarians <n=12) A l l Subjects (n-29) Trimming f a t from meat: Always N/A* N/A 4 4 Occasionally N/A • N/A 5 5 Never N/A N/A 3 3 Eating hot spices Yes 5 4 5 14 No 1 7 7 15 Frequency of eating hot spices 3-5/week 2 0 0 2 1/week 0 1 3 4 3/month 1 1 2 4 1/month 2 2 1 5 N/A 1 7 6 14 Fasting: Yes 1 7 1 9 Rarely 0 1 0 1 No 5 3 11 19 Frequency of f a s t i n g : 1/week 1 3 0 4 2-3/month 0 1 0 1 1/month 0 1 0 1 No regular pattern 0 3 1 4 N/A 5 3 11 19 Length of f a s t i n g : 12-24 hours 0 3 1 4 24 hours plus 1 5 0 6 N/A 5 3 11 19 Special d i e t : 0 0 0 0 Table 11 (cont'd) 54 Variable Ovo-lacto S t r i c t Non- A l l Vegetarians Vegetarians Vegetarians Subjects (n=6) (n=ll) (n=12) (n=29) Recent changes i n food s e l e c t i o n : Yes 0 3 4 7 No 6 8 8 22 In l a s t 6 months 0 0 3 3 In l a s t year 0 3 1 4 N/A 6 8 8 22 N/A denotes not applica b l e . 55 of the t o t a l sample reported t h i s p r a c t i c e , which was very common among the ovo-lacto vegetarians (83.3%). Only 6.9% of the population ate hot spices at l e a s t 3-5 times per week and 13.8% weekly. Most of the s t r i c t vege-tarians who abstained from hot spices were Seventh Day Adventists following the Recommendation of the Church ( P h i l l i p s et a l . , 1980). The p r a c t i c e of f a s t i n g was not"widespread among the ovo-lacto vege-tarians and the non-vegetarians. Only one subject from each of these groups reported f a s t i n g . However, i t was more common among the s t r i c t vege-tarians with 70.7% of the subjects f a s t i n g occasionally. Two s t r i c t vege-tarians and one non-vegetarian fasted for 12-24 hours, while one ovo-lacto vegetarian and three s t r i c t vegetarians fasted for 24 hours or longer. The frequency of f a s t i n g varied greatly. Of those who fasted (n=10) , four subjects did so weekly, one subject fasted two to three times per month, while four subjects had no regular pattern and one subject fasted monthly. None of the p a r t i c i p a n t s claimed to be on a reducing or any other thera-peutic d i e t at the time of the experiment. With food prices increasing and people becoming more aware of what they are eating, 24.1% of the p a r t i c i p a n t s made some changes i n t h e i r shopping habits i n the year p r i o r to the study. Among the non-vegetarians, 33.3% bought and ate l e s s beef, and one of these non-vegetarians increased h i s consumption of f r u i t s and vegetables. One s t r i c t vegetarian stopped buying canned goods and two increased t h e i r intake of grains. The frequency d i s t r i b u t i o n of the health status and habits v a r i a b l e s i s given i n Table 12. About ha l f the population studied reported weight changes i n the l a s t year and t h i s proportion was r e f l e c t e d i n the d i e t subgroups as w e l l . Of those, 34.5% stated to have gained weight, comprising 16.7% of the ovo-Table 12 Frequency D i s t r i b u t i o n of Health Status and Habits by Diet Groups Ovo-lacto S t r i c t Non- A l l Vegetarians Vegetarians Vegetarians Subjects Variable (n=6) (n=ll) (n=12) (n=29) Weight change i n l a s t year: Gained Lost N/Aa 1 2 3 3 3 5 6 1 5 10 6 13 Taking contraceptive p i l l s i n l a s t year: Taking a n t i b i o t i c s i n l a s t month: Digestive disease i n past: 1 Use of laxative i n past: 0 Use of la x a t i v e now: 0 Smoking: Yes 2 No 4 3 0 0 11 1 0 4 0 5 24 History of cancer i n family: Yes 1 No 5 Use of vitamins or supplements: Yes 5 No 1 6 5 8 4 3 9 15 14 12 17 N/A denotes not applicable. l a c t o vegetarians, 27.3% of the s t r i c t vegetarians and 50% of the non-vegetarians. Of the 20.7% who stated weight l o s s , we count 33.3% of the ovo-lacto vegetarians, 27.3% of the s t r i c t vegetarians and 8.3% (n=l) of the non-vegetarians. Using the Nomograph f o r Body Mass Index as a reference (Figure 3), one female non-vegetarian was c l a s s i f i e d as being 20% above desirable weight for her height. Two male ovo-lacto vegetarians, one female and two male s t r i c t vegetarians proved to be 20% underweight for t h e i r height. None of the p a r t i c i p a n t s took a n t i b i o t i c s for at least one month p r i o r to or during the study. The use of b i r t h control p i l l s was reported by four non-vegetarian women. Few p a r t i c i p a n t s admitted to having suffered from digestive problems i n the past. One ovo-lacto vegetarian (16.6%) was diagnosed as having an ameo-bi c i n f e c t i o n s i x years previously with no recurrence. Two non-vegetarians (16.6%) had experienced ulcer problems about three years previously but were symptom-free for the l a s t two years. One non-vegetarian (8.3%) had been t o l d that he had d i v e r t i c u l o s i s several years previously, but the diagnosis was not confirmed subsequently. A l l subjects were i n good health at the time of the experiment. The mean bowel frequency per day was 0.93±0.48 for non-vegetarians and 1.62±0.072 for vegetarians. The Mann-Whitney U test indicated that the difference i n the frequency of bowel movements was s i g n i f i c a n t (PO.05, two-t a i l e d t e s t ) . Of the sample studied, 13.8% were using l a x a t i v e s o c c a s i o n a l l y , although not during the period of c o l l e c t i o n of f e c a l samples. The laxatives c i t e d to be i n use were: 1. Milk of magnesia (by one non-vegetarian), 58 FIGURE 3 NOMOGRAPH FOR BODY M A S S INDEX ( K G / M 2 ) H E I G H T I N I C M M 2 5 50-4-55-60 65 -70 - F 75H 90-8 5 - ^ •130 i—135 •l'40 •145 •150 •155 •160 •1.65 •170 •175 •180 • 185 •190 • 195 • 200 • 205 •210 WOMEN (+20%) "OVERWEIGHT" "DESIRABLE" t i l "UNDERWEIGHT" ( -20%) W E I G H T K G -70 •60 -50 4^ 4 0 3 0 -MEN L B 340 320 3O0 280 260 240 220 200 190 180 170 160 •20" (+20%) "OVERWEIGHT" 1 5 0 140 III "DESIRABLE" 130 + 120-"UNDERWEIGHT" ( - 2 0 % 100-95-90-85-80-75-70-65 -60-55 -50 - i -150 40 ^-130 20 - HO ^-100 i h 9 0 :E-85 jj - 8 0 1 -75 70 65 £ - 6 0 ^ 5 5 10-+- 50 -h 45 3- "0 4-35 3- 30 + 25 In: Thomas et a l . (1976). 59 2. Flax seeds (by one s t r i c t vegetarian), 3. Swiss K r i s s "organic food preparation" (by 2 s t r i c t vegetarians). Smoking was not a prevalent p r a c t i c e since only 13.8% of a l l the par-t i c i p a n t s reported that they smoked. Two ovo-lacto vegetarians and two non-vegetarians smoked. None of the s t r i c t vegetarians smoked. The amount of ciga r e t t e s smoked ranged from 5 per month to 20 per day. History of cancer i n the family (parents, s i b l i n g s , grandparents, aunts, uncles, cousins, etc.) was reported by 51.7% of the p a r t i c i p a n t s , s p e c i f i c a l l y among the s t r i c t vegetarians (n=6/ll) and the non-vegetarians (8/12 subjects). The types of cancer c i t e d included breast, l i v e r , b r a i n , ovarian, colon and leukemia. Only two s t r i c t vegetarians and one non-vegetarian documented cancer i n t h e i r nearest family (father, mother, s i b l i n g s ) . Only one subject reported colon cancer but not i n the nearest family. The use of vitamins and supplements was very widespread i n the ovo-l a c t o vegetarian group (83.3%) compared to 36.4% and 25% in the s t r i c t vege-tarians and non-vegetarians, r e s p e c t i v e l y . Of the vitamins and vitamin com-binations described, there were multivitamins, multivitamins and minerals, ascorbic a c i d , vitamin B's, vitamin B's plus C, vitamin E, multivitamins plus vitamins B's and C, multivitamins plus B complex, and vitamin E plus B complex. L e c i t h i n was ingested d a i l y by two s t r i c t vegetarians (18.2%) and chloro-p h y l l t a b l e t s by two ovo-lacto vegetarians (33.3%). b. C o r r e l a t i o n Between Mutagenicity and Dietary P r a c t i c e s , Health Status and Habits of Groups Sampled In order to elucidate which f a c t o r s may be associated with f e c a l 60 mutagenicity, Spearman rank c o r r e l a t i o n test was conducted between the rank of mutagenicity with TA98 and TA100 (Tables 2 and 3) and some of the ques-tionnaire v a r i a b l e s . The r e s u l t s are shown i n Tables 13 and 14. A p o s i t i v e s i g n i f i c a n t c o r r e l a t i o n was established between increasing age and mutagenicity with TA100 (P=0.05) for the non-vegetarians. This c o r r e l a t i o n was not substan-t i a t e d f o r the other diet groups or when age was tested against mutagenicity with TA98. The influence of trimming f a t from meat on f e c a l mutagenicity was only analyzed for the non-vegetarians, since ovo-lacto and s t r i c t vegetarians were not concerned with t h i s v a r i a b l e . There was no c o r r e l a t i o n between mutagenicity and t h i s dietary p r a c t i c e . The s t r i c t vegetarians who ate hot spices (n=4) revealed high rates of mutagenicity with TA98 (PO.05). The frequency of eating hot spices was also correlated p o s i t i v e l y to the mutagenicity with TA98 for the same group, but was not seen i n ovo-lacto vegetarians who consumed a l o t of spicy foods (n=5). S u r p r i s i n g l y , the number of years that a person had been a vegetarian was co r r e l a t e d p o s i t i v e l y with mutagenicity using TA100 (PO.05) i n the s t r i c t vegetarian group and when a l l the vegetarians were combined together. This c o r r e l a t i o n was not present for the ovo-lacto vegetarians, possibly due to the small number of subjects i n t h i s group. The use of laxatives was s i g n i f i c a n t l y correlated to mutagenicity with TA98 for the s t r i c t vegetarians and for a l l vegetarians. However, few sub-Table 13 Spearman Rank C o r r e l a t i o n s Between Questionnaire V a r i a b l e s and Muta g e n i c i t y with TA100 A l l Subjects Ovo-lacto Veg. S t r i c t Veg. Non-•Veg. A l l Veg. V a r i a b l e s Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. C o e f f . Sign. Corr. Coeff. Sign. Age 0.2521 a n.s. -0.0617 n.s. 0.5210 n.s. 0.5747 0.05 0.1899 n.s. Sex 0.0377 n. s. 0.2070 n. s. -0.2096 n.s* 0.2418 n.s. -0.0377 n.s. Trimming of f a t from meat N/Ab N/A N/A N/A N/A N/A -0.1287 n.s. N/A N/A Hot spices -0.0330 n.s. -0.1309 n. s. -0.2995 n.s. 0.1717 n.s. -0.1685 n.s. Frequency of hot spices -0.1519 n.s. 0 n.s. -0.2064 n.s. -0.0321 n.s. -0.1175 n.s. F a s t i n g 0.3383 n.s. 0.1309 n.s. 0.1977 n.s. 0.4812 n.s. 0.1087 n.s. No. of years v e g e t a r i a n N/A N/A 0.6000 n.s. 0.6444 <0.05° N/A N/A 0.6154 <0.01 d Contraceptive p i l l s -0.1614 n.s. N/A N/A N/A N/A 0.1282 n.s. N/A N/A Bowel movement frequency -0.2439 n. s. 0.5296 n. s. -0.4452 n.s. -0.3249 n. s. -0.1458 n.s. Bowel movement i r r e g u l a r i t y -0.0498 n.s. 0.1690 n.s. 0.2027 n. s. -0.4354 n.s. 0.2229 n.s. Use of l a x a t i v e s -0.0837 n.s. N/A N/A -0.1294 n.s. -0.2187 n.s. -0.2206 n. s. L a x a t i v e now N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Smoking -0.1197 n. s. -0.2070 n.s. N/A N/A -0.0507 n.s. -0.1119 n.s. H i s t o r y of cancer -0.2476 n.s. -0.1309 n.s. -0.5208 n.s. -0.0769 n.s. -0.3906 n.s. Vitamins 0.0921 n.s. 0.1309 n.s. -0.0898 n. s. 0.0279 n.s. -0.0361 n.s. ^n.s. denotes not s i g n i f i c a n t (P>0.05). N/A denotes not a p p l i c a b l e . ^ 0 . 05 means >0.01 <0.05. <0.01 means >0.001 <0.01. Table 14 Spearman Rank C o r r e l a t i o n s Between Questionnaire V a r i a b l e s and Muta g e n i c i t y with TA98 A l l Subjects Ovo-lacto Veg. S t r i c t Veg. Non--Veg. A l l Veg. V a r i a b l e s Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. C o e f f . Sign. Corr. Coeff. Sign, Age 0.1049 a n. s. 0.0000 n.s. 0.3318 n.s. 0.0236 n.s. 0.2352 n.s. Sex -0.0545 n. s. -0.2070 n.s. -0.3893 n.s. 0.4118 n.s. -0.3528 n.s. Trimming of fat from meat N/Ab N/A N/A N/A N/A N/A 0.0991 n.s. N/A N/A Hot s p i c e s 0.0991 n.s. -0.6547 n.s. 0.6589 <0.05 c 0.0000 n.s. 0.1810 n.s. Frequency of hot s p i c e s -0.0320 n. s. -0.6179 n.s. 0.6298 <0.05 0.0170 n.s. 0.0550 n.s. F a s t i n g 0.3202 n.s. 0.6547 n.s. 0.0588 n.s. 0.3068 n.s. 0.2455 n.s. No. of years v e g e t a r i a n N/A N/A 0.6000 n. s. -0.0878 n.s. N/A N/A 0.2004 n.s. Contraceptive p i l l s -0.2275 n.s. N/A N/A N/A N/A -0.1799 n.s. N/A N/A Bowel movement frequency 0.0485 n.s. 0.4414 n.s. -0.2782 n.s. 0.2863 n.s. 0.0121 n.s. Bowel movement i r r e g u l a r i t y -0.0362 n. s. 0.6761 n. s. -0.2027 n.s. -0.2299 n.s. 0.1738 n. s. Use of l a x a t i v e s 0.2514 n. s. N/A N/A 0.7117 <0.05 -0.3944 n.s. 0.5371 <0.05 Laxative now N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Smoking -0.2112 n.s. -0.4140 n.s. N/A N/A -0.0901 n.s. -0.2991 n.s. H i s t o r y of cancer 0.1446 n. s. -0.3928 n.s. 0.4051 n. s. 0.2056 n.s. 0.2080 n.s. Vitamins 0.0419 n.s. 0.3928 n.s. 0.4193 n.s. -0.5595 n.s. 0.2896 n.s. n.s. denotes not s i g n i f i c a n t (P>0.05). ^N/A denotes not a p p l i c a b l e . c<0.05 means >0.01 <0.05. 63 jects (n=4) used a laxative and none at the time of the experiment. No s i g n i f i c a n t c o r r e l a t i o n was established with mutagenicity on TA98 or TA100 and sex, f a s t i n g , use of b i r t h c o n t r o l p i l l s , bowel movement f r e -quency, bowel i r r e g u l a r i t y , smoking, h i s t o r y of cancer and the use of vitamins and/or supplements. 2_. Questionnaire, Part II a. Comparison of Food Frequencies Between Diet Groups Since the aim of the food frequency segment of the questionnaire was to define which foods or food groups were correlated with mutagenicity, i t was important to f i r s t characterize the d i e t of the p a r t i c i p a n t s . This was achieved by comparing the frequency of intake of food groups among the di e t subgroups. The r e s u l t s from the Mann-Whitney U t e s t (two-tailed) are given i n Table 15. When the intake of ovo-lacto vegetarians was compared to that of the s t r i c t vegetarians, i t was found that they consumed s i g n i f i c a n t l y more dairy products, f i s h , eggs and animal f a t , as was expected from the d e f i -n i t i o n of the d i e t subgroups. They also ingested more sugar and sweets, baked goods, f r u i t j u i c e s , t o t a l f a t and more miscellaneous items such as tea, c o f f e e , wine, etc. (see Table 3, page 34). However, the ovo-lacto vegetarians ate l e s s nuts and legumes as well as hot cereals than the s t r i c t vegetarians. Compared to the non-vegetarians, the ovo-lacto vegetarians' intake was higher i n nuts and legumes, f r u i t s , f r u i t s and j u i c e s combined and f l o u r s , but lower i n meat, poultry and f i s h , which was expected. The s t r i c t vegetarians' d i e t d i f f e r e d from that of the non-vegetarians Table 15 Comparison of Food Frequencies Between D i e t Groups by the Mann-Whitney U Test (Two-Tailed) Food Items Ovo-lacto Veg. Ovo-lacto Veg. S t r i c t Veg. A l l Veg. n=6 n=6 n = l l n=17 vs vs vs vs S t r i c t Veg. Non-Veg. Non-Veg. Non-Veg. n = l l n=12 n=12 n=12 Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Dairy products 1A: Cheese 13.92 n i n b 9.00 a 6.64 . _.,d 11.35 6.32 < 0 - 0 1 0 9.75 n - S - 16.92 < 0 - 0 0 1 20.17 < 0 - 0 1 0 IB: Cream 11.67 n.s. 10.33 8.95 . n c r f i 13.32 7.55 9.08 n - s - 14.79 < 0 - 0 5 0 17.38 IC: Ice cream 11.08 6.58 7.14 . n n . 10.82 7.86 n - s - 10.96 n ' s - 16.46 < ( K 0 0 1 20.92 < 0 - 0 1 0 1 D : M i l k 1 4 - 3 3 -n mr, 8 - 3 3 6 - 0 9 r, ™ , 10-76 6.09 < 0 - 0 1 ° 10.08 n - S - 17.42 < 0 - 0 0 1 21.00 < 0 - 0 1 0 1: A l l 14.33 , n n , n 9.33 _ „ 6.00 „„„ 11.06 Meat & a l t e r n a t e s 6.09 < 0 - 0 1 0 9.5S n - s - 17.50 < 0 - 0 0 1 2o!58 < 0 - 0 1 0 2 A : B e e f 9-33 - - 3-50 <o.ooi ,6-?° <o.ooi <0.001 8.82 12.50 1 17.50 23.50 1:11 !42:S '«>•«» il% — S:S 9^7 JiS «™ i% O.001 Table 15 (cont'd) Food Items Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e 2D: V a r i e t y meats 9.00 _ £ 0 0 < 0 . Q l 0 £ 5 0 < Q ^ £ . 5 0 2E: Game 9.00 6.50 . 9.00 . n i n 12.00 „ 9.00 n - S - 11.00 < 0 - 0 5 ° 14.75 . < 0 - ° 1 0 19.25 < 0 M W 2F: P o u l t r y 11.25 n s 3.50 „. 6.00 . „ . 9.00 ... 7.77 12.50 < 0 - 0 0 1 17.50 < 0 - 0 0 1 23.50 < 0 - 0 0 1 2G: F i s h S seafoods 11.92 < 0 0 5 0 4.50 6 05 9.38 < Q J i Q l 7.41 12.00 17.46 22.96 2H:Eg8S Tsl < ° - ° 5 0 1:11 <o-ooi \l:bA 21: Nuts S legumes 5.67 < 0 0 5 0 13.75 < 0 0 5 0 17.82 < Q _ 20.26 < 0 _ 0 Q 1 10.82 7.38 6.67 7.54 2A-E: Meats 9.33 3.50 . 6.00 „- ... 9.00 ... 8.82 n " S - 12.50 < 0 ' 0 0 1 17.50 < 0 - ° 0 1 23.50 < 0 " 0 0 1 2A-F: Meats S p o u l t r y 11.00 " ^ 3.50 < Q 0 Q 1 6.00 < Q 0 0 l 9.00 < 0 Q Q l 7.91 12.50 17.50 zj.50 2A-H: Meat, p o u l t r y 14 33 < 0 _ 0 1 Q 3.83 Q 6.00 < Q J ) Q l 9.12 < Q Q O l & eggs 6.09 12.33 17.50 23.33 2: A l l 5.67 _ .,. 9.17 16.64 n m n 17.88 n 10.82 < 0 - 0 5 0 9.67 n - S " 7.75 < 0 - ° 1 0 10.92 < 0 - ° 5 ° 3: Vegetables 8.50 9.00 11.36 14.41 9.27 n - S - 9.75 n - S - 12.58 n " S ' 15.83 Table 15 (cont'd) Food Items Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e 4A: F r u i t s 4B: J u i c e s 4: A l l 3+4: F r u i t s & vegetables 5A: Bread, yeast & other 5B: C e r e a l , ready-to-eat 5C: Hot c e r e a l 5D: Other c e r e a l 5E: F l o u r s 5F: Baked goods 8.67 9.18 12.50 7.09 10.00 8.45 9.50 8.73 8.00 9.55 7.42 9.86 5.58 10.86 8.75 9.14 9.08 8.95 12.08 7.32 <0.050 <0.050 13.00 7.75 9.33 9.58 13.67 7.42 11.50 8.50 9.17 9.67 11.50 8.50 12.75 7.88 8.00 10.25 14.67 6.92 10.00 9.25 <0.050 <0.0 50 <0.005 16.00 8.33 9.41 14.38 15.41 8.88 13.73 10.42 12.-41 11.63 14.91 9.33 15.55 8.75 10.68 13.21 18.00 6.50 9.55 14.25 <0.010 <0.050 <0.050 <0.050-<0.001 18.82 9.58 13.26 17.46 18.68 9.79 16.82 12.42 15.15 14.79 17.59 11.33 18.44 10.13 13.62 16.96 20.71 6.92 13.59 17.00 <0.010 <0.0i0 <0.010 <0.001 5A-E 5: A l l 6.67 10.27 7.50 9.82 11.50 8.50 11.67 8.42 16.45 7.92 15.73 8.58 <0.010 <0.050 18.59 9.92 18.18 10.50 <0.0l0 <0.050 Table 15 (cont'd) Food Items Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e Mean Rank S i g n i f i c a n c e 6: Sugar & sweets 14.33 ^ n m n 10.50 8.14 12.85 n.s. 6.09 < 0 - 0 1 ° 9.00 n - S - 15.54 < 0 - ° 1 0 18.04 7A: Animal f a t 14.25 < 0 Q Q 1 9.42 n ^ 6.14 < 0 Q Q l 11.18 6.14 9.54 17.Jo Z U . 4 Z 7B: Vegetable f a t 9.83 6.17 9.23 12.03 8.55 n - S - 11.17 n - S - 14.54 n - s - 19.21 7C: Combination 9.42 6.08 7.00 10.56 8.77 n - S " 11.21 n - s " 16.58 <0.00l 21.29 <0.050 <0.001 1- A l l 12.33 „ n , „ 7.33 7.27 _ m n 11.18 n n l n 7.18 < 0 - 0 5 ° 10.58 n " S - 16.33 < 0 - 0 1 0 20.42 < 0 - ° 1 0 8: Miscellaneous 13.50 „ 8.33 6.55 11.06 < 0 0 1 0 6.55 <U .01U W Q 8 n.s. 1 7 0 Q u.uui 2 0 5 g n.s. denotes not s i g n i f i c a n t (P>0.05). b<0.010 means >0.001 <0.010. C<0.050 means >0.010 <0.050. d<0.001 means $0,000 <0.001. 68 by being s i g n i f i c a n t l y r i c h e r i n nuts and legumes, f r u i t s , f r u i t s and juices combined, ready-to-eat c e r e a l , hot c e r e a l s , f l o u r s , and i n bread and sub-s t i t u t e s i n general. Their diet was expectedly poorer i n dairy products, meats and alternates (except nuts and legumes), animal f a t and t o t a l f a t , and miscellaneous items such as sugar and sweets. A f a i r l y s i m i l a r pattern was drawn when a l l vegetarians combined were compared to the non-vegetarians. b. C o r r e l a t i o n Between Food Frequencies and Mutagenicity with TA100 and TA98 The Spearman c o r r e l a t i o n was then c a r r i e d out between mutagenicity and the frequency of intake of food items or food groups (Table 16). The food items which c o r r e l a t e d with TA100 did not n e c e s s a r i l y corre-l a t e with TA98. A p o s i t i v e s i g n i f i c a n t c o r r e l a t i o n was established between mutagenicity with TA100 and frequency of intake of: 1. Cream among the non-vegetarians (P<0.05), 2. Milk among the s t r i c t vegetarians (P<0.05), 3. Beef, lamb, pork, v a r i e t y meat, game, poultry, f i s h and seafoods and a l l meats plus alternates combined for a l l subjects. Only the intake of lamb and game was s i g n i f i c a n t l y correlated with TA100 among the non-vegetarians. Mutagenicity of extracts tested with TA100 was found to be negatively correlated with frequency of intake of: 1. Ready-to-eat cereals among the s t r i c t vegetarians (P<0.05), 2. Miscellaneous items among the ovo-lacto vegetarians (PO.05; see Table 3, page 34) . Table 16 Spearman Rank C o r r e l a t i o n Between Food Frequencies and Muta g e n i c i t y with TA100 (Two-Tailed Test) A l l Subjects Ovo-lacto Veg. S t r i c t Veg. Non-Veg. A l l Veg. Corr. Corr. Corr. Corr. Corr. Food Items Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. D a i r y products 1A: Cheese 0.2272 a n.s. -0.6000 n.s. 0.1848 n.s. 0.4912 n.s. -0.1663 n. s IB: Cream 0.1922 n. s. -0.0294 n. s. -0.2554 n.s. 0.7003 <0.050 -0.2174 n.s IC: Ice cream 0.3195 n.s. 0.0000 n.s. 0.2171 n.s. 0.3257 n.s. -0.0724 n.s ID: M i l k 0.2022 n.s. 0.0857 n.s. 0.6728 <0.050 -0.3363 n.s. 0.0578 n.s 1: A l l 0.2499 n.s. -0.0286 n.s. 0.5616 n.s. 0.1961 n.s. -0.0098 n. s Meats & a l t e r n a t e s < 2A: Beef 0.4996 <0.010c -0.1309 n.s. 0.3007 n. s. 0.4851 n. s. 0.0569 n. s 2B: Lamb 0.5364 <0.010 ' N/Ab N/A N/A N/A 0.7066 <0.010 N/A N/A 2C: Pork 0.4560 <0.050 d N/A N/A 0.3007 n.s. -0.1235 n. s. 0.2553 n.s 2D: V a r i e t y meats 0.4175 <0.050 N/A N/A N/A N/A 0.1378 n. s. N/A N/A 2E: Game 0.5853 <0.010 N/A N/A N/A N/A 0.8222 <0.010 N/A N/A 2F: P o u l t r y 0.4043 <0.050 -0.0976 n.s. 0.3007 n.s. 0.0726 n.s. -0.0566 n.s 2G: F i s h & seafoods 0.4744 <0.010 0.4058 n.s. 0.0405 n.s. 0.2159 n.s. 0.1321 n.s 2H: Eggs 0.2434 n.s. 0.1449 n.s. 0.0571 n.s. 0.1532 n.s. -0.0524 n. s 21: Nuts & legumes -0.1696 n.s. -0.0857 n.s. 0.2369 n.s. 0.4851 n.s. 0.2735 n.s 2A-E: Meats 0.5025 <0.010 -0.1309 n.s. 0.3007 n.s. 0.5333 n.s. 0.0569 n. s 2A-F: Meats & 0.4521 <0.050 -0.1234 n. s. 0.3007 n.s. 0.4386 n.s. -0.0280 n.s p o u l t r y 2A-H: 0.3882 <0.050 . 0.2571 n.s. -0.0594 n.s. 0.2942 n.s. -0.1618 n. s 2: A l l 0.0069 n. s. -0.0857 n.s. 0.2369 n. s. 0.2842 n.s. 0.2735 n. s Table 16 (cont'"d) Food Items Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. Coeff. Sign. Corr. Coeff. Sign. 3: Vegetables 0.2191 n.s. 0.4286 n.s. -0.0137 n.s. 0.1997 n.s. 0.2906 n. s. F r u i t s and j u i c e s 4A: F r u i t s -0.0737 n.s. 0.4286 n.s. 0.2688 n.s. 0.0385 n.s. 0.4402 n. s. 4B: J u i c e s -0.0264 n.s. -0.4119 n. s. 0.0411 n.s. -0.2487 n.s. -0.1208 n.s. 4: A l l -0.0960 n.s. 0.6000 n.s. 0.4374 n.s. 0.0070 n.s. 0.4365 n.s. 3-4: F r u i t s & Veg. 0.1111 n.s. 0.7143 n.s. 0.2688 n.s. 0.2067 n.s. 0.4365 n.s. Breads & a l t e r n a t e s 5A: Bread, yeast and others -0.1188 n.s. 0.2319 n.s. -0.1996 n.s. -0.0321 n.s. -0.2087 n.s. 5B: C e r e a l , ready-to-eat -0.2804 n.s. 0.1429 n.s. -0.6796 <0.050 0.2197 n. s. -0.3053 n.s. 5C: Hot c e r e a l -0.1563 n.s. 0.0857 n. s. -0.0572 n.s. 0.1248 n.s. 0.0651 n.s. 5D: Other c e r e a l s -0.0331 n. s. -0.1518 n.s. -0.1512 n. s. -0.1962 n.s. -0.1816 n.s. 5E: F l o u r s -0.3641 n. s. -0.0286 n.s. -0.0947 n.s. 0.1431 n. s. -0.0413 n.s. 5F: Baked goods -0.0898 n.s. -0.4286 n. s. -0.0938 n.s. 0.0211 n.s. -0.3225 n.s. 5A-E: -0.2843 n.s. 0.2571 n. s. -0.4510 n.s. -0.0600 n. s. -0.2097 n.s. 5: A l l -0.3346 n.s. -0.0286 n.s. -0.4601 n.s. 0.0911 n. s. -0.3115 n.s. 6: Sugar & sweets 0.1333 n.s. -0.7714 n.s. 0.1709 n.s. 0.2807 n.s. -0.1945 n.s. Fats 7A: Animal f a t 0.2416 n.s. -0.3043 n. s. 0.0647 n. s. 0.2425 n.s. -0.1654 n. s. 7B: Vegetable f a t 0.2759 n.s. -0.7143 n. s. 0.1689 n.s. 0.3697 n.s. 0.0147 n.s. 7C: Combination 0.2480 n. s. 0.3947 n.s. 0.0984 n.s. -0.3069 n.s. 0.0493 n.s. 7: A l l 0.1965 n.s. -0.3143 n.s. 0.2009 n. s. 0.1965 n.s. -0.0590 n.s. 8: Miscellaneous 0.2637 n.s. -0.8286 <0.050 0.2037 n.s. 0.4904 n.s. -0.1946 n.s. ^n.s. denotes not N/A denotes not s i g n i f i c a n t . a p p l i c a b l e . c<0.010 means <0.050 means >0.001 >0.010 <0, <0, .010. .050 No s i g n i f i c a n t c o r r e l a t i o n was found between mutagenicity with TA100 and intake of vegetables, f r u i t s , j u i c e s , f r u i t s and j u i c e s combined and intake of f a t regardless of type. A d i f f e r e n t p i c t ure emerged when food items were c o r r e l a t e d with muta-geni c i t y of f e c a l extracts using TA98. Most of the s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s with TA100 proved to be negative here (Table 17). Among the food items negatively c o r r e l a t e d with TA98, we f i n d the intake of: 1. Milk among s t r i c t vegetarians (P<0.05), 2. Poultry (P<0.05) i n the ovo-lacto vegetarian group, and the intake of beef, lamb, pork, v a r i e t y meats, game and poultry when a l l items are combined (P<0.01). None of these items are s i g n i f i c a n t l y corre-lated with mutagenicity with TA98 among the non-vegetarians, 3. F r u i t intake for a l l subjects (P<0.05), for s t r i c t vegetarians (P<0.01), and among a l l vegetarians (P<0.01). F r u i t s and j u i c e s combined showed the same c o r r e l a t i o n for the above groups, 4. The combined intake of f r u i t s and vegetables (P<0.05) among a l l vegetarians, 5. Ready-to-eat and hot cereals (P<0.05) and a l l breads and s u b s t i -tutes for the ovo-lacto vegetarians, 6. Vegetable f a t for ovo-lacto vegetarians (PO.05), s t r i c t vege-tarians (P<0.05), and for a l l vegetarians (P<0.01). The t o t a l f a t intake was also negatively c o r r e l a t e d with TA98 i n the s t r i c t vegetarian group, 7. Intake of miscellaneous items among s t r i c t vegetarians (P<0.05). Since no c l e a r picture about intake of nutrients and mutagenicity could be drawn from the food frequency segment of the questionnaire, i t was f e l t that looking at the nutrient intake of the subjects may be more informative. Table 17 Spearman Rank C o r r e l a t i o n Between Food Frequencies and Muta g e n i c i t y with TA98 (Two-Tailed Test) A l l Subjects Ovo-lacto Veg. S t r i c t Veg. Non-Veg. A l l Veg. Corr. Corr. Corr. Corr. Corr. Food Items Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. D a i r y products IA: Cheese 0. 1967 a n. s. -0.2571 n. s. IB: Cream 0. 2972 n. s. 0.1177 n.s. 1C: Ice cream 0. 3621 n. s. 0.0580 n.s. ID: M i l k -0. 0726 n. s. -0.7714 n.s. 1: A l l 0. 0893 n. s. -0.5429 n.s. Meats & a l t e r n a t e s 2A: Beef 0. 2893 n. s. -0.6547 n.s. 2B: Lamb 0. 2807 n. s. N/Ab N/A 2C: Pork 0. 2921 n.s. N/A N/A 2D: V a r i e t y meats 0. .1601 n.s. N/A ' N/A 2E: Game 0. 1834 n. s. N/A N/A 2F: P o u l t r y 0. 2109 n. s. -0.8783 <0.050 2G: F i s h & seafoods 0. 3027 n. s. -0.3479 n.s. 2H: Eggs 0. .3496 n.s. 0.4928 n.s. 21: Nuts & legumes -0. 2922 n. s. -0.4286 n.s. 2A-E: Meats 0. .2699 n. s. -0.6547 n. s. 2A-F: Meats & p o u l t r y 0.2197 n.s. -0.9258 <0.010 d 2A-H: 0. .3617 n.s. 0.4286 n.s. 2: A l l -0. 1314 n.s. -0.4286 n. s. 3: Vegetables -0. 0271 n.s. 0.0857 n. s. -0.2541 n. s. 0.3304 n.s. -0.1191 n. s, -0.0996 n.s. 0.3857 n.s. 0.1001 n.s 0.4578 n.s. -0.0820 n.s. 0.3040 n. s. -0.6590 <0.050° -0.4632 n.s. -0.3248 n.s. -0.2854 n.s. -0.1474 n.s. -0.1353 n.s o.oooo 6 n. s. 0.3081 n. s. -0.2853 n.s N/A N/A 0.1505 n.s. N/A N/A 0.0000 n. s. 0.1360 n.s. -0.0512 n.s N/A N/A -0.3352 n. s. N/A N/A N/A . N/A 0.0242 n.s. N/A N/A 0.0000 n.s. 0.0153 n.s. -0.3407 n. s -0.2703 n. s. -0.4812 n.s. -0.2145 n. s 0.4160 n.s. -0.1340 n. s. 0.4481 n.s -0.2232 n.s. 0.0282 n. s. -0.2409 n.s 0.0000 n.s. 0.2004 n.s. -0.2853 n.s 0.0000 n.s. 0.1125 n.s . -0.3406 n. s 0.2995 n.s. 0.0070 n.s. 0.2418 n.s -0.2232 n.s. 0.1195 n.s. -0.2409 n. s -0.3326 n.s . 0.0772 n.s. -0.1598 n.s Table 17 (cont'd) Corr. Corr. Corr. Corr. Corr. Food Items Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. C o e f f . Sign. F r u i t s and j u i c e s 4A: F r u i t s -0.3757 O.050 -0. .6000 n.s. -0. 7426 <0.010 -0.0491 n.s. -0.6380 O.010 4B: J u i c e s 0.0587 n.s. 0. ,6768 n.s. -0. 2877 n.s. 0.1201 n.s. 0.0031 n.s. 4: A l l -0.3745 <0.050 -0. 3143 n.s. -0.8018 <0.010 0.0561 n. s. -0.6343 <0.010 3-4: F r u i t s & Veg. -0.2159 n. s. -0. 1429 n. s. -0, .5695 n.s. 0.1123 n. s. -0.4831 <0.050 Breads & a l t e r n a t e s 5A: Bread, yeast and others 0.0684 n. s. -0. 6377 n.s. 0.4084 n.s. 0.1982 n.s. -0.0720 n.s. 5B: C e r e a l , ready-to-eat -0.1469 n.s. -0. ,8286 <0.050 0.4806 n.s. 0.0246 n.s. -0.0634 n.s. 5C: Hot c e r e a l -0.0247 n.s. -0. ,8286 <0.050 0, .1785 n.s. 0.2893 n.s. 0.0203 n.s. 5D: Other c e r e a l s 0.3254 n. s. 0.6375 n.s. 0. .1134 n.s. 0.2211 n.s. 0.3199 n. s. 5E: F l o u r s -0.3421 n.s. -0.5429 n.s. -0. .4596 n.s. 0.2427 n.s. -0.4568 n. s. 5F: Baked goods -0.0464 n.s. -0.0857 n.s. -0. ,2838 n.s. 0.1107 n.s. -0.2580 n. s. 5A-E: -0.2295 n.s. -0. .7714 n.s. 0, .2961 n.s. -0.0813 n.s. -0.2422 n. s. 5: A l l -0.1458 n. s. -0. .8286 <0.050 0 .3690 n.s. 0.1193 n.s. -0.1819 n.s. 6: Sugars & sweets 0.1405 n.s. -0.0857 n.s. -0, .4642 n.s. 0.4253 n. s. -0.1209 n.s. Fats -7A: Animal f a t 0.3243 n. s. 0 .1690 n.s. -0 .3882 n.s. 0.3715 n.s. -0.0171 n.s. 7B: Vegetable f a t -0.1421 n. s. -0 .8286 <0.050 -0 .7260 <0.050 0.1305 n. s. -0.6482 <0.010 7C: Combination 0.1860 n.s. -0, .3947 n.s. 0 .3386 n.s. 0.0954 n.s. 0.0045 n.s. 7: A l l 0.0648 n. s. -0 .4286 n.s. -0 .6986 <0.050 0.253i n.s. -0.4325 n.s. 8: M i s c e l l a n e o u s 0.0287 n.s. -0 .4286 n. s. -0 .6133 <0.050 -0.0070 n.s. -0.3982 n.s. a n . s . denotes not s i g n i f i c a n t . c<0.050 means >0.010 <0.050. eQne o f t h e s t r i c t v e g e t a r i a n s ate meat r a r e l y bN/A denotes not a p p l i c a b l e . d<0.010 means >0.001 <0.010. (see Appendix F ) . 74 This w i l l be discussed i n the following section of t h i s chapter. 3. Four Day Food Records a. Comparison of Nutrient Intake with the Dietary Standard for Canada Table 18 l i s t s the mean nutrient intake per day for each subject, including the supplements taken, obtained from the four day food record. When compared with the Dietary Standard for Canada (Health and Welfare Canada, 1975) (Appendix E), i t was found that a l l ovo-lacto vegetarians met at least two-thirds of the standard for a l l nutrients. With respect to c a l o r i e s , two female s t r i c t vegetarians (18.2%) and one non-vegetarian (8.3%) proved to have les s than two-thirds of the stan-dard. However, since physical a c t i v i t y patterns were not determined for the subjects and since none reported current weight l o s s , i t was f e l t that the di e t s were c a l o r i c a l l y adequate. Four s t r i c t vegetarians f a i l e d to meet at least two-thirds of the standard for calcium, p o s s i b l y r e f l e c t i n g the lack of dairy products or other high calcium foods i n t h e i r d i e t . Only one s t r i c t vegetarian met the standard for t h i s n u t r i e n t . Only one non-vegetarian showed i r o n intake below two-thirds of the standard. In the same subject, thiamine and c a l o r i e s also f a i l e d to meet two-thirds of the standard for these n u t r i e n t s . Niacin intake was lower than two-thirds of the standard i n the diet of two s t r i c t vegetarians whose calcium intake also f a i l e d to meet two-thirds of the standard. Table 18 Mean Nutrient Intake Per Day f o r Each Subject Derived from Four Day Food Records C a l o r i e s P r o t e i n Calcium Iron Subject ( k c a l ) (g) (mg) (mg) Vitamin A Thiamin R i b o f l a v i n N i a c i n Vitamin (I.U.xlO) (mg) (mg) (mg) (mg) C Fat (g) Carbohydrate (g) Crude F i b r e (g) Southgate Animal Vegetable Animal F i b r e Fat Fat P r o t e i n (g) (g) (g) (g) Vegetable P r o t e i n (g) 01 F 2544.1 69.5 1444.1 39.2 1144.7 52. .1 52.9 70. .9 1416. .4 59. .3 426. ,9 15. 3 115, .9 24.6 34, .7 20. .0 49.4 02 M 2456.5 60.6 824.5 16.6 1316.2 63. .8 63.8 75. .5 630, .5 116. .7 302. ,4 13. ,3 59. .3 70.3 46. .4 21. .6 39.0 03 F 1837.7 49.6 667.5 12.0 1146.8 51.0 51.3 51. .3 634. .7 96. .9 196. ,9 7. 0 28. .8 37.7 59, .2 22. .1 27.5 04 F 2535.0 85.0 1743.6 22.3 3548.5 54. .5 57.6 90. .7 312. .4 110. .0 308. ,5 13. 9 76. .2 73.2 36. .8 54. .0 31.0 05 F 2557.5 76.2 1305.5 13.5 643.4 38. ,7 39.8 49. .2 551. .4 100. .9 329. ,0 6. 3 27. .4 69.5 31. .4 46. .4 29.9 06 M 2988.0 81.9 1598.5 14.6 2612.7 1. ,4 - 2.8 13. .4 249. .2 130. .1 375. ,8 7. 6 28. .7 81.3 48. .8 48, .7 33.2 07 F 1310.1 39.1 508.9 12.6 1057.4 0. .9 1.4 13. .6 213. .3 26.0 239. ,3 15. ,7 33. .3 3.5 22 .5 4, .0 35.2 08 F 1584.3 33.8 352.1 13.2 347.1 1. .1 0.9 9. .0 227.0 50. .5 271. ,8 11. 5 47. .2 1.3' 49, .3 0, .3 33.5 09 F 2011.2 50.4 682.3 14.8 914.5 1. .9 4.3 10 .7 364, .5 87, .0 276. ,0 , 9. .8 72, .5 10.1 77. .0 8, .4 42.1 10 M 2864.1 67.3 812.5 22.4 2189.3 2. .6 3.2 17. .4 405. .6 92, .8 462. .2 .17. 0 174. .6 5.8 87. .0 4, .5 62.8 11 F 2155.1 74.1 502.9 19.2 1133.8 1, ,7 1.9 15, .3 112 .2 77, .8 312. ,8 I l 2 . ,1 46. .1 o.o b 77, .8 1, .0 73.1 12 M 2894.4 103.5 653.8 24.6 1191.9 2, .4 1.5 25. .5 103, .9 105, .9 427. .9 '16. 8 58, .9 4.0 101, .9 1, .0 102.5 13 F 1265.6 36.6 320.9 12.8 913.7 1, .4 2.6 7, .7 61, .7 34, .8 224. .6 10. ,6 98, .2 0.0 34, .8 0, .5 36.1 14 F 3356.4 85.9 719.6 .24.3 4186.7 2. .5 2.1 25, .0 291, .5 116, .3 505. .2 22. ,2 93, .8 6.8 109 .6 4, .5 81.5 15 M 2525.4 77.2 549.1 21.3 3660.3 2, .0 1.5 25, .1 248 .3 74 .4 404. .6 20. ,9 63, .1 0.0 77 .1 0 .0 77.8 16 M 2262.8 59.7 431.2 17.0 990.6 1, .8 1.03 21, .3 202 .6 73 .2 363. .8 16.0 51, .9 0.0 74 .5 0.0 59.7 17 F 2284.7 66.2 489.2 18.1 1498.8 1 .6 1.2 24 .7 207, .7 85, .2 342 .7 18. ,8 69 .5 2.8 82 .4 1 .4 64.8 18 F 1929.2 89.2 799.0 12.6 810.6 1, .4 1.9 20 .9 595 .5 103, .1 155, .3 4. ,5 15, .8 80.0 23 .1 70 .6 18.6 19 F 2407.2 89.7 793.0 12.7 586.4 1. .3 1.7 19, .4 200 .3 106 .8 273, .4 3. .3 12, .7 73.3 33 .5 63 .0 26.7 20 M 2361.3 91.0 871.8 16.0 1396.4 1. .0 1.8 24 .5 234 .9 85, .5 264, .3 2. .7 13, .1 65.7 19 .8 63, .0 28.3 21 M 3896.6 160.6 1435.0 19.4 2292.7 1 .6 3.0 39 .6 147 .3 237 .1 285, .1 9. ,7 31 .6 167.3 77 .1 123 .2 37.4 22 M 2415.8 79.7 799.9 15.1 2137.4 2, .2 2.2 26.0 142 .4 97, .9 307, .9 8. .6 29 .4 61.1 36 .5 47, .9 31.8 23 M 2208.3 80.6 1275.2 10.7 529.9 1 .1 1.9 14 .9 626 .2 105 .9 244, .7 5. .2 30 .1 60.1 45 .8 58 .9 21.8 24 M 2333.9 90.7 1105.0 12.1 728.9 1. .2 2.3 20 .9 163 .5 105, .4 230 .4 4. .1 16, .5 97.5 7 .9 73 .0 17.7 25 F 2212.8 98.8 537.8 14.5 912.9 1 .5 2.1 27 .2 104 .4 110, .1 150, .2 4. .9 17 .8 75.0 35 .1 74 .8 24.0 26 F 1937.9 77.6 1103.8 11.8 924.9 1, .1 4.9 15 .4 108 .4 76 .6 245, .7 5. .6 18 .4 60.8 15 .8 64 .8 12.8 27 F 2300.6 88.9 831.0 11.7 720.9 1 .4 1.6 30 .5 169 .8 107 .2 244 .5 7. .1 27 .3 52.1 55 .1 45 .8 43.8 28 F 1790.1 95.9 1580.2 11.5 2210.0 1, .4 2.7 15 .6 139 .8 75 .1 184 .3 5. .5 18 .6 43.7 31 .4 81 .0 15.0 29 F 1180.6 61.2 629.1 8.4 450.4 0. .7 1.1 13 .2 124 .8 60 .2 114, .2 3. .4 18 .2 30.0 30 .2 46 .7 14.5 Subjects 1-6 were ovo-lacto v e g e t a r i a n s , 7-17 s t r i c t v e g e t a r i a n s , and 18-29 non-vegetarians. 0.0 denotes not determined. In a l l , 23 subjects met at le a s t the two-thirds of the standard for a l l nutrients. The die t of three subjects was lower than two-thirds of the stan-dard for one n u t r i e n t , while one subject f a i l e d to meet the two-thirds of the standard for two nutrients and two subjects for three n u t r i e n t s . Vitamin A intake was adequate and even high i n many p a r t i c i p a n t s ' d i e t . The intake of thiamin, n i a c i n and vitamin C was also considerably high i n 5 out of 6 ovo-lacto vegetarians who also took multivitamins. Other nutrients such as crude f i b e r , Southgate f i b e r , animal p r o t e i n , vegetable p r o t e i n , animal f a t and vegetable f a t , were also included i n the analysis. However, no standard has been set for these nutrients at the present time. Although protein intake met the standard for most subjects, three s t r i c t vegetarians had lower amounts of protein than recommended. Even though they met two-thirds of the standard for t h i s n u t r i e n t , t h e i r c a l o r i c intake was also low and t h i s may have an e f f e c t on protein u t i l i z a t i o n i f t h e i r energy needs were not met. b. Comparison of Nutrient Intake Among Diet .Groups The Mann-Whitney U test was applied to compare the nutrient intake among the groups, and r e s u l t s are shown i n Table 19. When compared to the s t r i c t vegetarians, the ovo-lacto vegetarians had a s i g n i f i c a n t l y higher intake of thiamin, r i b o f l a v i n , n i a c i n and vitamins. This probably r e f l e c t s the intake of vitamin supplements by the ovo-lacto vegetarians. Five out of 6 ovo-lacto vegetarians used these supplements. Calcium intake was also higher i n ovo-lacto vegetarians than i n s t r i c t vege-Table 19 Comparison of Nutrient Intake Among Diet Groups by the Mann-Whitney U Test (Two-Tailed) Ovo-lacto Veg. Ovo-lacto Veg. S t r i c t Veg. A l l Veg. (n=6) (n=6) (n=ll) (n=17) vs vs vs vs S t r i c t Veg. Non-Veg. Non-Veg. Non-Veg. (n=ll) (n=12) (n=12) (n=12) Nutrients Mean Rank Sign. Mean Rank Sign. Mean Rank Sign. Mean Rank Sign. Calories 10.83 a n. s. 13.00 <0.050d 12.09 n. s. 16.29 n.s. 8.00 7.75 11.92 13.17 Protein 10.33 n.s. 5.17 <0.050 7.73 <o.oioc 10.71 O.010 8.27 11.67 15.92 21.08 Animal 14.50 <0.001b 4.67 <0.010 6.00 <0.001 9.41 <0.001 protein 6.00 11.92 17.50 22.92 Vegetable 4.67 O.010 13.33 <0.050 17.36 O.001 19.82 <0.001 protein 11.36 7.58 7.08 8.17 Carbohydrate 8.17 n.s. 14.00 <0.050 16.18 <0.010 19.29 <0.010 9.45 7.25 8.17 8.92 Fiber 5.50 <0.050 14.17 <0.010 18.00 <0.001 20.53 <0.001 10.91 7.17 6.50 7.17 Table 19 (cont'd) Nutrients Mean Rank Sign. Mean Rank Sign. Mean Rank Sign. Mean Rank Sign. Southgate 7.33 n.s. 14.00 <0.050 18.00 <0.001 20.47 <0.001. f i b e r 9.91 7.25 6.50 7.25 Fat 12.33 <0.050 10.33 n. s. 9.09 <0.050 13.41 n. s. 7.18 9.08 14.67 17.25 Animal fat 14.50 <0.001 8.67 n.s. 6.00 O.001 10.82 <0.010 6.00 9.92 17.50 20.92 Vegetable fat 5.33 <0.050 12.25 n.s. 16.32 <0.010 18.76 <0.010 11.00 8.13. 8.04 9.67 . Thiamin 13.17 <0.050 15.00 <0.010 15.23 <0.050 19.03 <0.010 6.73 6.75 9.04 9.29 Ribo f l a v i n 14.17 <0.010 15.17 <0.010 10.14 n. s. 15.79 n.s. 6.18 6.67 13.71. 13.88 Niacin 13.17 <0.050 13.67 <0.050 10.18 n.s. 15.29 n. s. 6.73 7.42 13.67 14.58 Vitamin C 13.67 <0.010 14.50 <0.010 13.09 n. s. 17.47 n.s. 6.45 7.00 11.00 11.50 CO Table 19 (cont'd) Nutrients Mean Rank Sign. Mean Rank Sign. Mean Rank Sign.: Mean\Rank Sign. Vitamin A 9.83 n.s. 12.67 n.s. 14.91 <0.050 18.00 <0.050 8.55 7.92 9.33 10.75 Calcium 14.00 <0.010 12.17 n.s. 7.09 <0.001 12.76 n.s. 6.27 8.17 16.50 18.17 Iron 8.33 n.s. 13.00 <0.050 16.00 <0.010 18.82 <0.010 9.36 7.75 8.33 9.58 n.s. denotes not s i g n i f i c a n t . b<0.001 means ^0.000 <0.001. C<0.010 means >0.001 <0.010. d<0.050 means >0.010 <0.050. 80 ta r i a n s . This was expected since intake of dairy products, r i c h i n calcium, d i f f e r e n t i a t e d . t h e ovo-lacto vegetarians from the s t r i c t vegetarians. Total fat intake as well as animal protein and animal f a t intakes were also s i g -n i f i c a n t l y increased i n ovo-lacto vegetarians compared to the s t r i c t vege-tarians. Since the l a t t e r generally avoid products of animal o r i g i n , the higher animal protein and animal f a t intakes of ovo-lacto vegetarians were expected. The d i e t s of the ovo-lacto vegetarians were found to be lower i n vege-table protein and vegetable f a t compared to those of the s t r i c t vegetarians. Crude f i b e r intake was also lower for the ovo-lacto vegetarians than for the s t r i c t vegetarians. However, no s i g n i f i c a n t difference was observed for intake of Southgate f i b e r between these two groups. The diets of the ovo-lacto vegetarians were also compared to those of the non-vegetarians. They were found to be r i c h e r i n thiamin, r i b o f l a v i n , n i a c i n and vitamin C, again r e f l e c t i n g the intake of supplements by the ovo-lacto vegetarians. Their d i e t s were also higher i n c a l o r i e s , vegetable p r o t e i n , i r o n , carbohydrate, crude and Southgate f i b e r , but s i g n i f i c a n t l y lower i n t o t a l p r otein and animal p r o t e i n . The intakes of the s t r i c t vegetarians d i f f e r e d from those of the non-vegetarians by containing more vegetable p r o t e i n , vegetable f a t , carbohy-drate, crude and Southgate f i b e r , thiamin, vitamin A and i r o n . The s t r i c t vegetarians had s i g n i f i c a n t l y lower intakes of t o t a l p r otein and animal p r o t e i n , t o t a l f a t , animal f a t and calcium than the non-vegetarians. c. C o r r e l a t i o n of Nutrient Intake with Mutagenicity When the nutrient intake was c o r r e l a t e d with TA100 by the Spearman 81 r e l a t i o n t e s t , the following r e s u l t s emerged (Table 20): 1. There was a negative s i g n i f i c a n t c o r r e l a t i o n (P<0.05) with carbo-hydrate when a l l subjects were analyzed. Since carbohydrate intake may influence f i b e r intake, i t was of in t e r e s t to see i f there would be a c o r r e l a t i o n with e i t h e r crude f i b e r or Southgate f i b e r alone. A negative c o r r e l a t i o n (PO.01) was established between Southgate f i b e r intake and mutagenicity with TA100 for a l l subjects. 2. Calcium intake was also negatively correlated with TA100 mutageni-c i t y for the non-vegetarians (P<0.05). 3. No s i g n i f i c a n t c o r r e l a t i o n was found with TA100 mutagenicity and intake of c a l o r i e s , p rotein and f a t (regardless of o r i g i n ) , thiamin, r i b o f l a v i n , n i a c i n , vitamin A, vitamin C, ir o n and crude f i b e r . When nutrient intake was corr e l a t e d with mutagenicity using TA98 as a ster s t r a i n , the following r e s u l t s were observed (Table 21): 1. A s i g n i f i c a n t negative c o r r e l a t i o n was established between mutageni-c i t y with TA98 and intake of Southgate f i b e r f o r a l l subjects (P<0.01). 2. Iron intake was also negatively correlated with TA98 mutagenicity when a l l subjects were analyzed (PO.05). 3. For the ovo-lacto vegetarians, a negative c o r r e l a t i o n was seen between intake of crude f i b e r (PO.05) as well as intake of ir o n (PO.01) with TA98 mutagenicity. 4. A negative c o r r e l a t i o n was observed between mutagenicity with TA98 and intake of t o t a l p r o t e i n (PO.05) and animal protein (PO.05) among non-vegetarians. It should be pointed out that the non-vege-tarians have the highest intake of these two nutrients of a l l the d i e t groups, although there i s a wide v a r i a t i o n among the subjects. Table 20 Spearman Rank Correlation Between Nutrient Intake by Diet Groups and Mutagenicity with TA100 (Two-Tailed) A l l ! Subjects Ovo-lacto Veg. S t r i c t Veg. Non-Veg. A l l Veg. Corr. Corr. Corr. Corr. Corr. Nutrients Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Calories -0.2341 a n.s. -0.4286 n.s. 0.0866 n.s. 0.1226 n.s. -0.1255 n.s. Protein 0.1575 n.s. -0.0286 n.s. 0.1321 n.s. -0.3187 n.s. -0.0369 n.s. Animal protein 0.2575 n.s. 0.3143 n. s. -0.2523 n.s. -0.1860 n.s. -0.2236 n.s. Veg. protein -0;3605 n.s. -0.3714 n.s. 0.0137 n.s. -0.0315 n.s. 0.0454 n.s. Carbohydrate -0.4170 <0.05b -0.3714 n.s. 0.1185 n.s. -0.1191 n.s. -0.0369 n.s. Crude f i b e r -0.3369 n. s. 0.1429 n.s. 0.2096 n.s. -0.0455 n.s. 0.1255 n.s. Southgate f i b e r -0.4633 <o.oic 0.2571 n.s. -0.5194 n.s. -0.1261 n.s. -0.1402 n.s. Fat 0.0382 n.s. -0.0857 n.s. -0.1777 n.s. 0.0245 n.s. -0.1107 n.s. Animal f a t 0.2091 n.s. 0.0857 n.s. -0.1492 n.s. 0.3503 n.s. -0.2172 n.s. Vegetable f a t -0.1611 n. s. 0.7143 n.s. -0.0683 n.s. -0.0876 n. s. 0.2636 n.s. Thiamin -0.2876 n. s. -0.0286 n. s. -0.0137 n.s. 0.2386 n. s. -0.0221 n.s. R i b o f l a v i n -0.2716 n. s. -0.0286 n.s. -0.3280 n.s. -0.1191 n.s. -0.2140 n.s. Niac in 0.0007 n.s. 0.1429 n.s. 0.1458 n.s. 0.0841 n.s. -0.0221 n.s. Vitamin C -0.1572 n.s. -0.0286 n.s. 0.3144 n.s. -0.2312 n.s. 0.0221 n.s. Vitamin A -0.1203 n.s. 0.5429 n.s. 0.2005 n.s. 0.1891 n.s. 0.1255 n.s. Calc ium -0.1533 n.s. 0.0857 n.s. 0.1731 n.s. -0.5954 <0.05 -0.0959 n.s. Iron -0.3245 n. s. -0.1429 n.s. 0.1048 n.s. 0.1296 n.s. -0.0074 n.s. n.s. denotes not s i g n i f i c a n t , P>0.05 <0.05 means >0.01 <0.05. °<0.01 means >0.001 <0.01. Table 21 Spearman Rank Correlation Between Nutrient Intake by Diet Groups and Mutagenicity with TA98 (Two-Tailed) A l l : Subjects Ovo-lacto Veg. S t r i c t Veg. Non-Veg. A l l Veg. Corr. Corr. Corr. Corr. Corr. Nutrients Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Coeff. Sign. Calories -0.1621 a n. s. -0.0857 n.s. 0.2050 n.s. -0.0947 n.s. 0.0149 n.s. Protein 0.0883 n. s. -0.3143 n.s. 0.3508 n. s. -0.6386 <0.05b 0.0896 n.s. Animal protein 0.1058 n.s. 0.2571 n. s. -0.3876 n.s. -0.6397 <0.05 -0.0573 n.s. Veg. protein -0.2775 n. s. -0.7143 n.s. 0.4282 n. s. -0.0421 n.s. 0.0012. n. s. Carbohydrate -0.2536 n. s. -0.4857 n.s. 0.1185 n.s. -0.0105 n.s. 0.0149 n.s. Crude f i b e r -0.3476 n.s. -0.8286 O.05 0.3918 n. s. -0.3263 n.s. -0.0149 n.s. Southgate f i b e r -0.4729 <0.01° -0.7714 n.s. -0.0364 n.s. -0.3614 n. s. -0.2687 n.s. Fat 0.0849 n. s. 0.2571 n. s. -0.0456 n. s. -0.1930 n.s. 0.1493 n. s. Animal fat 0.1459 n.s. 0.2000 n.s. -0.3964 n. s. -0.1228 n.s. -0.0575 n.s. Vegetable f a t -0.1701 n.s. 0.6571 n.s. 0.1276 n. s. -0.1298 n.s. 0.1364 n. s. Thiamin -0.2108 n. s. -0.6000 n.s. -0.0137 n. s. -0.1617 n.s. -0.0149 n.s. R i b o f l a v i n -0.2691 n.s. -0.6000 n. s. -0.2688 n.s. -0.5719 n.s. -0.1194 n.s. Niacin 0.0160 n.s. -0.6571 n.s. 0.5467 n.s. -0.3404 n.s. 0.1194 n.s. Vitamin C -0.1011 n. s. -0.2571 n.s. -0.5695 n.s. 0.3895 n.s. -0.1642 n. s. Vitamin A -0.1520 n.s. -0.0286 n.s. 0.3007 n.s. -0.3088 n.s. ' 0.0746 n.s. Calc ium -0.1021 n.s. -0.4857 n.s. -0.2187 n. s. -0.5053 n.s. -0.0746 n. s. Iron -0.4135 <0.05 -0.9429 O.01 0.2050 n.s. -0.2632 n.s. -0.1791 n. s. ,n.s. denotes not s i g n i f i c a n t , P>0.05. O.05 means >0.01 <0.05. °<0.01 means >0.001 <0.01. Leaf 84 missed i n numbering 85 Table 22 Pearson C o r r e l a t i o n Between Frequency of Intake from Questionnaire and from Four Day Food Record (One-Tailed) Food Items Co r r e l a t i o n C o e f f i c i e n t Significance Dairy products IA: Cheese 0.6429 <0.001 IB: Cream 0.6013 <0.001 1C: Ice cream 0.4418 <0.010 ID: Milk 0.6342 <0.001 1: A l l 0.7050 <0.001 Meat and alternates 2A: Beef 0.4841 <0.010 2B: Lamb 0.2819 n.s. a 2C: Pork 0.6838 <0.001 2D: Var i e t y meats 0.1544 n. s. 2E: Game N/Ab N/A 2F: Poultry 0.3892 <0.050 2G: F i s h and seafoods 0.3845 <0.050 2H: Eggs 0.7098 <0.001 21: Nuts and legumes 0.7498 <0.001 2A-E: Meats 0.7413 <0.001 2A-F: Meats and poultry 0.7813 <0.001 2A-H: Meats, poultry, eggs 0.8196 <0.001 2: A l l 0.3484 <0.050 3: Vegetables 0.3667 <0.050 F r u i t s and juices 4A: F r u i t s 0.5967 <0.001 4B: Juices 0.4479 <0.010 4A-B: A l l 0.6409 <0.001 3-4: F r u i t s and vegetables 0.4841 <0.010 Breads and alternates 5A: Breads, yeast, others 0.2442 n.s. 5B: Cereal, ready-to-eat 0.3976 <0.050 5C: Hot cereal 0.2318 n. s. 5D: Other cereal 0.4077 <0.050 5E: Flours 0.7603 <0.001 5F: Baked goods 0.5134 <0.010 5A-E: 0.4469 <0.010 5: A l l 0.5302 <0.010 6: Sugar and sweets 0.4639 <0.010 Fats 7A: Animal fat 0.7322 <0.001 7B: Vegetable fat 0.0634 <n. s. 7C: Combination 0.5067 <0.003 7: A l l 0.2614 n.s. 8: Miscellaneous 0.7843 <0.001 n.s. denotes not s i g n i f i c a n t . ^N/A denotes not applicable. 86 In summary, the food frequency questionnaire i s shown to be i n harmony with the four day food records and w i l l be used i n conjunction with the nutrient intake i n the discussion. 87 CHAPTER V DISCUSSION A. FECAL MUTAGENICITY Previous studies have demonstrated the presence of r e l a t i v e l y large amounts of mutagens i n feces (Bruce et a l . , 1977; Ehrich et a l . , 1979). It has been proposed that these mutagens are d i r e c t l y involved i n the etiology of colon cancer. In order to test t h i s hypothesis, the f e c a l mutagenicity was measured i n two population groups known to d i f f e r i n t h e i r s u s c e p t i b i l i -ty to colon cancer: vegetarians and subjects on a t y p i c a l meat-containing North American d i e t . S t a t i s t i c a l evaluation of the mutagenicity l e v e l s indicated that vegetarianism was c o r r e l a t e d with low l e v e l s of f e c a l muta-gens . Similar trends were reported from other laboratories while t h i s work was i n progress. Ehrich et a l . (1979) reported that 20% of 42 f e c a l samples from a white South A f r i c a n population (at high r i s k for colon cancer) were p o s i t i v e for mutagenic a c t i v i t y with TA100 compared to 2% from 82 urban blacks and none from 108 r u r a l blacks (both groups considered at low r i s k for colon cancer). Mutagenic a c t i v i t y with TA98 was also lower for the low r i s k groups, being 10%, 5% and 2% i n whites, urban blacks and r u r a l blacks, r e s p e c t i v e l y . In a d d i t i o n , Reddy et a l . (1980) have demonstrated that 22% of volunteers from a high r i s k New York population ingesting a high f a t , high meat, low f i b e r diet excreted f e c a l mutagens s e n s i t i v e to TA100 and/or TA98. In comparison, only 13% from a low r i s k Kuopio population consuming a di e t high i n f a t , low i n meat and high i n f i b e r exhibited f e c a l mutageni-c i t y l i m i t e d to TA98. None of the New York Seventh Day Adventists who were vegetarians showed any f e c a l mutagenic a c t i v i t y . 88 There were s i g n i f i c a n t f l u c t u a t i o n s i n mutagenicity within the three dietary groups. The vegetarian group contained the subject with the highest and the subject with the lowest l e v e l of f e c a l mutagens. Kuhnlein and Kuhnlein (1980) have also reported a s i g n i f i c a n t i n d i v i d u a l v a r i a b i l i t y among subjects for f e c a l mutagenicity even a f t e r 72 days of rigorously c o n t r o l l e d d i e t , and suggested that long-term habits or perhaps genetic t r a i t s could influence f e c a l mutagen excretion. It i s possible that, i n t h i s study, other l i f e s t y l e factors which were not evaluated or genetics may have contributed to f e c a l mutagenic a c t i v i t y i n the subjects. The test used i n t h i s study was the f l u c t u a t i o n test of Green and Muriel (1976). This t e s t i s more s e n s i t i v e than the conventional. Ames Sal-monella test and allowed to detect f e c a l mutagens at concentrations as low as 2 y l of extract per ml of culture suspension. This concentration i s equivalent to 1 mg of feces per ml of medium. Of the 29 f e c a l extracts s t u -died, 24 were s i g n i f i c a n t l y p o s i t i v e at t h i s concentration when tested with Salmonella typhimurium TA100 and 12 were s i g n i f i c a n t l y p o s i t i v e when tested with Salmonella typhirnurium TA98. The more widely used method for determining f e c a l mutagens i s the method of Bruce et a l . (1977). In t h i s method, ether extracts are prepared from freeze-dried f e c a l samples and analyzed with the standard Ames Salmonella t e s t . At extract concentrations equivalent to 60 mg wet weight per ml, only about 20% of the samples tested were p o s i t i v e . It i s possible that the lower percentage of p o s i t i v e samples detected with the Salmonella/Ames test i s due to the presence of mutagen i n h i b i t o r s i n f e c a l extracts. Bruce et a l . (1977) have documented the presence of such an i n h i b i t o r i n the ether extract, but allegedly separated the i n h i b i t o r from the main mutagen by a d d i t i o n a l ex-t r a c t i o n procedures. Inhibitory e f f e c t s on the a c t i v i t i e s of several muta-89 gens i n the Ames test caused by ether extracts of human feces have also been documented by Hayatsu et a l . (1981). In the f l u c t u a t i o n t e s t , the concen-t r a t i o n s of feces in aqueous extracts tested are roughly 100-fold lower than those used by Bruce et a l . (1977). It i s therefore expected that toxic or i n h i b i t o r y substances are less l i k e l y to i n t e r f e r e with the assay. With TA100, however, a decrease i n the number of revertant tubes at high concentrations of extract was noted. This was not a general toxic e f f e c t since there was no corresponding decrease of the b a c t e r i a l concentration i n tubes without revertants. High concentrations of extracts with TA100 may possibly carry an i n h i b i t o r y e f f e c t on metabolic pathways involved i n muta-genesis. Since a decrease i n the number of revertant tubes at high concen-t r a t i o n s was infrequently seen with TA98, k i l l i n g of the fast growing rever-tant c e l l s was not l i k e l y . The frequent occurrence of filamentous b a c t e r i a in/tubes containing high l e v e l s of extracts suggests that f e c a l extracts influence b a c t e r i a l metabolism. Several laboratories are now working to i d e n t i f y the mutagenic com-pound(s) i n f e c a l extracts. Bruce and Dion (1980) have reported that one f e c a l mutagen, active on TA100 without S9, had the following c h a r a c t e r i s t i c s : " acid, heat and UV l a b i l e , associated with a UV absorption t r i p l e t centered at 340 nm". It i s currently believed that there are probably many mutagenic and p o t e n t i a l l y carcinogenic compounds i n feces. The presence of more than one major f e c a l mutagen i s also indicated by c o r r e l a t i o n studies between mutagenicity l e v e l s with TA100 ( s e n s i t i v e to point mutagens) and TA98 ( s e n s i t i v e to frameshift mutagens) and between the pH of f e c a l samples and the mutagenicity. No c o r r e l a t i o n between f e c a l muta-ge n i c i t y with TA100 and TA98 was established among the vegetarians, i n d i c a -t i n g the presence of at l e a s t two separate compounds. Among the non-vege-90 t a r i a n samples, there was a c o r r e l a t i o n between mutagenicity with TA98 and TA100; however, only mutagenicity with TA98 was correlated with the pH, again suggesting the involvement of at least two mutagens. It has been proposed that high colonic pH may promote c o l o r e c t a l cancer (Thornton, 1981) and that a low pH (below n e u t r a l i t y ) could protect against cocarcinogen formation by providing a less tumorigenic environment (Macdonald et a l . , 1978; Malhotra, 1977). Furthermore, populations defined epidemio-l o g i c a l l y to be at low r i s k for colon cancer have also been found to have lower f e c a l pH than high r i s k populations (Walker et a l . , 1979; Ehrich et a l . , 1979) and bowel cancer patients (Macdonald, Webb and'Mahony, 1978). In t h i s study, however, no s i g n i f i c a n t differences i n f e c a l pH were found between non-vegetarians who had high l e v e l s of f e c a l mutagens and vege-tarians at low l e v e l s of mutagenicity. I n t e r e s t i n g l y , low f e c a l pH was associated with high mutagenicity with tester s t r a i n TA98 for the non-vege-tarians and when a l l subjects were combined together. Kuhnlein and Kuhnlein (1980) have also reported that low pH values of the f e c a l homogenates were associated with high mutagenicity using TA100 for subjects on c o n t r o l l e d formula d i e t s . Other factors such as f e c a l wet weight and percent dry weight did not d i f f e r s i g n i f i c a n t l y between the vegetarians and non-vegetarians, nor were they associated with mutagenicity. These findings are in agreement with the r e s u l t s of Ehrich et a l . (1979). In summary, vegetarians were shown to have s i g n i f i c a n t l y lower l e v e l s of f e c a l mutagens than non-vegetarians, and the presence of more than one mutagen i n the feces was indicated. 91 B. RELATIONSHIP BETWEEN FECAL MUTAGENICITY AND AGE, SEX AND MISCELLANEOUS DIETARY AND HEALTH DATA Vegetarians may have l i f e s t y l e habits or c h a r a c t e r i s t i c s , other than dietary intake, which d i f f e r e n t i a t e them from non-vegetarians. The Spearman rank c o r r e l a t i o n t e s t was therefore conducted between f e c a l mutagenicity with TA98 and TA100 and some questionnaire v a r i a b l e s (age, sex, etc.) to elucidate factors which may account for the difference i n f e c a l mutagens among the groups. Correlations between these factors and f e c a l mutagenicity were considered most meaningful when s i g n i f i c a n c e was PO.05 for a l l subjects or f o r more than one group. The factors associated with f e c a l mutagenicity with TA98 and/or TA100 were age, frequency of consumption of hot spices, and the length of time that a subject had been a s t r i c t vegetarian. Age was p o s i t i v e l y c o r r e l a t e d with mutagenicity on TA100 for the non-vegetarians only. This p o s i t i v e c o r r e l a t i o n probably resulted from the greater age d i s t r i b u t i o n i n the non-vegetarians compared to the other groups (see Table 10). The length of time for which a subject had been a s t r i c t vegetarian seemed to influence mutagenicity with TA100. However, age was not associated with mutagenicity f o r the s t r i c t vegetarians, as i t was for the non-vegetarians. There was a p o s i t i v e c o r r e l a t i o n between the frequency of consumption of hot spices ( c h i l l i e s , curry, etc.) with mutagenicity using TA98 i n the s t r i c t vegetarians. However, the number of s t r i c t vegetarians consuming hot spices was l i m i t e d to 4 out of 11 and were non-Seventh Day Adventists. Seventh Day Adventists tend to avoid hot condiments and spices ( P h i l l i p s et a l . , 1980). This c o r r e l a t i o n was not seen in the other groups and needs further i n v e s t i g a t i o n . 92 Although the average number of bowel movements per day was s i g n i f i c a n t l y higher i n vegetarians than i n non-vegetarians, no s i g n i f i c a n t c o r r e l a t i o n was observed between frequency of bowel movement and f e c a l mutagenicity with TA100 or TA98. The p o s i t i v e c o r r e l a t i o n found between the former use of laxatives and mutagenicity with TA98 i n s t r i c t vegetarians was possibly a spurious corre-l a t i o n , since very few s t r i c t vegetarians (n=3) took laxatives i n the past. Furthermore, bowel i r r e g u l a r i t y was not associated with mutagenicity with TA98. If f e c a l excretion of mutagens leads to colon cancer, i t i s reasonable to expect constipation to r e s u l t i n more contact of mutagens/carcinogens with i n t e s t i n a l c e l l s and therefore greater r i s k . However, Wynder and Shigematsu (1967) have shown that constipation was not correlated with the incidence of colon cancer. Only one subject reported a h i s t o r y of colon cancer i n the distant family, and no c o r r e l a t i o n was observed between his t o r y of cancer i n the family and f e c a l mutagenicity. Smoking, use of b i r t h control p i l l s , use of vitamins and supplements, gender, and f a s t i n g were not linked to mutagenicity with e i t h e r TA98 or TA100. In summary, age was p o s i t i v e l y correlated with TA100 for the non-vege-tarians only; the frequency of consumption of hot spices i n the s t r i c t vege-tarians was p o s i t i v e l y correlated with f e c a l mutagens s e n s i t i v e to TA98; and the length of time that a subject was a s t r i c t vegetarian was p o s i t i v e l y correlated with mutagenicity on TA100. None of the questionnaire parameters tested correlated s i g n i f i c a n t l y with f e c a l mutagenicity for a l l subjects. 93 C. DIETARY DATA AND FECAL MUTAGENICITY Dietary intake was assessed by computing intake of food groups using a food frequency questionnaire and by computing intake of nutrients from a four day food record. The four day food record was taken immediately p r i o r to the f e c a l c o l l e c t i o n . Spearman rank c o r r e l a t i o n s were c a r r i e d out between mutagenicity on either TA98 or TA100 and food groups or nutrient intakes. The c o r r e l a t i o n s were done on f i v e groups: s t r i c t vegetarians, ovo-lacto vegetarians, non-vegetarians, a l l vegetarians together, and a l l subjects together. Correla-tions were considered most meaningful when they re l a t e d to food items or nutrients consumed by a l l subjects and when there was a s i g n i f i c a n t r e s u l t with more than one group. Using these c r i t e r i a , i t was found that the most s i g n i f i c a n t dietary parameters were f r u i t s and j u i c e s i n the food group category and t o t a l carbo-hydrate, f i b e r and i r o n i n the nutrient category. Each of these w i l l be discussed i n turn. The lack of f r u i t s and vegetables i n the diet has been suggested as one of the factors leading to colon cancer (Graham et a l . , 1978). In t h i s study, the f r u i t intake as well as the combined consumption of f r u i t s and j u i c e s have been negatively c o r r e l a t e d to mutagenicity with TA98 for a l l subjects, for the s t r i c t vegetarians, and when a l l vegetarians are analyzed together. When f r u i t and vegetable intakes were combined, a negative c o r r e l a t i o n with mutagenicity using TA98 was also observed for a l l vegetarians. Although vitamin C i s a foremost component of f r u i t s and i t s intake i s thought to suppress f e c a l mutagenicity (Bruce et a l . , 1977), no c o r r e l a t i o n was observed between t h i s parameter and mutagenicity with e i t h e r TA98 or 94 TA100. Carbohydrate intake was negatively correlated with mutagenicity using TA100 for a l l subjects. This was expected since ovo-lacto vegetarians and s t r i c t vegetarians with low l e v e l s of f e c a l mutagens s e n s i t i v e to TA100 have a s i g n i f i c a n t l y higher intake of carbohydrate than the non-vegetarians with higher l e v e l s of mutagens. There was a negative c o r r e l a t i o n between mutagenicity with TA100 and the intake of ready-to-eat cereals for the s t r i c t vegetarians. There was also a negative c o r r e l a t i o n established f o r the mutagenicity with TA98 and consumption of ready-to-eat c e r e a l and a l l breads and substitutes f o r ovo-lacto vegetarians. Since these foods contain f i b e r , the s i g n i f i c a n t corre-l a t i o n s r e i n f o r c e the theory that f i b e r may i n h i b i t f e c a l mutagenicity. This statement i s substantiated i n the case of the ovo-lacto vegetarians, since crude f i b e r intake was negatively c o r r e l a t e d with mutagenicity with TA98. A negative c o r r e l a t i o n between intake of Southgate f i b e r and mutageni-c i t y with TA100 and TA98 was also observed when a l l subjects were analyzed. These r e s u l t s corroborate those of Bruce et a l . (1979), who reported a decrease i n f e c a l mutagenicity with supplementary f i b e r intake. The negative c o r r e l a t i o n observed between i r o n intake and mutagenicity with TA98 for the ovo-lacto vegetarians may be r e l a t e d to the consumption of ready-to-eat cereal and a l l breads and substitutes which generally c o n t r i -bute to ir o n intake i n a vegetarian d i e t (National Research Council-National Academy of Sciences, 1974). A s i m i l a r c o r r e l a t i o n between i r o n intake and mutagenicity with TA98 was seen among a l l subjects. This probably r e f l e c t e d the f a c t that vegetarians had s i g n i f i c a n t l y higher intakes of i r o n than non-vegetarians but lower l e v e l s of mutagenicity with TA98 than the l a t t e r group. In addition to these major f i n d i n g s , i t i s of i n t e r e s t to note those 9.5 cases where c o r r e l a t i o n s were anticipated but not found i n t h i s study. Animal protein intake, e s p e c i a l l y meat, has been linked to increased incidence of colon cancer (Gregor et a l . , 1969; Reddy, Narisawa and Weisburger, 1976; Armstrong and D o l l , 1975). With TA100, a p o s i t i v e corre-l a t i o n was observed between intake of beef, lamb, pork, v a r i e t y meat, poultry and a l l meat and alternates groups and mutagenicity for a l l subjects. This possibly r e f l e c t e d the high mutagenicity le v e l s with TA100 and the high intake of these products by the non-vegetarians. In the case of TA98, a negative c o r r e l a t i o n was seen between mutagenicity and intakes of t o t a l pro-t e i n and animal protein for the non-vegetarians but not with meat and a l t e r -nate food items. The non-vegetarians obviously had the highest intakes of t o t a l p rotein and animal protein. There was no c o r r e l a t i o n of mutagenicity on e i t h e r TA100 or TA98 with the e n t i r e category of meats and poultry within the non-vegetarian group. This strongly suggests that these food items do not contribute to f e c a l mutagenicity. Bjelke (1974) and Sporn (1977) believe that vitamin A may have a pro-t e c t i v e e f f e c t against colon cancer. However, no c o r r e l a t i o n was observed between vitamin A intake and mutagenicity with either TA98 or TA100 i n any of the d i e t groups. Total c a l o r i c intake has been associated with increased r i s k f o r colon cancer ( H i l l et a l . , 1979). In t h i s study, however, no association was found between high l e v e l s of f e c a l mutagenicity with either TA100 or TA98 and c a l o r i e s . The consumption of milk was p o s i t i v e l y c o r r e l a t e d with f e c a l mutageni-c i t y on TA100 for the s t r i c t vegetarians and negatively correlated with TA98 for the same group. S t r i c t vegetarians, by d e f i n i t i o n , generally abstain from milk and dairy products unless dictated by s o c i a l circumstances. The 96 food frequency questionnaire, on the other hand, reported the intake of food items for a monthly period. It i s therefore possible that the small amount of milk consumed by a few of the s t r i c t vegetarians may have become s i g n i -f i c a n t when Spearman c o r r e l a t i o n tested for factors a f f e c t i n g mutagenicity. Indeed, i f milk consumption enhanced f e c a l mutagenicity with TA100 i n some i n d i v i d u a l s , i t cannot be a t t r i b u t e d to i t s calcium content, since calcium was negatively correlated with mutagenicity using TA100 for non-vegetarians who consumed 15-78 servings of milk per month (see Appendix F ) . The p o s i t i v e association between intake of cream for the non-vegetarians and mutagenicity with TA100 could be interpreted as additional data i n favor of the high f a t , high mutagenicity theory. However, ovo-lacto vegetarians' intake of cream was f a i r l y s i m i l a r to that of the non-vegetarians, and t h e i r l e v e l s of mutagenicity with TA100 were low i n comparison. It i s poss i b l e , however, that other factors were responsible for the low l e v e l s of f e c a l mutagens i n ovo-lacto vegetarians. It should also be mentioned that no association was found between mutagenicity with TA100 or TA98 and animal or t o t a l f a t intake derived from the four day food records. In summary, the intakes of f r u i t s and j u i c e s , Southgate f i b e r and i r o n were found to c o r r e l a t e negatively with f e c a l mutagenicity on TA98 for a l l subjects. S i m i l a r l y , t o t a l carbohydrate and Southgate f i b e r intakes produced negative c o r r e l a t i o n s with f e c a l mutagenicity on TA100. Among the non-vege-ta r i a n s , negative c o r r e l a t i o n s were established between mutagenicity on TA98 and t o t a l p r o t e i n and between mutagenicity on TA100 and calcium. 97 CHAPTER VI CONCLUSIONS The findings of t h i s study show that non-vegetarians had a higher l e v e l of f e c a l mutagenicity than vegetarians. Factors inf l u e n c i n g mutagenicity with TA100 d i f f e r e d from those associated with mutagenicity using TA98, confirming the presence of more than one mutagen i n feces. This was further substantiated by the r e l a t i o n s h i p between pH and mutagenicity as well as by the association between mutagenicity with TA98 and TA100. Whether the mutagens detected i n t h i s study are s i m i l a r to those reported by other r e -searchers (Bruce et a l . , 1977; Reddy et a l . , 1980; Ehrich et a l . , 1979; Kuhnlein and Kuhnlein, 1980) remains to be determined. The f l u c t u a t i o n s i n mutagenicity among i n d i v i d u a l s i n the di e t groups were considerable. It i s possible that, i n t h i s study, other l i f e s t y l e f a c t o r s , which were not evaluated, or genetics may have contributed to f e c a l mutagenic a c t i v i t y i n the subjects. Certain food items and/or nutrients associated with mutagenicity on TA100 d i f f e r e d from those c o r r e l a t e d with TA98 and var i e d between the diet groups. Only intake of Southgate f i b e r seemed to have a protective e f f e c t against mutagenicity with both TA100 and TA98 for a l l subjects. The con-sumption of f r u i t s and ju i c e s and i r o n intake were negatively co r r e l a t e d with mutagenicity on TA98 for the whole group. For mutagenicity with TA100, a s i g n i f i c a n t negative c o r r e l a t i o n was established with t o t a l carbohydrate intake when data from a l l subjects were analyzed. Within the group of non-vegetarians, negative c o r r e l a t i o n s were found between mutagenicity on TA98 and t o t a l p r o t e i n and with mutagenicity on TA100 and calcium. With the demographic variables and health habits, no clear pattern emerged to 98' indicate factors which would predict lowered mutagenicity for a l l subjects. 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Names w i l l not be included i n the data a n a l y s i s . ***************** I, , have had the study explained to me and agree to p a r t i c i p a t e . I understand that I may decline to answer any i n d i v i d u a l question. I may also withdraw from the study at any time without fear of r e p r i s a l . Signature: Date: 112 APPENDIX C Sample of Food Record Sheet DATE: INITIALS: FOOD INTAKE RECORD PERIOD OF DAY TYPE OF FOOD AMOUNT OF FOOD CONSUMED HOW PREPARED COMMENTS BREAKFAST A.M. SNACK LUNCH AFTERNOON SNACK DINNER EVENING SNACK TODAY'S MEDICATION: VITAMIN SUPPLEMENT: ORAL CONTRACEPTIVES: LAXATIVES: OTHERS (SPECIFY) 113 APPENDIX D Questionnaire SECTION A: DEMOGRAPHIC INFORMATION Date of Interview Number Group Do not write in this space Q. 1 What are your i n i t i a l s ? Q. 2 CHECK the age category to which you belong 60+ CD Q. 3 CHECK the sex category to which you belong Q. 4 What is your native language? Q. 5 Were you born in Canada or elsewhere? 1. Canada 2. Elsewhere (specify): Q. 6 If born outside of Canada, how long have you l i v e d in Canada? 40-44 • 25-29 • 30-34 C Z 1 45-49 O 50-54 • 55-59 | / Male O Female • 114 SECTION B : DIETARY HABITS Q. 7 P l e a s e r e a d c a r e f u l l y e v e r y food i t e m on the f o l l o w i n g l i s t and no t e the f r equency o f an ave rage s e r v i n g t h a t i s u s u a l l y consumed. Be as p r e c i s e as p o s s i b l e . REMEMBER: 3 T S P . = 1 TBSP. 4 TBSP . = i CUP 16 TBSP . = 1 CUP 4 G Z . RAW MEAT = 3 OZ. COOKED 8 OZ. = 1 CUP N . B . I f i n t a k e o f c e r t a i n foods i s s e a s o n a l , p l e a s e i n d i c a t e i n the comments c o l u m n . FOOD SIZE OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS MILK P r o d u c t s 1. Homo m i l k 1 cup 2. C h o c o l a t e m i l k 1 cup 3. 2% m i l k 1 cup 4. Skim m i l k (fresh/powdered) 1 cup 5. Evaporated m i l k 1 cup 6. B u t t e r m i i k or sour m i l k 1 cup 7. Yoghurt: P l a i n F l a v o r e d i cup i cup 8. Ice cream J cup 9. Sherbet or i c e m i l k ! cup 10. Cream(sweet or sour,},!) i cup 11. Non-dairy creamer/whipped cream s u b s t . i cup FOOD SIZE OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 12. B u t t e r 1 t b s p . 1 3 . M a r g a r i n e What k i n d ? ( w r i t e i n comments) 1 t b s p . 14. Cream cheese 1 t b s p . 15 . Other cheese a . Skim cheese b . Chedda r , S w i s s , b l u e , s l i c e s , p r o -c e s s e d e t c . ) 1 o z . 1 o z . 16. C o t t a g e cheese J cup 17. E g g s ( f r i e d o r o m e l e t ) 1 medium 18. Eggs ( b o i l e d , poached or cooked w i t h o u t f a t ) 1 medium BEEF 19. C o m m e r c i a l l y p r e p a r e d hamburger 3 o z . (cooked) FOOD SIZE OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY • COMMENTS 20 . Ground hamburger (meat l o a f , s p a g h e t t i , hamburger , e t c a . Lean b . R e g u l a r 3 o z . (cooked) 3 o z . (cooked) 2 1 . Stew meat 3 o z . (cooked) 2 2 . Roast 3 b z . (cooked) 2 3 . S teak 3 o z . (cooked) 24 . Corned bee f o r p a s t r a m i 3 o z . 2 5 . P o u l t r y ( c h i c k e n , d u c k , t u r k e y , e t c . ) 3 o z . (cooked) 2 6 . a . Bacon ( s t r i p s ) b . S a l a m i , e t c . 3 3 o z . FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 2 7 . Sausage ( p o r k ) 3 p i e c e s 2 8 . Po rk (ham, back bacon) 3 o z . 2 9 . Roast p o r k , c h o p s , c u t l e t s 3 o z . 30 . Organ meats ( h e a r t , t o n g u e , g i z z a r d s , e t c . ! 3 o z . 3 1 . L i v e r ( b e e f , p o r k , l amb , e t c ' 3 o z . 32 . L a m b ( r o a s t , c h o p s , s t e w s , s t e a k ) 3 o z . 33 . V e a l ( r o a s t , c h o p s , s t e w s , s t e a k ) 3 o z . 34. Lunch meats ( b o l o g n a , s p i c e d mea t s , w i e n e r s ) 3 o z . o r 3 s l i c e s ' 35 . Game ( r a b b i t , f r o g l e g s , s q u i r r e l ) 3 o z . A2. Other pota-toes(boiled, mashed, baked, not pot. chips) Al. French fries (home-fried, deep fried or other fried potatoes) AO. Rice: White Brown 39. Other fish (fresh/frozen) (cod, sole, trout, bass, haddock) 38. Shellfish (shrimp, clams, lobster, cray-fish, scallops) 37. Canned fish (salmon, tuna, sardines, etc.) 36. Gravy(made from any meat or mix) FOOD i cup or 1 medium 10 pieces o c •o o c •a LO O N LO O N 2 tbsp. SIZE OF SERVING NEVER FREQUENCY OF INTAKE SELDOM FREQUENCY OF INTAKE 1/MONTH FREQUENCY OF INTAKE 2/MONTH FREQUENCY OF INTAKE 3/MONTH FREQUENCY OF INTAKE 1/WEEK FREQUENCY OF INTAKE 2 -4 /WEEK FREQUENCY OF INTAKE 5 -6 /WEEK FREQUENCY OF INTAKE 1 /DAY FREQUENCY OF INTAKE MORE THAN 1/DAY FREQUENCY OF INTAKE COMMENTS 611 FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 4 3 . M a c a r o n i ( n o o d l e s , p a s t a d u m p l i n g s , e t c ) J cup 44 . P a n c a k e s , c r e p e s , b l i n t -z e s , e t c . 2 sma11 (5" d i a m . ) 45 . Whi te b r e a d ( i n c l u d i n g l o a f r o l l s & b i s c u i t s ! 1 s l i c e or r o l l 46 . Brown b r e a d (whole whea t , r y e , pumper-n i c k e l ) 1 s l i c e 47 . Cornb read (cornmeal b i s -c u i t s , c o r n -b r e a d , e t c . ) 1 s l i c e or 1 b i s c u i t 48 . Bran ( c e r e a l o r b read! { cup o r 1 s l i c e 4 9 . P a r t b r a n ( c e r e a l or b read ! { cup o r 1 s l i c e 5 0 . Whole g r a i n c e r e a l s ( V i t a - B , R e d - R i v e r , G r a n o l a , A l p e n ) J cup FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 5 1 . Oatmeal (b read o r c e r e a l J cup o r 1 s 1 i c e 5 2 . Other c e r e a l ( c o o k e d , d r y , e g . Cream o f Wheat, c o r n f l a k e s , r i c e c r i s p i e s , e t c . R e a d y - t o - e a t ) 3/4 cup VEGETABLES 5 3 . Green and y e l l o w beans J cup 54 . B e a n s ( b a k e d , k i d n e y b e a n s , M e x i c a n , Navy , l i m a ) DRY i cup 55 . D r i e d peas ( b l a c k - e y e d peas s p l i t ( s o u p ) peas J cup 56 . Corn J cup o r 1 s m a l l cob 57 . O n i o n s ( l e e k s s c a l l i o n s , green o n i o n s , r e d and w h i t e ) t cup FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS . 5 8 . a . Cabbage ( r e d / g r e e n ) ( f r e s h o r cooked) b . Sauerkraut • J cup J cup 59 . Greens ( f r e s h o r cooked i n c l u d i n g l e t t u c e , c o l l a r d s p i n a c h , k a l e , t u r n i p ) 1 cup 60 . Green peas i cup 6 1 . Snow peas (eat pod) J cup 62 . Root v e g . ( c a r r o t s , b e e t s , t u r n i p , p a r s n i p ) J cup 6 3 . Tomatoes ( f r e s h o r canned cooked o r i n j u i c e ) 1 medium or J cup 64 . Bean s p r o u t s & a l f a l f a sprout s * c u p 6 5. V e g e t a b l e j u i c e s { cup 74. Other fruit (peach, pears, plums) 73. Dried fruits (prunes, raisins dates, etc.) 72. Melon (watermelon, canteloupe) 71. Bananas 70. Apples(or apple sauce) 69. Citrus fruit (orange, grape-fruit, tanger-ine, etc.) 68. Fruit juice a. Unsweetened natural juice b. Sweetened 67. Celery 66. Pickles (all types) FOOD 1 peach 2 plums 2 halves 2 or ' 1 cup J cup 1 wedge 1 small 1 medium J cup 1 medium orange i grapefruit J cup J cup 3 sticks SIZE OF SERVING NEVER FREQUENCY OF INTAKE SELDOM FREQUENCY OF INTAKE 1/MONTH FREQUENCY OF INTAKE 2/MONTH FREQUENCY OF INTAKE 3/MONTH FREQUENCY OF INTAKE 1/WEEK FREQUENCY OF INTAKE 2 - 4 /WEEK FREQUENCY OF INTAKE 5 -6 /WEEK FREQUENCY OF INTAKE 1/DAY FREQUENCY OF INTAKE MORE THAN 1/DAY FREQUENCY OF INTAKE COMMENTS . FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 75 . B e r r i e s ( f r e s h o r cooked 1 ! cup 76 . Nuts (peanuts w a l n u t s , p e c a n s , a lmonds , e t c . ) { cup 77. Peanut b u t t e : 1 t b s p . 78 . Sugar (b rown o r w h i t e ) on c e r e a l , d r i n k s 1 t b s p . 79 . Honey, j a m , marmelade, j e l l y o r p r e s e r v e s 1 t b s p . 80 . S y r u p ( m a p l e , m o l a s s e s , c o r n sy rup ) 1 t b s p . 8 1 . Doughnu t s , sweet r o l l s or D a n i s h p a s t r y 1 82 . C h o c o l a t e (bar o r c a n d i e s ) 1 8 3 . G r a n o l a ba r s 1 84 . O the r candy ( s p e c i f y ) 10 p i e c e s FOOD SIZE OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH -1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 85. P o t a t o c h i p s , c o c k t a i l snacks 10 p i e c e s 86. P i e , cake , c o o k i e s , pudding sweet d e s s e r t s 1 p i e c e 87. J e l l o type d e s s e r t s i cup 88. Beer 10 oz 89. Wine 3 oz. 90. L i q u o r o r c o c k t a i l s ( g i n , rum, s c o t c h , e t c 1 oz. 91. L i q u e u r (brandy, cognac, B e n e d i c t i n e , creme de menthe) 1 oz. 92. C o f f e e 1 cup 93. Tea a. B l a c k b. H e r b a l 1 cup 1 cup 94. S o f t d r i n k s 8 oz. FOOD S I Z E OF SERVING FREQUENCY OF INTAKE NEVER SELDOM 1/MONTH 2/MONTH 3/MONTH 1/WEEK 2-4/WEEK 5-6/WEEK 1/DAY MORE THAN 1/DAY COMMENTS 95 . F r u i t f l a v o r e d d r i n k s (o rangeade , Tang K o o l - a i d ) 8 o z . 96. P r e p a r e d s a l a d d r e s s i n g 1 t b s p . 97 . V e g e t a b l e o i ( f o r f r y i n g o r f o r s a l a d o i l ) 1 t b s p . 9 8 . S h o r t e n i n g 1 t b s p . 99 . D r i p p i n g s ( f rom b e e f , p o r k , b a c o n , e t c 1 t b s p . 100. B r o c c o l i J cup 101 . C a u l i f l o w e r J cup 102 . L e n t i l s (cooked) J cup 103. M i l l e t (cooked) J cup 104. Soy m i l k i cup 112. Others 111. Avocado 110. Texturized vegetable prote in 109. Wheat germ 108. Sesame seeds 107. Sunflower seeds 106. Miso 105. Tofu FOOD J medium { cup 1 tbsp. n o-in •o 1 tbsp. 1 tbsp. 1 square SIZE OF SERVING NEVER FREQUENCY OF INTAKE SELDOM FREQUENCY OF INTAKE 1/MONTH FREQUENCY OF INTAKE 2/MONTH FREQUENCY OF INTAKE 3/MONTH FREQUENCY OF INTAKE 1/WEEK FREQUENCY OF INTAKE 2 - 4 /WEEK FREQUENCY OF INTAKE 5 - 6 /WEEK FREQUENCY OF INTAKE 1 /DAY FREQUENCY OF INTAKE MORE THAN 1/DAY FREQUENCY OF INTAKE COMMENTS LZ\ 128 Q. 10 How o f t e n do you t r i m the f a t from meat and e a t . o n l y the l e a n ? 1. A l l the time 2. O c c a s i o n a l l y ( s p e c i f y ) 3. Never, Q. 11A Do you use hot s p i c e s ? ( e . g . , c u r r y , c h i l l i e s , t a b a s c o , e t c . ) 1. Yes 2. No Q. 11B I f y e s , how f r e q u e n t l y do you use these hot s p i c e s ? 1. E v e r y day 2. 3-5/week 3. 1/week 4. 3/month 5. 1/month Q. 12A Do you f a s t f o r s h o r t or l o n g p e r i o d s of time f o r r e l i g i o u s r e a s o n s or o t h e r s ? 1. Yes 2. No Q. 12B I f y e s , how l o n g do these p e r i o d s of f a s t i n g l a s t ? 1. L e s s than 8 hours 2. 8-12 hours 3. 12-24 hours 4. 24+ hours Q. 12C How f r e q u e n t l y do you f a s t ? 1. More than 1/week 3. Monthly 2. Weekly 4. No r e g u l a r p a t t e r n 129 Q. 13 Are you f o l l o w i n g a " s p e c i a l " d i e t a t p r e s e n t ? (not t y p i c a l o f y o u r u s u a l p a t t e r n ) 1. Yes ( s p e c i f y ) 2 . No Q. 14 I f you a re a v e g e t a r i a n , how l o n g have you f o l l o w e d t h i s way o f e a t i n g ? Q. 15 As a r e s u l t o f i n c r e a s i n g food p r i c e s , have you changed your food s e l e c t i o n s r e c e n t l y ? 1. Yes 2 . No Q. .16 I f y e s , when d i d t h i s change o c c u r ? 1. W i t h i n the l a s t week 2 . W i t h i n the l a s t month 3 . W i t h i n the l a s t 6 months 4 . 6-12 months ago Q. 17 What major changes d i d you make i n food s e l e c t i o n ? SECTION C: HEALTH HABITS Q. 18 How t a l l a re you? f e e t i n c h e s Q. 19 How much do you we igh? - l b s Q. 20 D u r i n g the p a s t y e a r , has y o u r w e i g h t changed? 1. Yes 2 . No Q. 21 I f y e s , s p e c i f y . 1.. G a i n e d kg OR l b s 2 . L o s t kg OR l b s 130 Q. 21A Do you t ake p i l l s o r m e d i c a t i o n s r e g u l a r l y ? 1. Yes 2 . No Q. 21B I f y e s : What k i n d How o f t e n What f o r 1. 1. 1. 2 . 2 . 2 . 3 . 3 . 3 . 4 . 4 . 4 . Q. 22A D u r i n g the p a s t mon th , have you t a k e n any a n t i b i o t i c s t o combat i n f e c t i o n ? 1. Yes 2 . No Q. 22B I f y e s , s p e c i f y Q. 23 Have you e v e r had a m e d i c a l d i a g n o s i s o f d i f f i c u l t i e s i n the d i g e s t i v e t r a c t ? S p e c i f y Q. 24 How o f t e n do you have a bowe l movement? 1. 1/day 2 . 2 / d a y 3 . More t h a n 2 / d a y 4 . E v e r y 2 days 5 . I r r e g u l a r 6 . O t h e r , s p e c i f y Q. 25 D u r i n g the p a s t y e a r , have you been t r o u b l e d by c o n s t i p a t i o n o r d i a r r h e a ? 1. Y e s , c o n s t i p a t i o n 2 . . Y e s , d i a r r h e a 3 . N e i t h e r 4 . B o t h c o n s t i p a t i o n and d i a r r h e a 26A Do you e v e r use l a x a t i v e s ? 1. Yes 2 . No 26B I f y e s , what k i n d ? 26C T h i s week? 1. Yes 2 . No 27 Do you smoke c i g a r e t t e s ? How many pe r day? C i g a r s ? P i p e ? O t h e r s ? 28A Do y o u have any h i s t o r y o f c a n c e r i n y o u r f a m i l y ? 1. Yes 2 . No 28B I f y e s , what t y p e ? 28C Which r e l a t i v e s ? 29A Do you t ake any v i t a m i n s o r food supplements? 1. Yes 2 . No 29B I f y e s , s p e c i f y \ ; 30 Comments o r o t h e r i n f o r m a t i o n you f e e l i s p e r t i n e n t t o t h i s q u e s t i o n n a i r e : Thank you 132 APPENDIX E Dietary Standard for Canada, Revised 1975 3U|Z f U0J| f 8UipO| ? uimsauBi sruogdsoiid f iuni3|B3 | " —-g !• 0 u! u ) Bl!A V UlLUBljA, ^ 3 uiuiej/v I5 "8 ujiUBijA 5 ujAEKoqia i 1 uiaiojf) . 2 •o g '5 a DC I e jt 5 c . -° t: .25 CD CD <=' 5> ^ g1 \Xn "2r> VZn "LO l^n "UTJ I"LV» un uri "U» i n LO "In i a in i n csi csi rsi ( s i r s ! csi esi esi esi csi rsi esi esi csi esi esi o a o o o a o o O O 0 C 3 <=> O O O C J CD O CD CD O CO V CD eo ro a o en esi L A o r— o csi O O O O CD CD CD CD O O CD CD a i • csi GO o r — CD CD CD O Ol o CD a CD CD as oo CD O O O CD O CD p L O ^ »— CD L O CO CD CO o o <n CD CD o o LO L O CO CD CD CO csi oo o a o o c o o c o o o o o o o o o o o o o o o e o o c o co co o CD c o c o p p o o CO CO csi ^ r-. fsi en o o o o o o o o o o o c o o o *T co fvj CD in n co O J csi»— CD »— r-- eo T— «— csi rsi csi csi csi rsi co csi f J (M C S I ^ -CO LO CSJ »— § ° o o O LO CO CSI o o o o O P -p p o o O CO co L O <sj ^ p — v S 5 S co o • » o o o o O LO ro csi CD CO o o CO P -O O o o as r-» CD O CD O CO CD CSI CSI p UTJ esi ^ p crj o «w p ~ -to •— L O p p f? CD if O V o o «=r o o o ro csi o o O CD oo CD O o o co r— p o co ro co o o o rs< csi p p oo co JS ca O O o o co csi o o CD o o o o o co r— o o co ro o o co o csi rsi Csi r-^ r-. csi •**; °. L O 5 p LO o o o o + + o o o o + + f5 co cd + CO o L O LO + + L O CO o o + + + + + + CO o + + u c C= o CD U *- ca l l .1 1 a l III III I it* i i i 111! i l l s ! mm co ro i> ^ 2 | | - i E 1 2 2 H e i is £ 3 .2 — S ^ ™ £ re ca 1 ? i S ro o In: Nutrient Values of Some Common Foods, 1979. APPENDIX F Raw Data: Food Frequency Intake by Food Items for Each Subject Grp 2D Grp 2G Grp 21 Grp 3 Subject Grp 1A Grp IB Grp IC Grp ID Grp 2A Grp 2B Grp 2C V a r i e t y Grp 2E Grp 2F F i s h and Grp 2H Nuts 6, Vege-Number Cheese Cream Ice cream M i l k Beef Lamb Pork meats Game P o u l t r y Seafoods Eggs Legumes t a b l e s Ovo-lacto veg. 1 12.0 0.0 0. .3 59. .3 0.6 0.0 0. .0 0.0 0.0 0 .3 0, .9 0.0 87. ,2 57.5 2 24.6 33 .0 2. .0 34. .6 0.0 0.0 0, .0 0.0 0.0 0 .3 2 .6 4.3 94, .3 132.5 3 2.6 0 .3 0. .3 12, .6 0.0 0.0 0, .0 0.0 0.0 0 .0 2, .0 12.0 86. ,3 119.4 4 37.3 4 .0 2. .3 66, .3 0.0 0.0 0, .0 0.0 0.0 0 .3 6. .0 16.0 30, ,9 141.2 5 41.0 0 .3 0. .6 25, .8 0.0 0.0 0, .0 0.0 0.0 0 .0 0, .0 16.0 15. .2 67.8 6 18.3 4 .0 12. .3 39, .6 0.0 0.0 0, .0 0.0 0.0 0 .0 0. .0 44.0 10, .9 100.9 : r i c t veg. 7 14.6 0 .3 2. .0 16 .9 1.5 0.0 0, .3 0.0 0.0 0 .3 2, .6 2.3 132, .3 154.6 8 0.0 0 .0 0. .0 4, .0 0.0 0.0 0, .0 0.0 0.0 0 .0 0. .0 0.0 131, .6 126.9 9 2.9 0 .3 0. .0 2, .0 0.0 0.0 0, .0 0.0 0.0 0 .0 0, .3 0.0 108, .6 160.5 10 2.6 0 .3 0. .0 2, .0 0.0 0.0 0, .0 0.0 0.0 0.0 0, .0 0.0 64, .6 160.2 11 2.3 0 .0 0. .6 2 .6 0.0 0.0 0 .0 0.0 0.0 0.0 0, .0 0.3 102, .0 92.9 12 0.9 0 .0 0. .6 1 .2 0.0 0.0 0, .0 0.0 0.0 0 .0 0 .0 0.3 131. .3 94.9 13 0.0 2 .0 2. .0 0 .6 0.0 0.0 0 .0 0.0 0.0 0 .0 0, .0 0.0 55, .6 82.6 14 0.9 0 .3 0 .6 1 .2 0.0 0.0 0 .0 0.0 0.0 0 .0 0, .0 0.6 89 .3 71.2 15 0.3 0 .3 4. .0 1 .2 0.0 0.0 0 .0 0.0 0.0 0 .0 0, .0 0.3 58, .3 79.2 16 0.0 0 .3 0. .0 0 .3 0.0 0.0 0 .0 0.0 0.0 0 .0 0, .0 0.6 84, .3 82.3 17 0.3 0 .0 0. .3 0 .0 0.0 0.0 0.0 0.0 0.0 0 .0 0, .0 0.6 105, ,0 98.6 Non-veg. 18 47.0 2.0 2.0 39.0 24 .9 2.0 4.3 0.0 0.3 4, .0 6, .3 4.3 8. .9 168.2 19 2.6 0.3 2.3 35.6 12.6 0.0 10.3 2.3 0.0 4, .0 6, .3 2.0 4, .9 35.5 20 2.6 0.3 4.0 35.3 12.6 0.0 10.3 2.3 0.0 4, .0 6, .3 0.3 4. .9 32.1 21 14.0 0.0 0.3 37.3 12.1 0.3 8.0 6.0 0.3 4, .0 2, .6 14.0 15. .2 130.2 22 14.3 12.0 6.0 36.5 20.3 2.0 10.0 6.0 0.3 12, .0 6, .0 14.0 16. .6 218.0 23 28.3 2.0 2.3 61.3 8.6 0.3 0.3 2.0 0.0 4, .0 18, .0 22.3 61. .6 127.9 24 39.3 4.0 6.0 45.5 23.8 2.0 6.3 0.6 0.3 12, .0 18 .0 24.0 13. .2 89.2 25 22.6 2.0 12.3 78.0 16.6 2.0 6.3 4.0 0.3 2, .0 4. .3 12.3 10, .9 90.6 26 35.3 2.0 12.0 47.3 22.6 2.0 4.0 0.3 0.0 12, .0 4, .3 2.3 10. .6 106.4 27 35.6 0.3 4.0 40.2 12.9 0.3 16.3 2.0 0.0 2, .0 4. .0 24.0 10, .6 92.3 28 10.0 0.0 2.3 47.6 22.9 0.3 10.0 2.3 0.0 12, .0 4, .3 12.3 2. .9 144.6 29 49.0 2.0 6.0 15.8 24.9 4.0 8.0 2.3 0.3 4 .0 6. .3 6.0 19. .5 127.6 Grp AB Grp 5A Grp 5B Grp 5C Grp 5D Grp 5F Grp 6 Grp 7A Grp 7B Grp 7C Grp 8 Subject Grp 4A F r u i t Breads, yeast C e r e a l , Hot Other Grp 5E Baked Sugars & Animal Vegetable Combi- M i s c e l l -Number F r u i t s j u i c e s and other ready-to-eat c e r e a l c e r e a l F l o u r s products sweets f a t f a t n a t i o n aneous Ovo-lacto veg. 1 116.3 22.3 37.3 78.3 18.0 0.3 35.0 33. .6 66.3 0.3 34 .0 2.0 65.0 2 88.0 33.0 35.3 12.9 10.0 2.0 26.0 4. .3 47.3 33.0 33 .3 0.0 70.0 3 114.3 33.0 26.6 12.6 8.3 2.0 16.0 2 .6 28.0 22.0 12 .3 0.0 12.9 4 180.0 12.3 35.6 59.0 14.6 2.0 12.0 14. .6 24.6 33.0 26 .0 12.3 68.3 5 59.3 33.3 16.3 6.3 6.6 4.0 4.0 35. .3 66.6 33.0 33 .0 0.0 100.9 6 40.9 33.3 35.6 12.9 4.3 12.0 0.3 39. .3 48.6 33.0 16 .0 12.0 20.6 S t r i c t veg 7 94.3 35.0 37.3 10.0 24.3 2.0 12.0 4, .3 24.9 0.3 37 .0 22.0 33.6 8 190.0 33.0 33.0 26.0 66.3 4.0 24.0 6, .3 16.0 0.0 22 .3 0.0 33.0 9 210.3 12.3 18.0 35.0 2.6 2.0 33.3 12. .0 16.0 0.3 66 .0 0.0 14.0 10 202.3 12.3 28.0 35.0 2.6 2.0 33.3 12, .0 16.0 0.3 66 .0 0.0 14.3 11 87.0 4.6 35.6 74.0 18.3 2.0 12.3 4. .9 4.6 0.0 8 .3 2.3 2.3 12 66.0 12.6 35.3 74.0 18.0 2.0 22.3 14, .6 6.3 0.0 16 .3 0.3 4.3 13 46.0 22.0 39.3 82.0 28.3 2.0 22.3 6. .6 14.6 . 0.0 6 .3 2.3 12.3 14 85.3 2.0 35.6 12.3 26.3 2.0 2.3 0 .9 0.9 0.0 45, .3 0.0 2.3 15 83.3 0.6 35.3 12.9 26.3 2.0 2.3 0, .9 1.2 0.0 4, .6 0.3 0.3 16 98.0 0.9 35.3 40.0 32.0 12.0 2.3 4, .0 6.3 0.0 2, .3 0.6 2.0 17 71.3 2.6 35.6 78.0 39.0 4.0 4.3 0, .6 0.9 0.0 4 .3 0.0 0.3 Non-veg. 18 24.6 4.0 12.9 2.0 2.0 2.0 0.3 4, .3 66.9 8.0 66 .0 16.3 86.3 19 8.9 44.3 35.6 2.6 6.0 12.0 0.0 43, ,0 53.3 35.0 4 .6 8.0 16.3 20 7.2 44.3 35.6 2.6 6.0 12.0 0.0 14, .0 53.3 35.0 4 .6 8.0 62.2 21 40.6 2.3 35.6 1.2 4.6 2.0 0.3 6, .6 16.9 0.6 55 .0 4.3 39.9 22 94.0 35.3 51.0 103.0 14.0 12.0 0.3 8 .3 63.0 35.0 66 .3 22.6 82.0 23 123.0 33.3 47.3 30.0 36.0 12.0 0.6 18 .0 82.3 33.3 78 .0 12.3 112.0 24 66.6 2.3 12.0 39.0 6.0 2.0 0.3 41, .3 51.3 35.0 22 .6 8.0 138.0 25 38.0 33.6 26.3 35.6 4.3 2.0 0.0 8, .3 34.9 14.0 45.3 2.6 149.0 26 26.3 16.0 34.3 1.2 4.0 12.0 0.3 35, .6 6.9 0.3 45 .0 24 .3 61.3 27 68.3 4.0 39.3 6.6 6.3 2.0 2.0 4, .6 8.0 12.0 45 .0 6.0 39.9 28 82.3 35.3 22.9 45.6 2.3 2.0 0.0 2, .6 4.6 2.0 35 .0 35.0 13.2 29 58.0 22.0 35.6 8.3 6.0 2.0 0.3 2 .6 12.6 2.3 66 .3 0.9 78.6 APPENDIX G Raw Data: Daily Nutrient Intakes for Each Subject Derived from Four Day Food Records Subject C a l o r i e s P r o t e i n Calcium Iron Vitamin A Vitamin E Thiamin R i b o f l a v i n N i a c i n Vitamin C Number Day k c a l g mg mg IU x 10 mg mg mg mg mg Ovo-lacto  veget. 01 1 2908,50 60. .30 968.00 28, .39 705. .50 204.03 51, .88 51 .55 74. .75 677.80 2 2382. .70 61 .80 1607 .70 37.05 974 .40 203, .71 51, .83 54 .41 73. .17 1611.50 3 1708. .20 41. .50 1492 .40 44, .24 1013. .10 130, .63 51, .60 52 .55 66, .34 1697.40 4 3177. .20 114. .30 1708 .10 47 .04 1885. .70 513, .11 53 .11 53 .07 69. .18 16 78.90 02 1 2452. .10 61. .70 742, .30 17, .58 1013. .50 140, .85 101, .56 101 .40 118, .62 618.50 2 2337. .20 54. .80 787 .80 15, .10 1961. .50 138, .60 51, .53 51 .45 60, .67 647.00 3 2839. .90 66.80 990, .50 20, .11 1119. .80 185.46 51 .16 51 .34 61, .64 669.80 4 2197. .00 59, .00 777 .50 13, .50 1170. .00 157.40 50, .76 50 .91 60, .95 587.00 03 1 1643. .60 63. .40 279.40 10, .92 532.00 171. .80 51 .37 50 .50 63. .28 590.50 2 1918. .40 37, .70 764, .20 11, .77 1640. .10 I l l , .12 50, .93 51 .04 56, .46 715.20 3 1861. .10 42. .60 560 .10 13, .40 86 7. .10 105, .12 50 .81 50 .80 58, .70 598.70 4 1927. .50 54, .50 1066, .30 11 .82 1548. .00 135, .05 50.64 53 .00 57, .34 634.50 04 1 3021. .90 99. .90 1991, .00 24, .72 1910.00 376.49 54, .80 ' 57 .97 88, .53 305.80 2 2327. .60 76. .10 1494, .30 22, .47 5027. .70 456. .60 54, .56 57 .00 90. .08 351.30 3 2554. .90 89. .10 1885, .00 19 .10 4 781. .50 331, .50 54, .47 58 .30 94, .37 274 .40 4 2235. ,50 74. .90 1604, .10 23.03 24 75. .10 76. .40 54, .36 57.02 89. .84 318.10 05 1 2718. .70 86.40 2037, .80 11, .15 600. ,70 93, .71 0, .97 2 .51 8, .26 151.40 2 2749. ,20 58. ,30 699, .40 11 .93 507. .30 116.01 51.01 51, .41 56. .92 601.90 3 2002. .20 68. .00 701, .80 9, .48 332. ,50 99. .06 51.48 52 .67 68. .75 671.10 4 2759. .70 92. ,20 1782 .90 21, .60 1133. .10 150. .83 51, .39 52, .45 62. .69 781.20 06 1 2144. .50 69. ,00 1493, .50 7, .15 4110. .00 66. .45 1, .27 3, .03 11, .75 183.00 2 3644. .80 71. .40 1048 .00 19, .60 2099, .20 335. .89 1 .29 2, .93 18. .22 384.90 3 3377. ,40 105. .70 2190, .00 14, .04 806. .20 325.09 1.66 2, .94 16 .93 162.60 - i c t 4 2785. .20 81. .30 1662, .40 17, .61 3435. .50 238. .65 1, .38 2 .26 6, .88 266.40 >et. 07 1 1373. .80 53. .40 896, .80 10. .56 967. .70 82, .65 0 .57 0 .76 4, .98 172.00 2 1104. .70 29. .40 247, .60 9, .26 517. .80 94, .82 0 .91 0 .58 15, .11 196.10 3 1668. .60 48. .10 560, .80 18, .04 1403, .90 68, .96 1 .48 1.49 26, .00 288.70 4 1093. .20 25. .60 330, .20 12, .51 1340, .20 90, .04 0 .69 2 .65 8, .30 196.20 08 1 2055. .50 53. .90 393, .00 16.76 618, .10 185, .55 1 .37 1 .19 13, .45 487.50 2 1766. .60 33. .20 429, .80 15 .84 137.40 81, .95 1 .13 0 .80 7, .39 113.90 3 1341.40 22. .10 310.20 9 .41 272, .90 78, .67 0 .90 0 .65 5, .98 165.90 4 1173. .50 25, .90 275 .40 10 .83 359, .90 68 .29 0 .88 0 .78 9, .36 140.80 Subject C a l o r i e s P r o t e i n Calcium Iron Vitamin A Vitamin E Thiamin R i b o f l a v i n N i a c i n Vitamin C Number Day k c a l g mg mg II) x 10 mg mg mg mg mg 09 1 1747. .20 45. ,30 421. .80 15.03 741. .60 164. ,28 2, .18 1.40 11. .95 395.80 2 1958. .50 54. ,50 775. .30 16. ,76 1453. .40 191.46 2. .15 3. .20 9. .25 392.90 3 2279. .90 47. ,50 472. ,80 13. ,11 1180. .20 154. ,97 1 .72 1, .50 14. ,65 361.10 4 2059, .10 54. .30 1059. ,20 14. .32 282, .70 151, .27 1, .50 11. .20 6. .77 308.10 10 1 3431. .40 79. ,80 810. ,10 26, .47 2807. .70 295. ,67 2, .65 2. .46 17. .42 349.00 2 3293. .90 81. ,10 988.80 32. .71 3437. .30 415.49 3, .77 2. .73 22. .14 446.20 3 2891, .20 56. ,60 541. ,60 17. .26 1371, .70 136.84 2, .05 2. .21 18, .36 401.20 4 1839. .80 51. .70 909. ,60 13. .20 1140. .30 182. .85 1, .78 5. .46 11, .56 425.80 11 1 2324, .80 77. .30 453. ,10 18.00 1168, .80 176.09 1, .65 1. .34 22.03 146.30 2 1863. .90 75. .60 544. ,30 20.47 2037, .50 . 263. ,84 1, .91 1.04 17. .37 96.40 3 2579. ;oo 73.00 588. .10 17. .46 877, .00 187. .28 1, .65 1. .18 11.87 144.00 4 1852. .70 70. .30 426. ,20 20. .80 452.00 300. .53 1 .56 3. .94 9. .74 62.00 12 1 2677. .60 88. .60 638. ,80 20. .40 980, .80 270. .37 2, .41 1. .72 27. .15 136.50 2 3435. .50 136. .80 738. .00 31. .49 2901.80 381. .65 3, .10 1. .65 36. .16 126.90 3 2562. .20 87. .20 643. ,00 17. ,14 489, .50 206.43 1 .72 1. .26 15. .74 103.50 4 2902. .30 101.40 595. ,30 29. .46 395, .40 474. .95 2, .33 1. .54 22. .77 48.50 13 1 1171. .60 39.40 239.40 11. .59 767, .00 119. .24 1 .36 1. .18 8. .84 88.00 2 1147. .90 42. .20 375. ,20 16. .23 2062 .50 105. .90 1, .82 1. .35 7. .69 87.50 3 1166. .00 31. ,80 253. ,10 10. .93 75.80 115. .75 0, .93 0. .89 7. .91 22.00 4 1576. .90 32. .80 415. ,90 12. .29 749, .60 99. .92 1 .35 7.09 6. .19 49.30 14 1 3057. .40 70. .70 599. .80 23. .24 7951, .50 261. .45 2.41 2. .52 20. .89 239.00 2 3054. .00 92. .30 620. .70 27. .26 7786, .60 262. .65 2 .84 1 .74 26. .80 213.20 3 3631. .80 71. ,20 399.00 21. .82 674, .70 300. .87 2, .25 1. .96 27. .09 531.10 4 3682, .20 109. ,60 1258.80 24. .81 334, .10 479. .97 2 .51 2. .15 25. .02 182.60 15 1 2064. .50 63. ,60 459. .40 17. .20 10,831.00 160. .07 1 .58 1. .94 23. .22 134.00 2 1911, .40 64. ,00 423. .90 19. ,30 1757.00 96, .86 1.89 0. .81 17. ;79 90.00 3 3384, .50 98. ,80 660. .70 23, .79 472 .70 354. .21 2, .39 1. .79 34. .90 445.00 4 2741.00 82. .30 652. .20 24. .90 1580, .50 313. .80 1 .97 1. .47 24. .66 324.50 16 1 1942, .70 62.40 386, .60 15, .69 1146.80 275, .79 1 .83 1. .05 18. .02 133.60 2 2750. .50 83. .70 619. .10 24. .86 1846 .80 100, .15 2 .45 1. .06 21. .38 193.50 3 2933.10 58. .40 327. .80 18.01 410 .80 311, .83 1 .70 1, .16 31. .05 175.20 4 1424, .90 34. .20 391. .10 9, .46 558 .10 241, .58 1 .17 0, .85 14. .86 308.30 17 1 3214, .60 97. .60 684, .90 26, .70 2597 .50 523.00 1 .95 1 .68 42, .64 125.00 2 1839, .90 47. .80 321. .70 14.47 892 .80 235 .46 1 .50 0, .64 15, .82 184.80 3 2145, .80 57. .20 481, .50 14.05 1143, .60 267, .43 1 .41 1 .38 21. .32 249.70 4 1938, .70 62, .20 468 .60 17.09 1361.40 298 .72 1 .59 1 .09 19. .13 271.40 Subject C a l o r i e s P r o t e i n Calcium Iron Vitamin A Vitamin E Thiamin R i b o f l a v i n N i a c i n Vitamin C Number Day k c a l g mg mg IU x 10 mg mg mg mg mg Non-veget. 18 1 1227. .00 59.00 530, .40 7.35 315. .00 156. .15 0, .57 1. .19 4, .00 19.00 2 2390, .30 106, .30 493 .90 14.52 143. .10 134. .00 1.43 1. .65 36, .29 83.30 3 1979. .20 98. .70 841 .00 13.59 345. .20 127. .92 2.02 2, .09 16, .68 2123.30 4 2120. .50 92. .80 1330 .60 15.02 2438, .90 137, .54 1. .52 2, .62 26. .41 156.20 19 1 2887. .20 111. .20 904, .00 16.46 978. .50 288, .17 2, .36 2, .02 30. .70 650.90 2 3136. .50 122. ,00 1194 .00 15.31 722. .10 341, .26 1. .33 2, .36 25, .20 62.60 3 2260.20 89. .80 454, .30 15.15 421. ,20 110, .73 0. .95 1, .33 18. .56 69.10 4 1345. .00 35, .80 620, .10 6.02 223, .60 17, .80 0. .60 1, .09 3. .32 18.40 20 1 1985. ,10 119 .60 913, .20 17.93 301. .10 155, .90 1. .02 1 .81 25, .05 585.40 2 2001. .00 97.40 793 .60 21.04 947. .00 172, .76 1. .29 1, .92 24 .27 234.50 3 1950. .50 62.00 850, .60 9.98 168. .10 76, .36 0.65 1 .48 16, .38 48.40 4 3508, .60 85. ,10 929, .80 14.92 4169. .40 215, .28 1. .16 2. .12 32 .13 71.90 21 1 3765 .30 179, .60 1345, .90 21.30 912 .20 335.42 1. .29 2, .73 31. .20 119.00 2 2442. .50 95. .60 1181, .50 13.08 5843, .40 310, .20 1, .12 2, .61. 23, .52 71.60 3 4323. .00 162.00 1570, .60 20.38 558. .80 808. .31 2. .43 3, .06 64. .54 192.50 4 5055. .50 205. .30 1642, .10 23.03 1856. .40 500, .70 1. .52 3. .45 39, .06 206.20 22 ' 1 2112. .60 89. .70 1148, .00 18.95 4312. ,60 255, .33 2. .29 3 .82 27. .47 141.40 2. 1982. .50 60, .50 361 .30 13.25 857. .10 220. .24 1. .98 1, .28 24, .70 172.50 3 3042. .20 91. ,80 593, .60 15.35 1163. ,00 475. .63 3. .15 1 .85 25. .59 152.90 4 2526. .00 76. .60 1096, .80 12.67 2216. ,70 199. .64 1. .26 1, .92 26, .16 102.90 23 1 2733. .50 80. .90 1551.40 12.44 577. .40 222, .99 1. ,22 2, .57 12. .67 583.00 2 2577. .20 112. .90 1569, .50 13.06 652, .80 110, .21 1.40 1 .96 30. .67 770.80 3 1742. .20 73. ,30 733, .90 10.41 593.00 155. .80 1. .10 1, .35 8. .18 627.40 4 1780. .20 55. .30 1246, .10 6.95 296.60 217. .94 0. .77 1. .60 8, .14 523.40 24 1 2120. .50 93. .00 1566, .20 9.49 251.40 163. .52 1. .14 2, .45 15. .27 149.80 2 1847. .00 82, .90 870.40 12.63 1340.00 105.00 0. ,99 1, .64 16, .61 267.10 3 3454. .90 103. .00 802, .40 15.37 264.40 417. .60 1. .66 3. .37 35. .08 105.90 4 1913. .00 83. .70 1180, .90 11.03 1059.80 60.90 1. .15 1 .75 16, .62 131.30 25 1 2490. .30 115. .90 723 .40 14.48 398, .50 94, .10 1. .80 2, .52 28, .90 137.50 2 2676. .00 109. .20 670, .90 19.78 1686.00 427, .41 2, .74 2 .13 26, .13 105.00 3 1507. .70 101.00 292, .30 11.33 966.40 68, .34 0, .63 1, .18 25.48 50.90 4 2177. ,20 69. .00 464 .70 12.39 600, .60 155, .58 1. .39 2, .62 28, .17 124.00 Subject C a l o r i e s P r o t e i n Calcium Iron Vitamin A V i t a m i n E Thiamin R i b o f l a v i n N i a c i n Vitamin C Number Day k c a l g mg mg IU x 10 mg mg . mg mg mg 1 2427. ,10 84.40 1276. ,90 16.68 1865.40 174.38 1.14 6. .00 14.55 49.70 2 2053.60 73.10 866.50 10.02 945.20 101.63 0.81 5. .61 18.43 174.10 3 1702. ,50 79.60 1256. ,20 11.17 490.80 154.92 1.14 2. ,18 17.26 90,30 4 1568. .40 73.40 1015. ,70 9.36 398.10 170.15 1.13 5. .73 11.47 119.50 1 1650. .50 67.60 327. ,10 13.76 907.40 200.09 1.73 0. .93 22.22 104.30 2 2463. .50 80.50 799. .50 8.35 665.00 179.95 1.24 1, .83 25.70 199.50 3 2889. .00 109.60 1221. .60 13.97 892.60 429.74 1.32 1. .86 43.47 169.90 4 2199. .50 97.70 975. .70 10.91 418.80 277.61 1.12 1. .72 30.72 205.60 1 2614. .80 111.00 1561. .10 13.93 277.20 182.44 1.30 2, .84 16.87 111.30 2 1458. .20 75.40 1523, .50 13.00 323.80 181.85 1.85 2, .54 10.35 186.30 3 1407. .60 94.50 1779, .20 9.52 142.20 121.66 1.10 3, .08 10.07 120.90 4 1679. .70 102.90 1456, .80 9.59 140.90 249.00 1.29 2, .37 25.00 140.50 1 1246. .10 62.40 832, .50 9.62 264.30 58.56 0.67 1, .31 13.94 177.70 2 1313, .40 54.90 574 .10 7.81 305.90 185.98 0.60 0 .93 17.51 118.30 • 3 1119, .40 63.20 680.00 8.45 947.10 172.62 0.86 1 .20 5.51 83.20-4 1043, .40 64.10 429.80 7.68 284.20 126.24 0.67 1 .14 15.76 119.90 Both Both Crude Southgate Animal Vegetable Animal Vegetable types of types of Subject Fat Carbohydrate f i b e r f i b e r f a t f a t p r o t e i n p r o t e i n p r o t e i n f a t Number Day g g g g g g g g g g Ovo-lacto veget. 01 1 83. .00 494. ,00 12. .60 52.87 2. .00 54.00 4.00 48. .50 6. .00 27.00 2 67. .40 328. ,30 10.65 130.41 18. .20 34. .90 23.60 33. .20 4. .00 14.00 3 31. ,70 289. ,30 10. .74 121. ,52 2. .00 21. .70 13.00 20.40 2. .00 8.00 4 55. ,40 596. ,10 27. .21 158. .97 9. .20 35. .90 20.20 85. .00 2.00 8.00 02 1 120. ,60 305. ,10 9. .61 32. .58 37. .00 64. .60 2.00 40. .20 0 .00 0.00 2 112. ,00 299.10 16. .42 75.68 82, .00 16. .00 18.00 28. .80 8. .00 14.00 3 130. ,50 349. ,90 16. .23 92. .75 49. .50 52. .00 12.00 37. .80 10.00 20.00 4 103. 50 255. ,30 10. .79 36. ,35 32. .00 43. .00 8.00 35 .50 5. .00 15.00 03 1 90. ,20 147. ,40 5. .78 15, .73 12, .50 77. .70 42.50 20. .90 0, .00 0.00 2 100. ,50 220. 20 6. .61 46.42 68 .50 21. .50 14.00 17, .70 6.00 10.50 3 95. ,40 202. ,80 7. .17 27. .25 32, .20 63. .20 9.40 33. .20 0, .00 0.00 4 101. .50 217. ,30 8. .34 25. .61 62.00 35. .00 26.00 27, .00 1, .50 4.50 04 1 141. ,70 348. ,70 12, .76 133. .31 64.00 47. .70 52.90 37, .00 10, .00 30.00 2 95. .80 311. ,60 11. .26 39. .71 41, .50 24. .10 33.20 35, .90 6, .40 22.00 3 104. .70 285. ,10 12. .73 82. .70 57, .70 27. .00 55.30 29, .80 4. .00 20.00 4 97. .80 288. ,70 19. .02 49. .03 37, .50 57. .30 31.20 41. .70 2. .00 3.00 05 1 98.10 376. .40 6. .12 27, .95 32, .30 19. .40 40.80 21, .30 10. .50 6.00 2 123. .40 372. .20 4.07 18.23 34 .40 12.00 21.10 13, .80 10 .00 40.00 3 55. .10 253. .80 5. .20 21, .10 25, .80 4. .00 39.40 10 .50 3, .00 12.00 4 126, .90 313. .40 9. .72 42.34 67, .30 56. .60 49.80 34, .40 8.00 3.00 06 1 68. .50 301. .00 5, .40 14, .85 20 .50 28, .00 51.50 10, .00 7, .50 20.00 2 172. .00 461. .80 10. .75 41. .93 114.00 32.00 29.00 35, .40 7, .00 26.00 3 151. .20 410. .70 6, .43 28, .54 81 .80 63, .40 66.10 38, .60 1, .00 6.00 4 128, .50 329. .60 7, .73 29, .34 76.00 40, .50 55.00 21, .80 4, .50 12.00 S t r i c t veget. 07 1 41. .20 211. .30 11.40 26 .39 16 .00 20.40 16.00 34 .40 0 .00 1.80 2 13.60 232. .50 12 .65 31, .97 0.00 13, .60 0.00 27 .40 0 .00 0.00 3 29. .30 305. .20 16, .57 37.09 3.00 8, .30 3.00 26 .10 0 .00 0.00 4 19. .90 208. .30 22.04 37, .81 0 .00 19 .90 2.00 23 .60 0 .00 0.00 Subject Number Day Fat g Carbohydrate g Crude f i b e r g Southgate f i b e r g Animal f a t g Vegetable f a t Animal p r o t e i n g Vegetable p r o t e i n g Both types of p r o t e i n Both types of f a t g 08 09 10 11 12 13 14 15 16 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 99.40 30.90 38.10 33.60 73.80 79.30 103.70 91.30 113.90 97.90 86.70 72.60 89.80 72.90 95.90 52.70 122.50 119.20 88.80 93.00 53.70 13.70 32.70 38.90 101.20 99.00 117.70 147.40 53.40 41.00 110.20 93.10 55.10 75.60 95.00 67.00 271.30 363.60 245.60 206.70 246.90 268.30 311.50 277.20 546.00 539.60 492.30 270.70 299.10 261.20 386.90 303.80 356.00 510.40 377.50 467.60 153.20 234.90 209.80 300.60 511.40 468.70 549.80 490.70 349.00 343.30 503.50 422.50 324.10 468.00 475.10 188.10 10.70 14.27 11.36 9.87 11.48 9.52 10.59 7.75 20.85 19.56 19.11 8.57 12.38 12.47 16.48 7.21 14.28 23.21 16.51 13.31 11.02 9.77 9.51 11.95 25.54 23.91 17.28 21.99 13.22 24.65 17.58 28.09 11.76 27.51 11.90 12.67 43.68 67.28 43.03 34.90 72.15 74.80 70.38 72.49 180.33 248.48 188.20 81.42 53.03 43.31 43.09 45.10 51.06 78.09 44.47 61.90 31.11 119.75 118.88 122.89 59.90 139.21 110.21 65.89 56.50 46.57 71.12 78.25 50.38 59.34 49.26 48.49 0.00 0.00 0.00 0.00 0.00 0.00 15.10 2.00 0.00 0.00 11.00 2.00 0.00 0.00 9.80 0.00 0.00 0.00 22.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 27.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 69.20 29.90 38.10 33.60 42.30 36.40 41.20 44.00 67.50 66.30 47.50 39.10 69.80 60.50 86.10 36.60 88.70 102.90 60.30 75.90 42.10 6.00 32.70 38.70 90.70 95.00 112.70 115.40 53.40 39.00 105.00 93.10 55.10 73.60 95.00 67.00 0.00 0.00 0.00 0.00 0.00 2.00 1.60 14.00 0.00 0.00 0.00 8.00 0.00 0.00 8.80 2.80 0.00 0.00 10.00 0.00 0.00 0.00 0.00 6.00 0.00 0.00 0.00 18.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 44.30 30.50 20.90 24.70 34.00 28.00 25.80 15.30 39.20 49.60 29.00 24.20 55.70 56.30 64.20 48.60 54.40 108.90 69.70 76.00 29.00 28.00 31.80 24.30 56.60 83.70 70.20 90.60 59.60 56.00 97.10 82.30 62.40 75.70 58.40 34.20 6.00 0.00 0.00 0.00 2.20 8.00 14.00 9.00 30.00 24.00 25.00 11.00 1.60 0.00 0.00 1.50 2.60 0.00 0.00 1.50 2.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 8.00 1.00 0.00 0.00 8.00 0.00 0.00 16.00 .0.00 0.00 0.00 2.10 4.00 20.00 18.00 17.00 12.00 20.00 21.50 8.00 0.00 0.00 4.50 13.00 0.00 0.00 4.50 6.00 0.00 0.00 0.00 0.00 0.00 5.00 5.00 0.00 2.00 5.00 0.00 0.00 2.00 0.00 0.00 Both Both Crude Southgate Animal Vegetable Animal Vegetable types of types of Sub j ec t Fat Carbohydrate f i b e r f i b e r f a t f a t p r o t e i n p r o t e i n p r o t e i n f a t Number Day g g g g g g g 1 7 g g 17 1 123. .50 492. .20 21. ,54 103. .65 0. .00 113, .70 0 .00 84 .60 2.00 6.00 2 60. .10 288. .40 17. ,06 38.91 0. .00 60, .10 0 .00 47, .80 0.00 0.00 3 85. .20 305, .30 15.48 83. .23 3. .10 48.00 5 .10 42 .60 4 .00 7.00 4 72. .10 284. .70 21. ,19 52, .34 0. .00 71, .10 0, .00 61 .20 0.00 0.00 Non-veget. 18 1 86. ,00 58. .90 1. .91 5.65 48. .50 16 .50 51, .00 6, .00 2.00 21.00 2 90. .40 284. .20 4. .31 16, .79 42. .50 47, .90 79, .50 26, .80 0.00 0.00 3 114. ,90 144. .30 5. ,98 21, .76 69. .10 16, .80 71, .80 11, .90 6.00 26.00 4 120. .90 133. .90 5. .63 18 .87 101. .00 0, .20 69, .00 16, .60 6.00 3.30 19 1 145. .30 299. .00 6.23 17, .53 85.00 53, .00 66, .00 38, .00 7.20 7.30 2 167. .60 289. .10 3.80 13.69 60. .00 53, .60 63, .30 30, .70 10.00 30.00 3 75. ,00 283. .40 2.27 15.03 45. .70 5, .50 61, .60 15 .50 12.70 23.80 4 39. .30 222. .00 0. .90 4.60 27. .30 3, .00 16.80 13, .00 0.00 0.00 20 1 80. .30 189, .30 1. .05 7, .08 76. .30 4, .00 99, .40 17 .20 0.00 0.00 2 67. .50 214, .10 2, .51 20, .20 61, .50 6 .00 72, .40 25 .00 0.00 0.00 3 60. .30 232, .50 2, .57 14.40 35. .80 5 .00 17 .80 19 .50 24.70 19.50 4 133. .70 421, .40 4. .57 10, .80 20, .10 24, .00 22, .40 11 .50 10.00 39.00 21 1 254. .10 204, .40 4. .20 20, .64 149. .20 96, .40 132 .70 27 .40 18.00 8.00 2 118. .10 269, .50 2. .80 16, .20 45. .50 31 .00 50 .50 25, .50 5.20 22.40 3 285. .20 304, .00 24. .78 59, .10 49. .20 218 .00 57.00 100 .00 5.00 18.00 4 290. .80 362, .30 7. .20 30 .51 220. .50 49 .30 146 .00 56, .30 3.00 15.00 22 1 75. .80 295, .70 7. .96 26, .17 41, .80 17 .00 46 .30 28 .90 14.50 17.00 2 90. .10 227, .00 7. .16 25, .90 49, .30 27 .30 26 .50 29 .00 5.00 7.50 3 162. .50 303, .90 10. .91 33 .95 71, .00 69.00 51 .50 35 .80 4.50 22.50 4 63. .30 404, .90 8. .21 31, .40 37.00 20 .30 50.00 26 .60 0.00 0.00 23 1 118. .50 355, .60 6. .24 24, .05 61, .60 17 .90. 51 .70 16 .20 13.00 39.00 2 117. .00 279, .80 9. .13 31 .27 71, .00 46 .00 87 .30 25 .60 0.00 0.00 3 83.50 176, .60 2. .98 11, .64 24, .00 46 .50 24 .50 11 .80 19.00 7.00 4 104. .70 166, .80 2. .33 53, .36 45, .70 32 .00 34 .70 14 .20 6.40 27.00 24 1 85. .10 248, .90 4.40 13, .55 63 .60 11.00 75.00 14 .50 3.50 10.50 2 90. .20 177, .60 5. .11 19 .85 81, .20 9.00 62 .90 20 .00 0.00 0.00 3 158. .70 296, .30 2. .39 15.70 95, .30 14 .00 69 .70 23 .10 9.00 33.00 4 87. .60 198, .70 4. .56 16, .85 59, .30 19 .50 63 .10 19.00 0.00 0.00 Both Both Crude Southgate Animal Vegetab le Animal Vege tab le types of types of Subject Fa t Carbohydrate f i b e r f i b e r fa t f a t p r o t e i n p r o t e i n p r o t e i n fa t Number Day g g g g g g g g g g 25 1 100.70 177.80 5.85 20.07 62.50 36.70 78.50 33.40 4 .00 1.50 2 164.60 150.70 4.42 18.57 86.10 39.00 71.40 21.80 5.00 8.50 3 50.90 140.10 4.54 19.09 59.90 3.00 78.70 18.30 4 .00 1.50 4 110.80 132.10 4 .97 13.38 52.00 58.80 48 .60 20.40 0.00 0.00 26 1 92.20 332.70 6.25 19.13 44.30 31.90 54.50 14.90 8.00 3.00 2 96.00 223.80 6.52 16.70 34.00 8.00 52.00 12.50 2.00 3.00 3 60.20 224.90 5.52 18.34 20.20 20.70 40 .70 12.60 24.00 13.00 4 58.00 201.30 3.92 19.44 35.80 18.20 45 .80 15.60 12.00 4 .00 27 1 78.90 191.70 7.31 26.04 20.20 31.60 19.60 37.90 2.00 6.00 2 106.00 254.00 3.86 26.85 29.00 45 .00 24.00 33.50 23.00 32.00 3 118.20 341.10 11.28 29.65 17.60 76.60 22.00 67.60 0.00 12.00 4 125.60 191.10 6.01 26.74 50.00 31.20 66.40 23.50 3.00 18.00 28 1 131.10 250.00 6.20 20.15 69.50 45 .60 90.50 19.70 0 .00 16.00 2 59.90 158.10 7.83 24.65 34.30 1.60 61 .80 13.60 0.00 24.00 3 48,50 148.80 3.96 12.28 30.90 1.60 86.40 8.10 0.00 16.00 4 60.90 180.20 4 .02 17.31 18.00 18.90 85.80 17.10 0.00 24.00 29 1 73.00 82.80 4.04 12.03 43.50 29.50 52.50 9.90 0.00 0.00 2 68.80 120.50 3.90 13.32 23.80 42 .00 41.30 13.60 0.00 3.00 3 52.. 50 100.40 2.51 7.79 21.50 3.00 37.00 12.20 0.00 6.00 4 46.40 153.00 3.20 7.55 26.60 19.80 4 7.30 16.00 0.00 0.00 

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