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Relations of trans fatty acids, cis n-6 and n-3 fatty acids in newborn cord plasma, maternal plasma and… Elias, Sandra Leanne 1999

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RELATIONS OF TRANS FATTY ACIDS, CIS N-6 AND N-3 FATTY ACIDS IN NEWBORN CORD PLASMA, MATERNAL PLASMA AND DIET By Sandra Leanne Elias B.H.Ecol., The University of Manitoba, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Department of Human Nutrition) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 1999 © Sandra Leanne Elias, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of l4uvv\av\ N U T Y I - T I O K V The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT ABSTRACT Trans fatty acids (TFA) are found in partially hydrogenated oils used in margarines, and in processed foods. Concern has been raised that TFA may inhibit desaturation of 18:2n-6 and 18:3n-3 to 20:4n-6 and 22:6n-3, respectively, which are necessary for normal growth and development. The effect of diet-derived TFA on human growth, however, is unclear. This study estimated intakes and major sources of dietary TFA in 60 women at 28 and 35 weeks gestation using a food frequency questionnaire. Maternal blood was collected at 35 weeks gestation. Cord blood was collected at birth to assess the relation of maternal diet and plasma TFA on cord TFA, n-6 and n-3 fatty acids. The mean estimated TFA intake was 3.8 and 3.4 g/day at 28 and 35 weeks, respectively (1.3% of total energy). The major dietary source of TFA was baked foods. TFA were present in maternal and cord plasma triglycerides (TG), mean ± S.E., 4.0 ± 0.2 and 2.9 ± 0.2%, phospholipids (PL), 2.4 ± 0.1 and 0.7 ± 0.0%, and cholesteryl esters (CE), 1.6 ± 0.1 and 2.0 ± 0.1% of total fatty acids, respectively. Cord plasma T G TFA was inversely related to 22:6n-3 (r = -0.36) and cord plasma T G and C E TFA were inversely related to 20:4n-6 (r = -0.27). Cord plasma T G and C E TFA were inversely related to length of gestation (r - -0.28 and r = -0.25, respectively), whereas T G 20:4n-6 was positively related to length of gestation (r = 0.5) and infant birthweight (r = 0.4). Conjugated linoleic acid (CLA) was inversely related to length of gestation in TG (r = -0.44) and in cord plasma C E to length of gestation (r = -0.52), birthweight (r = -0.35) and infant length (r = -0.35). In summary, this study found a mean TFA intake of <4g/d, ii A B S T R A C T with baked foods representing the major food source of dietary TFA. Evidence of an inverse association between cord plasma TFA, length of gestation, and with 20:4n-6 and 22:6n-3 in some lipids, suggests the need for larger prospective studies to determine relations between maternal dietary fat quality and birth outcomes, considering potential confounding dietary and lifestyle variables. TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT ii A C K N O W L E D G E M E N T S xiv TABLE OF CONTENTS iv LIST OF ABBREVIATIONS viii, LIST OF TABLES . viii LIST OF FIGURES x LIST OF APPENDICES xiii 1 INTRODUCTION 1 2 LITERATURE REVIEW 3 2.1 Definition and Classification of Fatty Acids 3 2.2 Formation of Trans fatty Acids 6 2.3 Dietary Sources of Trans Fatty Acids 7 2.4 Variation of Trans Fatty Acid Levels in Food Sources 8 2.5 Estimated Consumption of Trans Fatty Acids 10 2.6 Trans Fatty Acid Absorption and Metabolism 12 2.7 Fatty Acids and Growth and Development 16 2.8 Health Concerns About Trans Fatty Acids 20 2.8.1 Cardiovascular Disease 20 2.8.2 Cancer 24 2.9 Transfer of Fatty Acids to the Fetus 25 3 STUDY OVERVIEW 28 4 P U R P O S E . . . . 2 8 3.1 Objectives 28 3.2 Hypotheses 29 5 ETHICS 29 6 S U B J E C T S AND METHODS 30 6.1 Study One . . 3 0 6.1.1 Participant Selection Criteria 30 6.1.2 Participant Recruitment 30 6.1.3 Study Questionnaire 31 6.1.4 Blood Collection 31 6.1.5 Birth Outcome Measures 31 IV TABLE OF C O N T E N T S 6.2 Study Two 32 6.2.1 Participant Selection Criteria 32 6.2.2 Participant Recruitment 32 6.2.3 Participant Interviews 33 6.2.4 Study Questionnaires 34 6.2.5 Blood Collection 35 6.2.6 Birth Outcome Measures 36 7 DATA ANALYSIS 37 7.1 Demographic and Health Data Handling 37 7.2 Analysis of the Food Frequency Questionnaires 38 7.3 Fatty Acid Analysis 39 7.4 Statistical Analysis 41 8 R E S U L T S 43 8.1 Participant Recruitment and Characteristics 43 8.1.1 Study One 43 8.1.2 Study Two 45 8.2 Estimated Energy and Dietary Fat Intakes 51 8.3 Dietary Sources and Contribution of Trans Fatty Acids by Food Group 57 8.4 Maternal Plasma Fatty Acids 64 8.4.1 Triglyceride . . . 64 8.4.2 Phospholipid 65 8.4.3 Cholesteryl ester. . 66 8.5 Relations Between Trans Fatty Acids and C/'s n-6 and n-3 Fatty Acids in Maternal Plasma 68 8.6 Cord Plasma Fatty Acids 73 8.6.1 Triglyceride 73 8.6.2 Phospholipid 74 8.6.3 Cholesteryl ester 75 8.7 Relations Between Trans Fatty Acids and Cis n-6 and n-3 Fatty Acids in Cord Plasma 76 8.8 Relations Between Selected Fatty Acids in Maternal and Cord Plasma 81 8.9 Relations Between Trans Fatty Acid Intake Level of Trans Fatty Acids in Maternal Plasma Lipids 85 8.9.1 Triglyceride 85 8.9.2 Phospholipid . . 85 8.9.3 Cholesteryl ester 85 8.10 Relations Between Trans Fatty Acid Intake and Level of Trans Fatty Acids in Cord Plasma Lipids 85 8.11 Dietary Trans Fatty Acid Intake and Birth Outcomes 87 8.12 Relations Between Cord Plasma Lipid Fatty Acids and Birth Outcomes 87 8.12.1 Triglyceride 87 v, TABLE OF CONTENTS 8.12.2 Phospholipid .'. : 93 8.12.3 Cholesteryl ester 93 9 DISCUSSION 105 9.1 Dietary Trans Fatty Acid Intakes 105 9.2 Dietary Fat Intakes: Proportion of Energy from Total, Trans, Polyunsaturated, Mononunsaturated and Saturated Fatty Acids 108 9.3 Dietary Sources of Trans Fatty Acids 111 9.4 Maternal Plasma Fatty Acids 114 9.4.1 Relations Among Selected Fatty Acids in Maternal Plasma 114 9.4.2 Relations Between Selected Fatty Acids in Maternal Plasma and Dietary Trans Fatty Acid Intake 117 9.5 Cord Plasma Fatty Acids 117 9.5.1 Relations Among Selected Fatty Acids in Cord Plasma ..117 9.5.2 Relations Between Selected Fatty Acids in Cord Plasma and Maternal Dietary Trans Fatty Acid Intake 120 9.6 Relations Between Selected Fatty Acids in Maternal and Cord Plasma 121 9.7 Relations Between Cord Plasma Fatty Acids and Birth Outcomes 123 9.8 Conclusions and Future Directions 124 BIBLIOGRAPHY 128 APPENDICES 137 Appendix 1: Informed consent: Study One 137 Appendix 2: Research study questionnaire: Study One 140 Appendix 3: Recruitment letter: Study Two 143 Appendix 4: Informed consent: Study Two 145 Appendix 5: Socio-demographic and health questionnaire: Study Two .149 Appendix 6: Food frequency questionnaire: Study Two 155 vi \ LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS T C total cholesterol LDL-C low-density lipoprotein cholesterol HDL-C high-density lipoprotein cholesterol Lp(a) lipoprotein (a) LPL lipoprotein lipase TFA trans fatty acids CLA conjugated linoleic acid T G triglycerides PL phospholipids C E cholesteryl esters g grams kcal calorie FFQ food frequency questionnaire 18:2n-6 linoleic acid 18:3n-3 alpha-linolenic acid 20:4n-6 arachidonic acid 20:5n-3 eicosapentaenoic acid 22:6n-3 docosahexaenoic acid vii, LIST OF TABLES LIST OF TABLES 8.1 Characteristics of women in Study One 44 8.2 Birth outcome measures in Study One 45 8.3 Participant recruitment results for Study Two 47 8.4 Socio-demographic characteristics of women in Study Two 48 8.5 Characteristics of women in Study Two 49 8.6 Birth outcomes of infants born to women in Study Two 50 8.7 Diet and health characteristics of women in Study Two 51 8.8 Estimated dietary trans, polyunsaturated, monounsaturated and saturated fatty acids, total fat and energy intakes as determined by a food frequency questionnaire at 28 and 35 weeks gestation 55 8.9 Distribution of women by quartiles of estimated trans, polyunsaturated, monounsaturated and saturated fatty acids and total fat intake (g/d) as determined by a food frequency questionnaire 56 8.10 Quantity of trans fatty acids consumed by food group in the second and third trimester 61 8.11 Fatty acid composition of maternal plasma triglyceride (TG), phospholipid (PL) and cholesteryl ester (CE) at 35 weeks gestation (g/100g). 67 8.12 Relations between trans fatty acids and selected cis n-6 and n-3 fatty acids in maternal plasma at 35 weeks gestation and newborn cord plasma at birth 69 8.13 Fatty acid composition in cord plasma triglyceride (TG), phospholipid (PL) and cholesteryl ester (CE) of term gestation infants at birth (g/100g). . 77 v u i LIST OF TABLES 8.14 Relations between trans fatty acids, cis n-6 and n-3 fatty acids in maternal plasma at 35 weeks gestation and cord plasma lipids at birth 81 8.15 Relations between trans fatty acid (TFA) intake at 28 and 35 weeks gestation and trans fatty acids in maternal plasma at 35 weeks gestation and cord plasma at birth 86 8.16 Relations between selected fatty acids in cord plasma triglyceride at birth and birth outcomes 88 8.17 Relations between selected fatty acids in cord plasma phospholipid at birth and birth outcomes 94 8.18 Relations between selected fatty acids in cord plasma cholesteryl ester at birth and birth outcomes 99 i x LIST OF FIGURES LIST OF FIGURES 2.1 Saturated, monounsaturated and polyunsaturated fatty acids 5 2.2 Trans and cis bonds 5 8.1 Scatter plots of estimated dietary trans fatty acids and total fat intakes (g/d) of women at 28 and 35 weeks gestation, n=57 54 8.2 Scatter plots of estimated dietary trans fatty acid and total fat intakes as percent total energy (%en) of women at 28 and 35 weeks gestation, n=57 54 8.3 Estimated contribution of trans fatty acids by food groups to the total trans fatty acid intake of women in their second trimester, n=60 59 8.4 Estimated contribution of trans fatty acids by food groups to the total trans fatty acid intake of women in their third trimester, n=58 60 8.5 Top five individual foods classified by food groups that contributed to trans fatty acid intake of women at 28 weeks gestation by rank order, n=60 . . 62 8.6 Top five individual foods classified by food groups that contributed to trans fatty acid intake of women at 28 weeks gestation by rank order, n=60 . . 63 8.7 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in maternal plasma triglyceride at 35 weeks gestation, n=58 70 8.8 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in maternal plasma phospholipid at 35 weeks gestation, n=58 71 8.9 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in maternal plasma cholesteryl ester at 35 weeks gestation, n=58 72 LIST OF FIGURES 8.10 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in cord plasma triglyceride at birth, n=70 78 8.11 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in cord plasma phospholipid at birth, n=70 79 8.12 Scatter plots of selected cis n-6, n-3 fatty acids and trans fatty acids in cord plasma cholesteryl ester at birth, n=70 80 8.13 Scatter plots of selected fatty acids in triglyceride in maternal plasma at 35 weeks gestation and cord plasma at birth, n=46 82 8.14 Scatter plots of selected fatty acids in phospholipid in maternal plasma at 35 weeks gestation and cord plasma at birth, n=46 83 8.15 Scatter plots of selected fatty acids in cholesteryl ester in maternal plasma at 35 weeks gestation and cord plasma at birth, n=46 84 8.16 Scatter plots of length of gestation and selected fatty acids in cord plasma triglyceride at birth, n=70 89 8.17 Scatter plots of infant birthweight and selected fatty acids in cord plasma triglyceride at birth, n=70 90 8.18 Scatter plots of infant length and selected fatty acids in cord plasma triglyceride at birth, n=44 91 8.19 Scatter plots of infant head circumference and selected fatty acids in cord plasma triglyceride at birth, n=45 92 8.20 Scatter plots of length of gestation and selected fatty acids in cord plasma phospholipid at birth, n=70 95 xi; LIST OF FIGURES 8.21 Scatter plots of infant birthweight and selected fatty acids in cord plasma phospholipid at birth, n=70 96 8.22 Scatter plots of infant length and selected fatty acids in cord plasma phospholipid at birth, n=44 97 8.23 Scatter plots of infant head circumference and selected fatty acids in cord plasma phospholipid at birth, n=45 98 8.24 Scatter plots of length of gestation and selected fatty acids in cord plasma cholesteryl ester at birth, n=70 100 8.25 Scatter plots of infant birthweight and selected fatty acids in cord plasma cholesteryl ester at birth, n=70 101 8.26 Scatter plots of infant length and selected fatty acids in cord plasma cholesteryl ester at birth, n=44 102 8.27 Scatter plots of infant head circumference and selected fatty acids in cord plasma cholesteryl ester, n=45 103 8.28 Scatter plots of birth outcome measures and conjugated linoleic acid (CLA) in cord plasma at birth 104 xii LIST OF APPENDICES APPENDICES APPENDIX 1 Informed consent: Part One 137 APPENDIX 2 Research study questionnaire: Part One 140 APPENDIX 3 Recruitment letter: Part Two 143 APPENDIX 4 Informed consent: Part Two 145 APPENDIX 5 Socio-demographic and health questionnaire: Part Two . 149 APPENDIX 6 Food frequency questionnaire: Part Two 155 xifi A C K N O W L E D G E M E N T S ACKNOWLEDGEMENTS I would like to express my gratitude to the following persons who have helped me over the past two and half years. Special thanks to Dr. Sheila Innis for allowing me the opportunity to work with her and for her guidance and advice along the way. To Roger, Janette and Carolanne who guided me patiently, through the process of fatty acid analysis. To Lorraina, Carolanne, Vikki, Patty and Angela, thanks for your support, and friendship and a special thank you to Tim for your patience, love and support. I would also like to thank NSERC for their financial support and the women who participated in the study. xiv INTRODUCTION 1 Introduction Trans fatty acids (TFA) are formed during the process of hydrogenating vegetable oils which converts naturally occurring cis fatty acid isomers into a variety of trans fatty acid isomers, as well as cis positional isomers and saturated fatty acids. The hydrogenation process improves the oxidative stability and shelf life of unsaturated vegetable oils. Hydrogenated and partially hydrogenated oils are used in margarines, shortenings, snack items and in a variety of baked and prepared foods made with these fats. Dietary trans fatty acids, however, have been shown to have adverse effects on certain risk factors for cardiovascular health, which include elevated low-density lipoproteins (LDL), decreased high-density lipoproteins (HDL) (Mensink 1990, Zock 1992), and elevated lipoprotein (a) (Lp (a)) (Aro 1997, Nestel 1992, Sundram 1997). It has also been suggested that dietary TFA may have adverse effects on fetal growth and development (Carlson 1997). Findings from studies using animals and human fibroblasts have suggested that TFA inhibit n-6 and n-3 fatty acid metabolism, specifically the desaturation of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) to arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), respectively (Zevenbergen 1988, Rosenthal 1984). Arachidonic acid (20:4n-6) is an important fatty acid in cell membranes, in cell signaling and as a precursor for synthesis of eicosanoids. Docosahexaenoic acid (22:6n-3) is also found in high levels in cell membranes, particularly the non-myelin membranes of the central nervous system, including the brain (Innis 1991). Both 20:4n-6 and 22:6n-3 are, therefore, important in fetal growth. At the present time there is a paucity of information on T F A and fetal development. Some evidence to show placental transfer of TFA has, however, been published. For example, Koletzko and Muller (1990) reported that levels of TFA in term 1 INTRODUCTION infant cord and maternal blood were similar, suggesting transfer of TFA. Other studies have also reported similar levels of TFA in maternal and fetal plasma, supporting the hypothesis that TFA are transported from the mother to the fetus (Ayyagari 1996, Berghaus 1998, Billeaud 1998). Thus, if TFA have an inhibitory effect on n-6 and n-3 fatty acid metabolism, it seems possible that maternal dietary TFA, and subsequent fetal TFA exposure, may adversely affect the growth and development of the fetus. The amount of'dietary TFA required to decrease plasma 20:4n-6 and 22:6n-3 to levels likely to have adverse effects on growth and development may be much greater than dietary TFA intakes. To date, however, no studies have quantified maternal dietary TFA intake or looked at the relations between maternal dietary TFA intake and fetal plasma levels of TFA. This study determined the level of TFA, n-6 and n-3 fatty acids in cord blood in relation to maternal TFA intake and maternal blood levels of T F A during gestation. 2 LITERATURE REVIEW 2 LITERATURE REVIEW 2.1 Definition and Classification of Fatty Acids Fatty acids are carbon chains with a carboxyl group (COOH) at one end and a methyl group (CH 3) at the other end. The number of carbons in a fatty acid can range from 4 to up to 30 carbons or more. When only single bonds are present between adjacent carbons, the fatty acid is saturated. When one or more double bonds are present between two adjacent carbons, the fatty acid is unsaturated. A monounsaturated fatty acid contains one double bond in the carbon chain, whereas a polyunsaturated fatty acid contains more than one double bond in the carbon chain (Figure 2.1). Fatty acids are commonly referred to by the number of carbons present in the carbon chain, followed by a colon and a number indicating the number of unsaturated bonds. A saturated fatty acid, therefore, will have the number of carbons present in the carbon chain followed by a colon and a '0' indicating no unsaturated bonds. In unsaturated fatty acids, the number of unsaturated bonds is followed by an V or 'n' and a number to indicate at which carbon from the methyl end the first unsaturated bond is positioned. For example 18:2n-6 would indicate 18 carbons in the fatty acid with 2 double bonds, of which the first double bond would be 6 carbons from the methyl end of the fatty acids. The position of the double bond in an unsaturated fatty acid may also be indicated by the number of carbons away from the delta (A) end, also known as the carboxyl end of the fatty acid. For example A918:1 would indicate a fatty acid with 18 carbons and one double bond 9 carbons from the carboxyl or A end. If there is more than one unsaturated bond in the carbon chain, the unsaturated bonds are separated by a methyl group. Methyl groups, however, do not separate unsaturated bonds of 3 ; LITERATURE REVIEW conjugated linoleic acid (CLA); rather the double bonds are adjacent to each other in the carbon chain (Figure 2.1). When the hydrogen atoms of a double bond are on the same side of the double bond, the bond is referred to as a 'cis' bond. When the hydrogen atoms are on opposite sides of the double bond, the bond is referred to as a 'trans' bond (Figure 2.2). For example, 18:2n-6, an 18 carbon fatty acid with 2 double bonds may have one double bond in the cis position and the second double bond in the trans position. These would be referred to as geometric isomers of 18:2n-6. Positional isomers of unsaturated fatty acids also occur, in which the double bond shifts from the original carbon position to another carbon in the fatty acid. Trans fatty acids are fatty acids that contain one or more trans bonds and may be geometrical or positional isomers of unsaturated fatty acids. 4 LITERATURE REVIEW Figure 2.1 Saturated, monounsaturated and polyunsaturated fatty acids Saturated fatty acid C H 3 - CH2 (16)— COOH Stearic acid, 18:0 Monounsaturated fatty acid CH 3 - CH 2 ( 7 ) - CH = CH - CH 2 ( 7 ) - COOH Oleic acid (18:1n-9 or A918:1) Polyunsaturated fatty acid CH 3 - CH 2 ( 4 ) - CH = CH - CH 2 - CH = CH - CH 2 ( 7 ) - COOH Linoleic acid (18:2n-6 or A 9' 1 218:2) CH 3 - CH 2 (5)- CH = CH - CH = CH - CH 2 ( 7 ) - COOH Conjugated linoleic acid (18:2n-7 or A 9 , 1 118:2) Figure 2.2 Trans and cis bonds H 1 H H 1 1 1 R - C = C - R 1 1 1 R - C = C - R 1 H Trans bond Cis Bond 5 LITERATURE REVIEW 2.2 Formation of Trans Fatty Acids The major dietary source of TFA is hydrogenated and partially hydrogenated vegetable oils. Hydrogenated and partially hydrogenated oils are formed when a vegetable oil is mixed with hydrogen atoms under high temperature and pressure in the presence of a catalyst such as nickel. The hydrogen atoms react with the fatty acids at the cis bonds and with complete hydrogenation cis unsaturated bonds are changed to saturated bonds (Stender 1995). The initial binding of the catalyst to the double bond is not generally stable and may result in the formation of trans double bonds. As the hydrogenation process continues, the fatty acid profile of the vegetable oil changes. Once hydrogenated, vegetable oils that initially contained 18:1, 18:2n-6 and 18:3n-3 will have significant levels of trans 18:1, 18:2 and 18:3 isomers with lower levels of cis 18:2n-6 and 18:3n-3 than the original oil. Trans 18:1 (elaidic acid) and trans 18:2 are usually the major TFA formed during hydrogenation (Stender 1995). A variety of trans 18:1 and 18:2 isomers may be formed including positional isomers of trans 18:1 and 18:2 and geometrical isomers such as cis, trans 18:2, trans, cis 18:2 or trans, trans 18:2 isomers. The formation of the trans double bond results in a linear and more rigid carbon chain that is energetically more favorable and less susceptible to oxidative degradation than its cis configuration (Stender 1995, Welch 1992). In addition, the melting point of TFA is lower than for cis fatty acids, but lower than for saturated fatty acids of equal carbon length. For example, the melting points of 18:0 (stearic acid), 18:1n-9 (oleic acid) and trans 18:1n-9 (elaidic acid) are 70°C, 44°C and 13°C, respectively. Because of the higher oxidative and thermal stability, food manufacturers use hydrogenated oils 6 LITERATURE REVIEW rather than cis polyunsaturated and monounsaturated oil as the major oil source in a variety of baked and prepared foods. 2.3 Dietary Sources of Trans Fatty Acids An assessment of TFA availability in the United States (U.S.), reported that significant sources of TFA in the diet are margarines, spreads, food service and industrial fats and oils (Hunter 1991). Food service fats and oils were described by Hunter and Applewhite (1991) as oils used in deep-frying fish, chicken and french fries, while industrial fats and oils are those used in the production of salted snacks, cookies, crackers and par-fried French fries. The level of TFA in foods in the Canadian market, i.e. salted snacks, cookies and crackers are similar to the levels reported for similar foods in the U.S. (Carlson 1997, Ratnayake 1991). According to Hunter and Applewhite (1991), many margarines and spreads have been modified in the last decade to contain lower levels of TFA. These products, however, remain a significant dietary source of TFA. The mean total content of TFA in stick and tub margarine in the U.S. in the early 1990's was estimated at 27% and 17.3% of their total fat, respectively, (Carlson 1997) while in the same time period Canadian stick and tub margarine contained 34.2% and 20.4%, respectively (Ratnayake 1991). In addition to hydrogenated fats and oils, meats, dairy products and to a lesser extent, certain plants also contribute TFA to the diet. Bacterial hydrogenation of fatty acids in the rumen of ruminants (cattle, goat and sheep), leads to the presence of TFA in both the animal body fat and their milk (Keweloh 1996). The content of T F A in the body fat of ruminants ranges from 4-11 % of the total fatty acids. The TFA level in milk fat is dependent on the animal's diet and can range from 2% to 9% of total fatty acids, 7 LITERATURE REVIEW (Keweloh 1996). In contrast to hydrogenated oils and shortenings in which the major trans isomer is elaidic acid (trans 18:1n-9) (Etherton 1995) the major trans isomer present in milk, butter, beef fat is vaccenic acid (trans 18:1 n-11) (Sommerfeld 1983). Trans fatty acids are not generally found in plants. Small amounts of TFA, however, have been detected in some plant seed oils as well as in plant leaves. Six species of plants have been identified as sources of TFA, including pomegranates, peas, cabbage, lettuce, leeks and spinach. The most common trans isomer in plants is 3-trans hexadecaenoic acid (16:1,3t) (Sommerfeld 1983). Human milk can also be a source of TFA, depending on the mother's diet. A case-control study using eight lactating women demonstrated that as hydrogenated fat increased in the diet, the level of TFA, specifically isomers of 18:1 and 18:2, increased in breast milk (Craig-Schmidt 1984). Chen et al (1995) measured the TFA levels in 198 Canadian human milk samples. The major trans isomer present in these Canadian human milk samples was 18:1 with a mean of 5.9 (range 0.1-15.4%) of total fatty acids. The mean total TFA of human milk samples reported by Chen et al (1995) was 7.2% (range 0.1-17.2%) of total fatty acids. Studies in Spain have reported mean TFA level in human breast milk of 1.23% total fatty acids (Boatella 1993). This is probably reflective of a low mean dietary TFA intake in Spain of 2.4 g/person/day, as reported in 1988 by the United Kingdom Nutritional Consultative Panel (San Juan 1996) than in Canada. Canadian data on TFA intake, however, has not been published. 2.4 Variation of Trans Fatty Acid Levels in Food Sources There is considerable variability in the TFA content of foods containing hydrogenated and partially hydrogenated fats or oils. The variability of TFA in foods is 8 LITERATURE REVIEW evident not only between different foods such as crackers and cookies, but also within similar foods, such as two types of cookies. Enig et al (1984) reported a wide variation of TFA within a variety of similar foods in the United States. The average amount of TFA, as a percentage of total fatty acids in the food ranged from 0.2 to 23.6% in breads and rolls, 8.1 to 32.7% in breading mixes and fried crusts, 1.9 to 29.0% in crackers, from trace amounts to 35.1% in french-fried potatoes, and from trace amounts to 32.1% in pastries and pastry crusts. Ratnayake et al (1991) and Ratnayake et al (1993) also reported a wide variation within food categories, such as cereals (9.2-33.7%), cookies (7.6-38.7%), crackers (13.8-35.4%), muffins (16.5-24.2%), print margarines (26-50%) and tub margarines (0-36%) in Canada. The amount of TFA in a variety of foods purchased in Vancouver, British Columbia, Canada has also been analyzed. Foods analyzed included bread (whole wheat and white), breaded chicken, breakfast cereals, cookies, muffins, pie shells and tub margarines. The average amount of TFA as a percentage of total fatty acids within these food categories, as found by others (Enig 1984), also varied widely. The percentage of TFA ranged from; in breads 1.0-36.3%, breaded chicken 11.9-56.7%, breakfast cereals 0.2-24.3%, cookies 1.4-45.7%, muffins 1.7-36.2%, pie shells 1.9-45.6% and tub margarines 1.1-44.4% (Innis 1999). There are several explanations for the variability in the trans fatty acid content of foods containing hydrogenated oils. First, the production of hydrogenated oils can result in a variable content of trans fatty acids. Temperature, gas pressure, type and amount of catalyst, and agitation may all affect the resulting trans fatty acid content of the oil. Second, food producers may use single hydrogenated, partially hydrogenated or non-hydrogenated fats or oils, or many possible combinations of both hydrogenated, partially hydrogenated and non-hydrogenated fats and oils to achieve the desired final product 9 LITERATURE REVIEW characteristics. Finally, the use of hydrogenated and non-hydrogenated fats and oils in food products can also be expected to vary with national and regional domestic availability, and costs of various edible oils. 2.5 Estimated Consumption of Trans Fatty Acids Several methods have been employed to estimate the TFA intake in the U. S. and Europe (ASCN/AIN Task Force on Trans Fatty Acid, 1996). No data have been published to describe the TFA intake of Canadians. Due to the widespread cultural and ethnic diversity in the U.S. and Canada and the apparent similarities in the TFA content in the food supply, it is reasonable to consider that TFA intakes in Canada and the U.S. may be similar. The amounts of TFA in the U.S. diet has been estimated using several methods, including food disappearance data, food availability data, diet records, food frequency questionnaires (FFQ), and adipose tissue fatty acid analysis (ASCN/AIN Task Force on Trans Fatty Acid, 1996). Estimations of dietary TFA intakes obtained from food disappearance and availability data have given the highest values for TFA intakes, with estimates ranging from 8.1 -12.8 g/day/capita. This represented approximately 10% of total fat intake, and 2-4% of total energy intake (Enig 1990, Hunter 1991). Several problems exist when using food disappearance and availability data to estimate dietary intakes that may result in an over-estimation of actual per capita TFA intake. These problems include foods used but not consumed, for example oil discarded post-frying, restaurant, catering and home food wastage, and the wide variation in dietary intakes among individuals (Carlson 1997). Estimates of TFA intakes based on diet records and FFQ tend to give lower estimates than estimates based on food disappearance and 10 LITERATURE REVIEW availability data. Estimates of TFA intake based on diet records and FFQ, however, seem to be the most consistent among studies. For example, mean estimates of TFA intakes using diet records or FFQ have ranged from 2.6 - 5.3 g/person/d, representing a mean 5-7% of total fat intake (Allison 1999, London 1991, Troisi 1992, van den Reek 1986). Errors that occur when obtaining estimates of intake from dietary records, however, include problems associated with memory, accuracy of recall or recording and estimating portion sizes (Gibson 1990). These errors may, therefore, result in lower estimates of dietary TFA than actual intakes of TFA. Trans fatty acid intakes have also been estimated from analysis of TFA in adipose tissue fatty acids. The analysis of adipose tissue TFA reflects long-term (months to years) TFA intake (London 1991). Katan et al. (1986) and van Staveren et al (1986) measured TFA levels in subcutaneous fat of Dutch women. The latter studies (Katan 1986, van Staveren 1986), found that the trans 18:1 isomers were the most prevalent trans isomers in adipose tissue, and that the levels of trans 18:1 in adipose tissue correlated with dietary intake of trans 18:1 isomers. Similar findings were also reported for women in the U.S. (London 1991) and for Canadian adults (Chen 1995). The choice of database used to analyze the TFA content of foods can be a considerable source of error in the estimates of TFA intakes among and within different studies. Some databases use TFA values of specific food brands whereas other databases use average values for a food using TFA values from more than one brand. This may result in two distinctly different values for the same food . As reported in Innis and Green (1999) the use of an average TFA value for a food or a food group rather than the actual value for the food eaten, can lead to gross underestimation or overestimation of the true TFA intake. Consequently, it is important to obtain 11 LITERATURE REVIEW information on the actual food brands consumed when assessing an individual's dietary TFA intake. If the TFA content of a food brand has not been determined, an alternative analyzed food with the same amount and type of fat can be used (Innis 1999). Another source of variation in estimating TFA intake is the time in which the dietary data is collected. A significant change in the restaurant and par-frying operations during 1989 and 1990 was predicted to increase the TFA availability to consumers. These operations began to substitute partially hydrogenated vegetable oils for edible tallow in their deep frying methods. This switch in oils was implemented to lower the saturated fatty acid content of deep fried or par-fried foods (Hunter 1991). Data collected and analyzed prior to the late 1980's may, therefore, give lower estimates of TFA intakes than data collected and analyzed following 1989. 2.6 Trans Fatty Acid Absorption and Metabolism The absorption and metabolism of TFA has been reported to be similar to that of cis unsaturated fatty acids. Geometrical isomerization does not appear to alter the hydrolysis of fatty acids from triglycerides via lipoprotein lipase (Emken 1979). Once digested and absorbed, TFA may be oxidized for energy or acylated to triglycerides, phospholipids or cholesteryl esters. Trans fatty acids have been suggested to have an inhibitory effect on the A 6 and A 5-desaturase enzymes. The A 6 desaturase enzyme is responsible for the desaturation of linoleic acid (18:2n-6) to y-linolenic acid (18:3n-6) and a-linolenic acid (18:3n-3) to 18:4n-3. A A 6 desaturation is also involved in the conversion of 24:4n-6 to 24:5n-6, and of 24:5n-3 to 24:6n-3. It has been suggested that the enzyme responsible for this latter desaturation may be different than the A 6 desaturase responsible for the conversion of 18:2n-6 to 18:3n-6 and of 18:3n-3 to 12 LITERATURE REVIEW 18:4n-3 (Sprecher 1995). The A 5 desaturase enzyme is responsible for the desaturation of 20:3n-6 to arachidonic acid (20:4n-6), and the desaturation of 20:4n-3 to eicosapentaenoic acid (20:5n-3). Cook and Emken (1990) and Rosenthal and Doloresco (1984) reported that trans isomers were potent inhibitors of A 5-desaturation activity in glioma cells and human fetal skin fibroblasts, respectively. Rosenthal and Doloresco (1984) incubated radioactively labeled 20:3n-6 with trans isomers of 16:1, 18:1, 18:2 or 20:1 in media containing normal human skin fibroblasts. The cells were harvested at 1, 2, 4, 6, 24 and 48 hours post-incubation. The amount of radioactively labeled 20:3n-6 incorporated into cellular lipids and the amount of 20:3n-6 desaturated to 20:4n-6 was then determined. The lowest amounts of radiolabeled 20:4n-6 was present in the media containing trans 18:1 isomers, followed by the media containing trans 18:2 isomers. A dose-dependent inverse relation was seen between the levels of trans 18:1 isomers and the amount of 20:4n-6 present in the media. At levels of trans 18:1 isomers (0.6 uM), of one-fourth the concentration of 20:3n-6, significantly decreased levels of 20:4n-6 were present in the media when compared to cell incubated without trans 18:1 isomers. The effect of trans 18:2 isomers on synthesis of 20:4n-6 was minimal at concentrations up to ten times that of 20:3n-6. Rosenthal and Doloresco (1984), thus concluded that trans 18:1 isomers were more effective at inhibiting the A 5-desaturase enzyme than trans 18:2 isomers. In a similar study by Cook and Emken (1990) glioma cells were incubated with radiolabeled 18:2n-6 and 18:3n-3 and trans isomers of 18:1 or 18:2 at 40-100 uM concentrations. Cell cultures containing 18:2n-6 and 18:3n-3 were harvested after 4 and 2 hours incubation, respectively. The amount of 18:2n-6 and 18:3n-3 incorporated 13 LITERATURE REVIEW into cell tissues and the products of desaturation and chain elongation, 20:3n-6, 20:4n-6 and 20:5n-3 were then determined. The glioma cells rapidly incorporated 18:2n-6 and 18:3n-3 into tissue phospholipids and triglycerides. Incubation with trans 18:1 isomers at 40 jiM, decreased the percent 20:4n-6 formed from 18:2n-6 by 60% relative to the cells incubated with 18:2n-6 without trans 18:1 isomers. When glioma cells were incubated with trans 18:2 isomers, the formation of 20:4n-6, but not that of 20:3n-6, from 18:2n-6 was decreased, suggesting that trans 18:2 inhibits the A 5, but not the A 6-desaturase. Inclusion of trans 18:1 or 18:2 isomers in the media, however, had no effect on the metabolism of 18:3n-3. The results of this study suggest that trans isomers of 18:1 and 18:2 at concentrations of 40 uM have an inhibitory effect on the A 5 desaturation of 18:2n-6. The concentrations of 18:2n-6 and 18:3n-3, however, were not reported in the studies by Cook and Emken (1990). Thus, consideration of the relative amounts of 18:2n-6 and 18:3n-3, with respect to the effects TFA have on n-6 and n-3 fatty acid desaturation is not possible. Extrapolation of the results of in vitro studies to the multi-organ system, or whole body, is difficult. For example, in vivo, a variety, not individual fatty acids are present, together with a variety of hormonal and dietary factors which can influence A 5 or A 6 desaturation. Further, intracellular concentrations of unesterified fatty acids for desaturation in the endoplasmic reticulum are not yet clear. If inhibition of A 5 or A 6 desaturase occurs in the presence of TFA, the extent of inhibition is likely to be determined in part to the relative concentrations of TFA and of the substrates 18:2n-6 and 18:3n-3. Further, the inhibitory effect of the desaturation of 18:2n-6, but not of 18:3n-3, suggests that 18:2n-6 competes for the desaturase enzyme over 18:3n-3. If inhibition of the desaturase enzyme occurs in the presence of TFA, lower levels of both 14 LITERATURE REVIEW 20:4n-6 and 22:6n-3 would be expected. The effect of inhibiting the desaturation of 18:2n-6 and 18:3n-3 to 20:4n-6 and 22:6n-3, respectively, to alter physiological function, however, is unknown. Several in vivo studies (Anderson 1975, Zevenbergen 1988) have examined the effect of varying dietary levels of TFA and 18:2n-6 on levels of 20:3n-6 and 20:4n-6 in rat liver. Anderson et al (1975) fed rats a diet containing 20% energy from fat with trans 18:2 isomers at levels of 1.2, 2, 5, 10 or 19%, and 18:2n-6 at 3% of total dietary fatty acids for six weeks. Fatty acid analysis was completed on total lipid extracts of liver tissue. The proportion of 20:4n-6 in the liver lipids decreased with increasing trans 18:2 isomers, specifically the 18:2 isomers with two trans bonds. This geometrical isomer, however, is produced in very small amounts in the diet, therefore, any effect, if present in humans is likely to be minimal. Similarly, Zevenbergen et al (1988) fed rats a diet with 40% energy from fat containing 0.4, 0.8, 2, 3, 5, or 7.1 % of energy from 18:2n-6, and 20% of energy from trans 18:1 and 18:2 isomers. The fatty acid composition of liver mitochondrial membrane lipids was measured after 13-14 weeks of dietary treatment. Rats fed 0.4, 0.8 and 2% 18:2n-6 had slightly lower 20:4n-6 levels than the rats fed a TFA-free diet, however, the differences were not statistically significant. No effect of TFA on 20:4n-6 levels was found in rats fed 5 or 7% energy from 18:2n-6. Despite lower levels of 20:4n-6 in the rats fed 2% or less energy from 18:2n-6, mitochondrial function, as measured by oxygen uptake and ATP synthesis, was not different from that of rats fed 5 and 7% energy from 18:2n-6. These findings suggest that trans 18:1 and 18:2 isomers have little effect on mitochondrial function even though trans 18:1 and 18:2 isomers may decrease 20:4n-6 in membrane phospholipids. 15 LITERATURE REVIEW Lawson et al (1983) studied the effect of feeding pregnant rats and their newborn pups with a diet containing approximately 42% energy from total fat with or without TFA. Trans 18:1 in the control diet and the TFA diet accounted for 2.4% and 33.2% of total fatty acids, respectively. The level of 20:4n-6 in the newborn pup liver phospholipid was 35% lower in the group born to rats fed a diet with TFA than pups born rats fed a diet with no TFA. The level of 18:2n-6, 18:3n-3, 18:3n-6 and 20:3n-6, however, were higher in the TFA group compared to the control group. The level of liver phospholipid 20:5n-3, was also higher in the TFA group than in the control group despite equal dietary amounts of 18:3n-3 in both groups. This suggests that rather than inhibiting the A 5 desaturase enzyme T F A favored the desaturation of 18:3n-3 rather than 18:2n-6. In summary, the in vitro studies of Cook and Emken (1990) indicates that the trans isomers of both 18:1 and 18:2 can decrease production of 20:4n-6 from 20:3n-6. Rosenthal and Doloresco (1984), however, reported that trans 18:1, but not trans 18:2 isomers decreased synthesis of 20:4n-6 from18:2n-6 by cultured human fibroblasts. Studies with rats have reported that diets containing trans, trans 18:2 result in lower liver total lipid 20:4n-6 (Anderson 1975). Similarly, others have found lower liver phospholipid 20:4n-6 in rats fed diets containing a mixture of trans 18:1 and 18:2 (Zevenbergen 1988). The relevance of the findings by Anderson et al (1975), Cook and Emken (1990) and Zevenbergen et al (1988) to humans, is unclear. 2.7 Fatty Ac ids and Growth and Development Arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) are important fatty acids with many functions. Arachidonic acid is required as an integral component of cell membranes and is involved in cell signaling, a mechanism that stimulates the 16 LITERATURE REVIEW production of certain proteins. In addition to cell membrane functions, 20:4n-6 is also important as a precursor to eicosanoids, a group of compounds that have a variety of functions, for example in inflammation, immune response and blood clotting. Docosahexaenoic acid, like 20:4n-6, is also important as a component of cell membranes, particularly within parts of the retina and the brain (Innis 1991). Because of the importance of these fatty acids in cell structure and function, it would be expected that an increase in both 20:4n-6 and 22:6n-3 would be present in situations of growth, such as during gestation. Several studies have examined the relations between 20:4n-6 and 22:6n-3 in newborns and birth outcomes, such as length of gestation and birthweight. Foreman van Drongelen et al (1995) reported a positive relation between arterial cord vessel wall 20:4n-6 and length of gestation in pre-term infants. Similarly, Koletzko and Braun (1991) and Leaf et al (1992) reported a positive relation between birthweight and umbilical cord plasma 20:4n-6 in pre-term infants. Leaf et al (1992) also reported an association between 22:6n-3 and birthweight, head circumference and length of gestation in preterm infants. Woltil et al (1998) suggested that cord red blood cell 20:4n-6 was representative of in utero growth, rather than postnatal growth because lower levels of 20 :4n-6 was present in term and small for gestational age infants than in pre-term infants. This, however, may suggest that certain fatty acids, such as 20:4n-6, are transferred at higher amounts by the placenta at certain periods within gestation rather than continuously throughout gestation. For example, 20:4n-6 is a constituent of cell membranes and, therefore, important during phases of growth when cell membranes are being made. 17 LITERATURE REVIEW Several authors have reported a higher percentage of 20:4n-6 and 22:6n-3 in cord plasma than in maternal plasma (Al 1995, Otto 1997) suggesting that these fatty acids are either transported to or produced by the fetus. Although A 5 and A 6 desaturase activity has been detected in human fetal liver tissue (Rodriguez 1998), the relative significance of these enzymes to 20:4n-6 and 22:6n-3 produced in the fetus is not known. The fetus may, however, obtain 20:4n-6 and 22:6n-3 from the mother and therefore desaturase activity in the fetal liver may not contribute significantly to levels of 20:4-6 and 22:6n-3 in the fetus. In addition to the importance of 20:4n-6 and 22:6n-3 in the developing fetus, 20:4n-6 and 20:5n-3, the precursor to 22:6n-3 are precursors to biologically active compounds called eicosanoids. The eicosanoids produced by 20:4n-6 are involved in a variety of functions such as vasoconstriction, platelet aggregation and more recently have been suggested to have a role as a promoter of parturition (Olsen, 1991). In contrast, the eicosanoids produced by 20:5n-3 are less involved in vasoconstriction and platelet aggregation, therefore, causing diminished platelet aggregation and prolonged bleeding. A study was conducted on women from the Faroese Islands because they tended to have longer pregnancies and infants with higher birthweights than women in Denmark. Olsen et al (1991) suggested that the higher birthweights and longer lengths of gestation in the Faroese women were due to higher intakes of marine fats such as whale and fish. Marine fats are rich sources of n-3 fatty acids, such as 20:5n-3 and 22:6n-3. It was speculated that n-3 fatty acids would decrease the production of the prostaglandins that are involved in parturition, specifically the prostaglandins produced by 20:4n-6. The shift in the balance of prostaglandins produced would thereby prolong gestation and subsequently increase birthweight. Olsen et al (1992) reported that 18 LITERATURE REVIEW Danish women randomized to a fish oil supplement had a mean gestation of 283 days and infant birthweight of 3571 g, while women who received an olive oil supplement had a slightly lower mean gestation of 279 days and infant birthweight of 3445 g. The women who received a placebo had a mean gestation of 282 days and infant birthweight of 3504 g. Although there was a significant difference in mean duration of gestation between the fish oil and olive group, the infants born to women in both treatment groups and the control group were born within the parameters of a term pregnancy (38-42 weeks). An important observation, however, was that 12% of women receiving fish oil (n=32), 8% of women receiving olive oil (n=11) and 12% of women in the control group (n=16) had a duration of gestation longer than term (>42 weeks). A longer duration of gestation provides more time for the fetus to grow, but is also associated with decreased amniotic fluid levels and increased complications for delivery. Therefore, a duration of gestation greater than term (>42 weeks) does not necessarily benefit the fetus or mother and may potentially create more problems than benefits. Inhibition of the A 5 and A 6 desaturation of 18:2n-6 and 18:3n-3 by TFA can be expected to result in an inverse relation between the levels of 20:4n-6, 20:5n-3 and 22:6n-3 and the levels of TFA in the newborn. This relation, however, assumes that the mother's diet is devoid of dietary sources of 20:4n-6 and 22:6n-3 such as meat, eggs and fish and that a similar inhibition of 20:4n-6 and 22:6n-3 synthesis occurs in the mother. Several investigators have examined the relations between TFA and 20:4n-6 and 22:6n-3 in newborns and children aged 1 -15 years (Decsi 1995, Koletzko 1992). The percent TFA in cholesteryl ester (CE) but not phospholipid (PL) showed an inverse correlation with the percent 20:4n-6 (r = -0.38, p<0.05) and 22:6n-3 (r = -0.53, p<0.01) 19 LITERATURE REVIEW in 29 newborn premature infants (Koletzko 1992). In contrast, Decsi and Koletzko (1995), reported a significant inverse relation between trans 18:1 isomers and 20:4n-6, and between total TFA and 20:4n-6 in plasma PL but not C E of 53 healthy children (Desci 1995). Koletzko (1992) reported an inverse relation between birthweight and trans 18:1 isomers in plasma C E (r = -0.40, p<0.05) and plasma PL (r = -0.42, p<0.01) but not in plasma triglyceride (TG) of pre-mature infants. No statistically significant relations between the length of gestation and plasma TFA, however, have been reported in the literature. The statistical relation between TFA in plasma PL and C E and birthweight, however, can not be interpreted as evidence of a cause and effect relation. The absence of an inverse relation between TFA and 20:4n-6 in plasma PL (Koletzko 1992) provides no support for a hypothesis that TFA inhibits the synthesis of 20:4n-6. No studies on the possible associations between TFA in plasma TG, PL or C E and birthweight have been reported on term infants. It should be noted that data reported on the relations between TFA and 20:4n-6 and 22:6n-3 are inconsistent and have been conducted primarily by one group of researchers in Germany. To establish whether the significant findings reported by Koletzko (1992), and Desci (1995) can be generalized to other groups of individuals, similar studies need to be conducted in other regions of the world. 2.8 Health Concerns About Trans Fatty Acids 2.8.1 Cardiovascular Disease It has been suggested, based on epidemiological studies, that the development of certain diseases, including heart disease, may begin in utero (Barker 1998). Plasma cholesterol levels are typically low in the human fetus, but levels rapidly increase 20 LITERATURE REVIEW following birth (Van Biervleit 1986). It is not known whether T F A in the fetus plasma affect cholesterol levels in the fetus or following birth. In adults, elevated low-density lipoprotein cholesterol (LDL-C) is considered a risk factor for heart disease (Etherton 1995). Dietary TFA intakes have been positively correlated with plasma LDL-C, leading to the suggestion that dietary TFA may be associated with an increased risk of cardiovascular disease (Katan 1995). Epidemiological studies have identified a positive relation between hydrogenated fat intake and incidence of cardiovascular disease (Booyens 1988, Kromhout 1995). Although a relation between dietary TFA intake and incidence in heart disease has been reported (Booyens 1988, Kromhout 1995), the results of several case-control studies (Aro 1995, Bolton-Smith 1996, Roberts 1995) have found no relations between TFA intake and cardiovascular disease. Roberts et al (1995) found no relation between 18:1 or 18:2 trans isomers in adipose tissue and the incidence of sudden cardiac death. Similarly Aro et al (1995) found no relation between plasma trans 18:1 isomers and the incidence of acute myocardial infarction in European men. Bolton-Smith et al (1996) also reported no relation of TFA intake with diagnosed heart disease despite an inverse relation between TFA intake and high-density lipoprotein cholesterol (HDL-C). The relations between TFA intake and incidence of cardiovascular disease may be due in part to the effect TFA have on plasma cholesterol levels, specifically on increasing LDL-C and lowering HDL-C, and possibly through raising Lp(a). A number of studies have reported that replacing dietary cis unsaturated fatty acids with TFA, either as trans 18:1 n-9 (elaidic acid), or as a variety of trans isomers resulted in increased plasma total cholesterol (TC) and LDL-C (Judd 1994, Laine 1982, Lichtenstein 1993, Zock 1992). Several investigators have reported that TFA lowered HDL-C in addition to 21 LITERATURE REVIEW elevating T C and LDL-C in comparison to dietary cis unsaturated fatty acids (Mensink 1990, Zock 1992). Others have reported that when saturated fat is replaced with TFA, there was a decrease in HDL-C with no effect on LDL-C (Aro 1997) or an increase in LDL-C (Sundram 1997). The mechanism by which TFA alters cholesterol profile, specifically HDL-C may involve cholesteryl ester transfer protein (CETP), a protein that transfers cholesterol from HDL-C to LDL-C and very low-density cholesterol (VLDL-C). Khosla et al (1997) demonstrated that the C E T P activity of 11 normo-cholesterolemic cebus monkeys fed a diet rich in elaidic acid (frans-18:1 n-9) was higher than the C E T P activity of the monkeys fed a diet rich in saturated fatty acids. Similarly, an increase in C E T P activity was seen in humans who were fed an elaidic-rich diet (Abbey and Nestel 1994). Aro et al (1997), however, reported no significant effect of dietary TFA compared to dietary saturated fat on C E T P activity in a group of 80 healthy adults. In the study by Aro et al (1997) TFA contributed 8.7% of energy whereas Abbey and Nestel (1994) used 7% of energy as trans 18:1 n-9. Possibly, an increase in CETP activity may be more likely to occur with a diet containing trans 18.1 n-9, rather than a mixture of trans isomers. Another possible mechanism to explain the effect TFA have on HDL-C is an alteration in lecithin: cholesterol acyltransferase (LCAT) activity. The LCAT enzyme catalyzes the transfer of a fatty acid from the 2 position of phosphatidylcholine to free cholesterol to form cholesteryl ester (CE). The LCAT enzyme is able to bind to HDL particles and after activation by apo-A-l, its action increases the amount of C E in HDL-C. The addition of LCAT, isolated from human or rat plasma, to substrates containing trans isomers resulted in a 30-50% lower LCAT activity when compared to the LCAT activity with cis unsaturated fatty acid isomers (Subbaiah 1998). This suggests that 22 LITERATURE REVIEW LCAT prefers cis rather than trans fatty acid isomers as a substrate, however, levels of TFA in plasma PL are low with dietary TFA appearing in plasma T G . There are no published data oh the levels of TFA in the 2-position of plasma as HDL phosphatidylcholine, i.e. the LCAT substrate. Thus, interpretation of the results of the in vitro studies of Subbaiah et al (1998) with respect to the effects of dietary TFA on plasma cholesterol is difficult. In summary, the available data does not allow a conclusion on the mechanisms involved in altering plasma lipoprotein cholesterol levels when TFA-rich diets are consumed. More recently, interest has been focused on the possible effect of T F A on Lp(a), an LDL-C particle linked to apoprotein(a). Lp(a) has been found to be a strong risk factor for developing cardiovascular disease (Mackinnon 1997). It should be noted that Lp(a) has been described as a genetically determined risk factor for atherosclerotic vascular disease that remains constant in any given individual, irrespective of diet and exercise (Mackinnon 1997). Sundram et al (1997) recently reported elevated serum Lp(a) levels when a diet with 7% of energy from TFA, of which 39% TFA were trans 18:1 n-9, was fed to a group of Malayasians. Aro et al (1997) and Nestel et al (1992) also reported elevated serum Lp(a) in humans fed TFA either as 8.7% or 7% of total energy, with 22 and 27% of TFA as trans 18:1 n-9, respectively. Clevidence et al (1997), however, reported no effect of TFA, either at 3.8% or 6.6% of energy, on Lp(a) plasma levels. The dietary TFA were again a mixture of trans isomers, but the percent trans 18:1 n-9 was not reported. The findings reported by Sundram et al (1997), Aro et al (1997) and Nestel et al (1992) are the first to show a negative effect of diet, specifically TFA, on Lp(a) levels in humans. 23 LITERATURE REVIEW In summary, trans 18:1 has been the most common trans isomer used when studying the effect of trans isomers on plasma cholesterol levels. Although trans 18:1n-9 is the most prevalent trans isomer in the diet, many other mono and polyunsaturated trans isomers are also present in foods containing partially hydrogenated oils and shortenings. It is possible that plasma lipoprotein cholesterol levels may be affected by only certain trans isomers or perhaps by a certain concentration of one or more individual trans isomers, for example isomers of 18:2. With respect to human studies, this is further compounded by the differences in the amounts and proportion of TFA in partially hydrogenated oils and shortenings produced from different oils and by different manufacturers. For example, partially hydrogenated canola oil which has about 53% 18:1 n-9, 21% 18:2n-6 and 11% 18:3n-3 will have different amounts and isomers of TFA from partially hydrogenated soybean oil which has 23% 18:1 n-9, 54% 18:2n-6 and 8%18:3n-3. 2.8.2 Cancer A link between TFA and cancer has also been postulated (Bakker 1997, Kohlmeier 1997, Petrek 1997) but is not well established. Most studies in this area have explored a possible relation between TFA intake and breast cancer. Petrek et al (1997) measured adipose tissue fatty acids in 161 American women presenting with an early-stage of breast cancer. The level of TFA in adipose tissue was inversely associated with positive lymph node status. In contrast, cis 18:1 n-9 and saturated fatty acids were positively associated with positive lymph node status, an indicator of metastasis. Several studies in Europe have explored the possible relation between TFA and breast cancer in post-menopausal women. In one study, a significant relation 24 LITERATURE REVIEW between TFA in adipose tissue and incidence of cancer of the breast (r = -0.76) was found (Bakker 1997). Kohlmeier et al (1997) measured adipose fatty acids in 209 post-menopausal women in Europe who had been recently diagnosed with breast cancer. The mean levels of trans 18:1 and total TFA were higher in the women with breast cancer, compared with women with no history of breast cancer. Despite the finding of a positive relation between TFA in adipose tissue and breast-cancer, in several epidemiological studies (Bakker 1997, Kohlmeier 1997, Petrek 1997) it can not be concluded that dietary TFA have a direct effect on the cause or progression of cancer. Women with breast cancer may consume high levels of dietary TFA, but whether this has any significance to the development or progression of breast cancer can not be inferred from the results of epidemiological studies. 2.9 Transfer of Fatty Acids to the Fetus The placenta is an organ that has a wide range of functions including the transfer of respiratory gases, fluid, waste products and nutrients to and from the fetus (Page 1993). Fatty acids are able to enter the placenta as free fatty acids via the placental lipoprotein lipase and the fatty acid-binding protein present in the maternal facing membranes of the placenta (Campbell 1995a, Campbell 1995b, Coleman 1989). The placental lipoprotein lipase (LPL) is responsible for the hydrolysis of maternal plasma triglycerides releasing free fatty acids and monoglycerides. The free fatty acids are then able to bind to a fatty acid-binding protein for transport into the placenta. The placental LPL activity, however, does not explain how and if fatty acids from maternal plasma PL and C E enter the placenta. Phospholipase A2, an enzyme that hydrolyzes fatty acids from the second carbon of the phospholipid glycerol backbone, has been 25 ; LITERATURE REVIEW detected in human placental tissue (Jendryczko 1989, Jendryczko 1990). Higher levels of phospholipase A2 enzyme have been associated with pre-eclampsia and onset of labour, however, the role of this enzyme in hydrolyzing fatty acids, from phospholipid, for entry into the placenta, is unknown. The human is unable to synthesize certain nutrients such as 18:2n-6 and 18:3n-3, therefore, the fetus is dependent on the mother's diet or tissues for these fatty acids. A longitudinal study examining the n-6 and n-3 fatty acid status of women during normal pregnancy has reported that the percent 18:2n-6 of the fetal cord PL was correlated to the mother's plasma PL 18:2n-6 (r = 0.52, p<0.01) (Al 1995). No relation between maternal and cord plasma PL 18:3n-3, however, was reported. The level of 18:2n-6 in the fetal and maternal PL was approximately 7 and 22% of total fatty acids, respectively. An alteration in maternal dietary fat intake, either during or prior to pregnancy could theoretically modify the fatty acid pool available for transfer to the fetus. For example, if a women started consuming flaxseed oil, which contains about 54% 18:3n-3, several months prior to pregnancy, she would have more 18:3n-3 available for transfer to the fetus than if she had consumed a vegetable oil low in 18:3n-3. Connor et al (1996) supplemented pregnant women with fish oil containing 22:6n-3 and then measured the levels of plasma n-3 fatty acids in both the mother and newborn cord plasma. The percent 22:6n-3 in the cord plasma of the supplemented and control groups were 5.05% and 3.47% of total fatty acids, respectively. The percent 22:6n-3 in the maternal plasma, of the supplement and control groups at birth, were 2.35% and 1.47% of total fatty acids. The results of this study demonstrate that increasing the maternal intake of 22:6n-3 resulted in slightly higher levels of 22:6n-3 in the fetus. It has been suggested that the increased level of 20:4n-6 and 22:6n-3 in the fetus is a 26 LITERATURE REVIEW result of preferential, selective transfer by the placenta (Coleman 1995). This could explain why the level of 20:4n-6 and 22:6n-3 are lower in the maternal plasma than in cord plasma at birth (Hoving 1994, Innis 1991). No mechanisms to support selective transport of fatty acids across the placenta to the fetus, however, have been reported. Other fatty acids, specifically trans fatty acids have been reported in cord plasma at birth (Ayyagari 1996, Koletzko 1990, Koletzko 1992) suggesting that T F A enter the placenta and eventually the fetal circulation via the maternal circulation. Several human studies have found similar levels of TFA in maternal and cord plasma, confirming placental transfer of TFA (Berghaus 1998, Hoving1994, Koletzko 1992). There are no published data concerning possible relations between maternal diet, maternal plasma and cord plasma levels of TFA. The purpose of this study, therefore, was to examine whether there was a relation between the level of TFA in maternal diet, maternal plasma and cord plasma at birth. 27 STUDY OVERVIEW. P U R P O S E AND OBJECTIVES 3 STUDY OVERVIEW This research was conducted as two consecutive studies. Study One was a cross-sectional study using a convenience sample of pregnant women in early stage, normal labor in the Low Risk Labor and Delivery Unit at the BC Women's Hospital. Study Two was a prospective study with a sample of pregnant women from the Lower Mainland, recruited between 22 and 24 weeks gestation from pre-admission records provided by the BC Women's Hospital. 4 PURPOSE The purpose of Study One was to determine the range of TFA present in cord plasma. Study Two was designed to determine the amounts of TFA in the maternal diet, the level of T F A in maternal and cord plasma, and whether variability in levels of TFA in cord plasma can be explained by levels of TFA in the maternal diet and plasma. 4.1 Objectives • To determine the range of trans fatty acids and cis n-6 and n-3 polyunsaturated fatty acids in newborn cord plasma. • To determine if trans fatty acids in newborn cord plasma are inversely related to cis n-6 and n-3 polyunsaturated fatty acids and birth outcome measures (length of gestation, birthweight, length and head circumference). • To determine the range of TFA and cis n-6 and n-3 polyunsaturated fatty acids in maternal plasma. • To estimate dietary TFA intake in pregnant women during the second and third trimester and to identify the major sources of dietary TFA. 28 HYPOTHESES AND ETHICS • To identify relations between maternal TFA intake and level of TFA in newborn cord plasma. 4.2 Hypotheses • Trans fatty acids are not well transported across the placenta, therefore, the range of TFA in cord plasma will be less than the range of TFA in maternal plasma. • The range of TFA in maternal plasma is higher than the range of T F A in cord plasma • There is a positive relation between the amount of T F A in the maternal diet and the level of TFA in cord plasma. • There is no relation between the level of TFA and cis n-6 and n-3 fatty acids in cord plasma, or between TFA and birth outcome measures. • The major dietary source of TFA is from invisible fats, such as those present in baked, processed and convenience foods. 5 ETHICS The study protocol and procedures for both studies were approved by the University of British Columbia Clinical Research Ethics Board and the British Columbia Women's Hospital Research Coordinating Committee. 2 9 S U B J E C T S AND METHODS 6 SUBJECTS AND METHODS 6.1 Study One The purpose of Study One was to determine the range of T F A in cord plasma. This study provided data for Study Two, a larger study, to determine whether maternal dietary TFA intake and the range of TFA in maternal plasma are related to the range of TFA in cord plasma. 6.1.1. Participant Selection Criteria Eligible participants for this study were women delivering full-term infants (37-42 weeks), 20-40 years in age, in normal, early stage labor in the Low Risk Labor and Delivery Unit at the British Columbia Women's Hospital. Women known to have a history of tuberculosis, HIV/AIDS, peri-natal infection, substance abuse or conditions known to affect lipid metabolism, such as maternal diabetes, were excluded. Women delivering more than one fetus or delivering a fetus with known or suspected problems were also excluded. 6.1.2. Participant Recruitment Participants for this study were recruited between January and June 1997 in the Low Risk Labor and Delivery Unit at the British Columbia Women's Hospital. After giving informed consent (Appendix 1), the participants were asked to complete a brief questionnaire (Appendix 2). A self-addressed, stamped envelope was provided to those participants who chose to complete the questionnaire at a later time. 30 K S U B J E C T S AND METHODS 6.1.3. Study Questionnaire The questionnaire used in Study One was designed to collect information on each woman's age, highest education level attained, gross annual family income, smoking habits, dietary practices, frequency of fast food and commercial baked goods consumption, and type(s) of fat used in baking and as a table spread. In addition, length of gestation, infant birthweight and gender were collected after deliver of the infant from the medical chart. 6.1.4. Blood Collection A coded 3 mL EDTA vacutainer was given to each participant's nurse for collection of a newborn cord blood sample. The participant's nurse or physician collected the blood samples after the newborn cord was clamped. The blood samples were then placed in a -4°C refrigerator in the Low Risk Delivery Unit. Blood samples were transported on ice to the BC Research Center for Child and Maternal Health and centrifuged at 2000 g for 15 minutes. The plasma was separated from the red blood cells and frozen at - 8 0 ° C . 6.1.5. Birth Outcome Measures The length of gestation, determined using the date of the last normal menstrual period was recorded from the participant's medical chart. The infant gender and birthweight were also recorded from the participant's medical chart. 31 S U B J E C T S AND METHODS 6.2 Study Two The purpose of Study Two was to estimate dietary intakes of TFA in pregnancy, to determine the level of TFA in maternal and cord plasma, to determine if differences in levels of TFA in cord plasma can be explained by maternal diet and plasma TFA, and to determine if levels of T F A are inversely related to cis n-6 and n-3 polyunsaturated fatty acids in maternal and cord blood. 6.2.1. Participant Selection Criteria Eligible participants for this study were low-risk pregnant women between 20-40 years of age who had registered to deliver their infants at the British Columbia's Women's Hospital with expected dates of delivery between June 1 and July 19, 1998. Exclusion criteria included diabetes of any origin, a known pre-pregnancy history of high blood cholesterol and triglycerides, and chronic or communicable diseases including HIV/AIDS, hepatitis or tuberculosis. Women who had used elicit drugs or consumed three or more alcoholic drinks per week during pregnancy, or expecting more than one child were also excluded. 6.2.2. Participant Recruitment The Health Records Department generated a list of 804 pregnant women, who had registered to deliver their infants at the British Columbia's Women's Hospital between June 1 and July 19, 1998. This list was used to develop a mailing list of low-risk pregnant women between 20-40 years of age who lived in Vancouver, West Vancouver, North Vancouver, Burnaby or Richmond. Low-risk pregnancy, age and mailing address were provided on the pre-registration list. Women with a multiple 32 S U B J E C T S AND METHODS gestation, previous pregnancy complications, previous pre-term delivery, poor obstetrical history including hyper-emesis, abnormal weight and congenital anomalies, psychological and social problems, street drug and alcohol use, cardiac disease, renal disease, diabetes, epilepsy, respiratory conditions, lupus or other rheumatoid conditions, cholestasis, HIV/AIDS, and other medical or surgical problems were not included on the mailing list. A letter describing the study, the participant's role and the selection criteria was sent to 486 women between their 22 n d-24 t h week of gestation (Appendix 3). Within two weeks of mailing the letters, a follow-up telephone call was made. During the telephone call, the women who were interested in participating were asked to confirm their eligibility, as described in the selection criteria of the recruitment letter, and the requirements of the study were explained. 6.2.3. Participant Interviews Face to face interviews were scheduled with each study participant at both 28 and 35 weeks gestation either at the participant's home, or at the British Columbia's Women's Hospital. The participants were asked to read and sign an informed consent (Appendix 4) at the first interview. After signing the informed consent, the participants then completed a socio-demographic and health questionnaire (Appendix 5). After completing the socio-demographic questionnaire, a food frequency questionnaire (FFQ) was administered to each participant (Appendix 6). This questionnaire was administered to each participant at 28 weeks and again at 35 weeks gestation. At the second interview, a 3 mL venous blood sample was also collected at the Outpatient Laboratory of the British Columbia's Women's Hospital. 33 S U B J E C T S AND METHODS 6.2.4. Study Questionnaires Questionnaires in Study Two were designed to collect information on the socio-demographic background, health and diet of each participant. The socio-demographic and health questionnaire included questions on the participant's age, ethnicity, highest level of education attained, gross annual family income, number of individuals residing in the household that were dependent on the family income, height, weight, weight history, pregnancy history, smoking habits, alcohol consumption during pregnancy, vitamin and mineral supplements used during pregnancy, as wells as any medications taken during pregnancy, dietary practices and infant feeding plans (breast-feeding vs. formula feeding). For the purpose of collecting information on fat and energy consumption of the participants, a FFQ was developed using an unpublished FFQ that had been designed to measure total fat intake and dietary sources of fat. A FFQ was chosen rather than a 3-day food record to estimate TFA intake because a longer time period could be covered in which a variety of foods containing TFA could be consumed. The unpublished questionnaire was modified to include all known sources of dietary TFA and to include questions on brand names and methods of food preparation. Known food sources of TFA were identified from detailed fatty acid analysis of over 250 foods previously conducted in our laboratory, and from previous publications on sources of TFA in Canadian and U.S. foods. In addition, frequency of consumption and usual portion size was included on the FFQ. The FFQ contained 106 items, the completeness of foods listed on the FFQ was then validated using data from 24 hour dietary recalls and three day food records completed by 24 lactating women in the Okanogan Valley. Any additional foods containing TFA identified on these records were added to the FFQ. 34 S U B J E C T S AND METHODS The FFQ was administered in a face to face interview with each participant at each of 28 and 35 weeks gestation. Participants were asked to indicate their average total intake during the past 30 days, either on a daily, weekly or monthly basis for each food and beverage item listed in the FFQ. In addition, the participants were asked to give the usual brand or brands of each food and beverage consumed. Food pictures and plastic food models were used to help each participant determine usual portion sizes of the foods and beverages consumed. 6.2.5. Blood Collection A 10 ml maternal venous blood sample was collected from each participant during their 35 t h week of gestation at the British Columbia Women's Hospital laboratory, by a registered phlebotomist. Blood samples were then transported on ice to the BC Research Center for Child and Maternal Health and centrifuged at 2000 g for 15 minutes. The plasma was separated from the red blood cells and frozen at - 8 0 ° C . A research study protocol sheet, describing the procedure for collecting the newborn cord blood sample, was attached to each participant's admission sheet in the BC Women's Hospital Admitting area. A box of 3 mL EDTA vacutainers was placed in the Low Risk Delivery Unit. The participant's nurse or physician collected the blood sample after the newborn cord was clamped. The blood samples were then placed in a - 4 ° C refrigerator in the Low Risk Delivery Unit. Blood samples were transported on ice to the BC Research Center for Child and Maternal Health and centrifuged at 2000 g for 15 minutes. The plasma was separated from the red blood cells and frozen at - 8 0 ° C . 35 S U B J E C T S AND METHODS 6.2.6, Birth Outcome Measures The length of gestation, determined by the first day of the last normal menstrual period, was recorded from the participant's medical chart. Infant gender, birthweight, length and head circumference were also recorded from the participant's medical chart. 36 _ DATA ANALYSIS 7 DATA ANALYSIS 7.1 Demographic and Health Data Handling Data from the questionnaire in Study One were entered into a computer spreadsheet. Numerical values were assigned to all categorical variables such as highest level of education attained, gross annual family income, consumption of fried fast foods, consumption of commercial baked goods, smoking habits, infant gender, baking practices, type of baking fat used, type of fat used as a table spread, dietary practices and food avoidances. Continuous variables such as maternal age, length of gestation and birthweight were entered as the value provided by the participant or recorded from the participant's medical chart. Data from the socio-demographic and health questionnaire and birth outcome measures in Study Two were entered into a second computer spreadsheet. Numerical values were assigned to all categorical variables such as ethnicity, highest level of education attained, gross annual family income, smoking habits, alcohol consumption, supplement and medication use, dietary practices, whether the woman was planning to breast or formula feed the infant and infant gender. Continuous variables such as maternal age (calculated from their date of birth), number of individuals in the household dependent on the gross annual family income, height, present weight, pre-pregnancy weight, number of previous pregnancies, number of live births, number of miscarriages, number of alcoholic drinks consumed weekly, length of gestation, infant birthweight, length and head circumference were entered as provided by the participant or recorded from the medical chart. 37 DATA ANALYSIS 7.2 Analysis of the Food Frequency Questionnaire The dietary intakes provided in the FFQ were converted to daily intake values based on the number of servings and frequency of consumption, i.e. daily, weekly or monthly. For example, if a participant consumed four ounces of orange juice six times per week, their daily intake of orange juice would be calculated by multiplying the serving size (4 oz) by the frequency of consumption (6 days) and dividing by the time period covered (7 days), resulting in an average intake of 3.4 oz of orange juice per day. The daily intake values were then entered into a computer nutrient database (Food Processor II version 7, 1997, ESHA Research, Salem, Oregon). The Food Processor database was modified to include nutrient information for all foods and beverages containing dietary fat which were consumed by the study participants. The fatty acid composition of 250 different foods and beverages analyzed in this laboratory were included in the updated database. If a specific brand consumed by a participant was not present in the updated database, values were extrapolated from the closest brand of food or beverage product from the 250 items analyzed in this laboratory, based on label information on the type and amount of fat in the product. Once all foods were entered into the computer nutrient database, all dietary sources of T F A were identified for each participant. The dietary sources of TFA were then categorized into one of fifteen food groups. These food groups were baked foods, fast foods, margarine and shortenings, snack foods, breads, cereal/waffles/pancakes, pasta and other grain products, dairy products, peanut butter, meat, fish, oil/sauces/dressings, fruits and vegetables, chocolate/candy/icing, and soup. These food groups were developed using a modified list of previously identified food groups containing TFA (Enig 1990, Hunter 1991). The contribution of food groups to the total 38 DATA ANALYSIS TFA intake of the participants was calculated using three methods. First, the mean percent contribution of each food group to the total TFA intake was calculated for the study population. Second, for each food group, the mean T F A intake was calculated using only those participants who consumed foods within that food group, that is, for example, the mean TFA intake from margarine was calculated for only those participants who consumed margarine. Finally, the top five individual foods contributing to each participant's TFA intake were determined, and then each classified into one of the fifteen above food groups. Foods within a food group were then summed and the food groups ranked in order by their contribution to total TFA intake. 7.3 Fatty Acid Analysis Total lipids were extracted from 400 \iL plasma aliquot using chloroform-methanol-saline (6/3/1 v/v/v) (Folch, 1957). The phospholipid (PL), triglyceride (TG) and cholesteryl ester (CE) fractions were then separated by thin layer chromatography (TLC) using silica plates (Whatman PK6F Silica Gel 60 A, 20 x 20 cm, 500 um) with a solvent system of hexane-diethyl ether-glacial acetic acid (60/40/2 v/v/v) (Hrboticky 1990). Authentic T L C standards containing T G and C E were used on each TLC plate to identify the T G and C E bands. The PL bands, which remain at the origin of the TLC plate were then scraped into tubes. The fatty acids in the PL were converted to methyl esters using 1 m i BF3 and 2 mL hexane, at 100°C for 15 minutes. The bands on the TLC plate corresponding to T G and C E were visualized under ultra-violet light and the silica containing the bands scraped into labeled glass tubes. The T G and C E were extracted from the silica using chloroform/methanol. The fatty acids in the T G and C E were then converted to methyl esters using 1 mL BF3-benzene-methanol (25/25/55, 39 DATA ANALYSIS v/v/v) at 100°C for 30 minutes, and 1 mL BF3-benzene-methanol (35/30/55 v/v/v) at 100°C for 45 minutes, respectively. The fatty acid methyl esters were then separated and quantified by gas liquid chromatography (GLC) (Varian 3400, Georgetown, Ontario). The G L C separation was achieved using 100 m x 0.25 mm non-bonded, fused silica capillary SP2560 columns (Supelco, Bellefonte, Pennsylvania). Helium was used as the carrier gas at a column flow of 1 mL/min and an inlet pressure of 55 pounds per square inch. The inlet splitter was set at 10 to 1. Samples were injected at 100°C and the oven temperature programmed to remain at 100°C for 2 minutes, then increase to 190°C at 5°C/minute, stabilize for 20 minutes, rise to 255°C at 1°C/minute and held for 2 minutes. The column was then heated to 245°C at 25°C/minute and held for 22 minutes prior to returning to 100°C and stabilization for the injection of the next sample. A 15 minute stabilization period at 100°C, was set prior to each sample injection. The injectors and detectors were set at 240°C and 260°C, respectively. Fatty acid methyl esters were identified by comparison of retention time with that of authentic standards. The identification of fatty acids for this study did not include mass spectrometry of the samples. For the purpose of this research, the identified fatty acids were grouped as saturated fatty acids, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0, 24:0; monounsaturated fatty acids, 16:1, 18:1, 20:1, 22:1; polyunsaturated n-9 series, 20:3; n-6 series, 18:2, 18:3, 20:2, 20:3, 20:4, 22:4, 22:5; n-3 series, 18:3, 20:5, 22:5, 22:6; and trans isomers of 18:1 and 18:2. With the availability of new conjugated linoleic acid (CLA) isomer standards, CLA was identified in maternal plasma and the newborn cord plasma in Study Two, but not Study One. 40 DATA ANALYSIS Peak area counts and retention times were recorded by a G L C data system (IBM computer system using Varian "STAR" software, Georgetown, Ontario). Peak area counts for each identified fatty acid were entered into a computer spreadsheet and the relative percents of each fatty acid were calculated. The response of the detector to equivalent weights of fatty acids across the range of C14 to C22 was similar. The variance in the flame ionization detector response across the C14 to C22 did not at any time exceed 5% and was within the range of inter-assay reproducibility. The consistency of the flame ionization detector response was checked by injecting a calibration mixture containing equivalent amounts of saturated fatty acids 14:0 through to 22:0, as well as known authentic saturated, monounsaturated and polyunsaturated fatty acid methyl ester standard mixtures. The use of sodium methoxide, as a methylating agent, has been suggested to be possibly superior to B F 3 (Kramer 1997). The latter suggestion was due to concerns that BF3 may artificially elevate trans isomers during the methylation procedure by converting some c/'s isomers to trans isomers. To address this issue, three randomly chosen plasma samples from Study One were extracted and methylated using 1mL BF3-benzene-methanol (25/25/55, v/v/v) at 100°C for 30 minutes and 1 mL sodium methoxide at 50°C for 10 minutes. The analyses found no differences in the relative percent of any fatty acids, including TFA between the two methods. Thus, BFswas used as the methylating agent in all fatty acid analyses in both Study One and Two. 7.4 Statistical Analysis All data from Study One and Two were analyzed using the Statistical Package for the Social Science (SPSS Inc. version 7.5 for Windows, Chicago, Illinois). 41 DATA ANALYSIS Descriptive statistics were obtained for dietary energy and fat intakes, birth outcome measures, maternal plasma fatty acids and cord plasma fatty acids. Frequency counts were calculated for all categorical variables. When variables were not normally distributed, the data were transformed using the natural log, square roots or squared values. Non-parametric statistics were performed on non-normally distributed data. A paired sample t-test was used to compare means of total energy and fat intakes in the second and third trimester. Pearson correlation coefficients were calculated to determine potential relations between: - total energy and fat intakes in the second trimester and total energy and fat intakes in the third trimester - maternal TFA intake and TFA in maternal plasma T G , PL and C E - maternal TFA intake and TFA in cord plasma T G , PL and C E - T F A in maternal plasma T G , PL and C E and cis n-6 and n-3 polyunsaturated fatty acids - T F A in cord plasma T G , PL and C E and cis n-6 and n-3 polyunsaturated fatty acids - birth outcome measures (length of gestation, birthweight, infant length) and cord plasma fatty acids. - maternal plasma TFA, cis n-6 and n-3 polyunsaturated fatty acids in TG , PL and C E and cord plasma TFA, cis n-6 and n-3 polyunsaturated fatty acids in T G , PL and C E Results with a probability less than 0.05 were considered statistically significant. 42 RESULTS 8 RESULTS 8.1 Participant Recruitment and Characteristics 8.1.1. Study One A total of 33 women were approached for participation in Study One. Of the 33 approached, 24 (72.7%) agreed to participate. Of these 24 women, 16 completed the socio-demographic and health questionnaire. The mean age of the women who participated in Study One was 32.6 years (range 18-40) (Table 8.1). Over 68% of women (n=11) had completed post-secondary education. Most of the women (56.3%) had a gross family income greater than $50,000, only one woman had an annual family income less than $20,000. The average length of gestation and birthweight were 40.5 (range 38^2) weeks and 3625.5 (3045-4460) g, respectively (Table 8.2). Fourteen of the infants were female and 10 were male. One woman smoked during her pregnancy. None of the women followed a special diet during pregnancy, e.g. vegetarian. Three women indicated that they had avoided certain foods during pregnancy including red meat, offal, wheat, eggs and seafood. One woman had an allergy to onions and one woman was lactose intolerant. Nine women (56.3%) consumed commercial baked foods, such as muffins and cookies, one to two times per week and thirteen women (81.3%) consumed fast foods one to two times per week. The type of fat used by women as a table spread was butter, n=8, margarine, n=4, butter and/or margarine, n=2 and none, n=1. Six women baked on a weekly basis of which, one woman used butter, two women used vegetable oil and three women used a combination of butter, margarine and/or oil in their baking. 43 RESULTS Table 8.1 Characteristics of women in Study One1 n % Age (yr) <24.0 1 6.3 >25<30 1 6.3 >30<35 11 68.8 >35<40 3 18.8 Education Completed < Secondary School 2 .12.5 Secondary School 3 18.8 College/Technical School 5 31.3 Undergraduate Degree 4 25.0 Graduate Degree 2 12.5 Gross Annual Family Income $10,000 < $20,000 1 6.3 > $20,000 < $35,000 2 12.5 > $35,000 < $50,000 3 18.8 > $50,000 < $75,000 3 18.8 > $75,000 6 37.5 Did not report income 1 6.3 1 Of the 24 women who participated, only 16 women completed the socio-demographic and health questionnaire 44 RESULTS Table 8.2 Birth outcome measures in Study One1 n=24 % Birthweight (g) >3000 < 3500 > 3500 < 4000 >4000 Mean (range) 11 8 5 3625.5 (3045-4460) 45.8 33.3 20.8 Infant Gender Female Male 14 10 58.3 41.7 1 The mean length of gestation was 40.5 wks, study eligibility criteria was 38-42 weeks 8.1.2 Study Two Of 504 letters mailed, two letters were returned marked undeliverable. A follow-up telephone call was made to all women with the exception of those women who had called the Research Center upon receiving the letter (n=19). Follow-up phone calls were made to 483 women, of whom 33 women could not be reached due to incorrect or out of service phone numbers (Table 8.3). A further 65 women did not speak or understand English. Reasons for not participating in the study included time constraints, acute illness, other study commitments (n=18), or study ineligibility due to gestational diabetes, drug use, miscarriage, or a change in hospital (n=15). Of the 151 eligible women contacted, 70 women agreed to participate in the study and 60 women were interviewed at 28 weeks. The remaining 10 women who agreed to but did not participate, declined due to time constraints or illness. All but three of the 60 women completed the study, and one woman joined the study at 35 weeks gestation. The majority of women in the study (96.6%) were between 25 and 40 years of age, with a mean age of 32.5 years (range 20-40 years). Forty-nine women were Caucasian (80.3%), 10 were Asian (16.4%), 1 was First Nations (1.6%) and 1 was 45 RESULTS Black (1.6%) (Table 8.4). Most women (n=52) had completed post-secondary education that included college/technical school, undergraduate and graduate university degrees. Thirty-nine women (63.9%) had a gross annual family income of more than $50,000. Over 90% (n=57) of the women had a total of two or three persons living in their household, including themselves, who were dependent on the family income. About half of the women who participated (52.5%) had a pre-pregnancy body mass index between 20.0 and 25.0 with a mean of 23.5 (range 17.9-30.7). The average height of the women was 164.2 cm (Table 8.5) and the average pre-pregnancy weight was 63.3 kg. Most of the women gained between 7-11 kg (68.4%), from the beginning of their pregnancy to 35 weeks gestation. The heights and weights were self-reported by the participants. Over 50% (n=34) of the women had had a previous pregnancy and 34% (n=24) of women had had at least one previous live birth (Table 8.6). Over 95% of participants (n=44) had a full-term pregnancy, with an average length of gestation of 39.9 wks (range 36-42 wks) and birthweight of 3524 g (2035-4810 g). Thirty-two infants had a length at birth between 50-55 cm and 24 infants had a head circumference at birth between 35-37 cm. Of the 46 infants, 23 were female and 23 were male. Twelve of the women (19.7%) followed a lacto-ovo vegetarian, or semi-vegetarian diet and one woman was allergic to dairy products, wheat products and nuts. Over 80.3% of the women who participated in the study were taking a supplement intended for use during pregnancy (prenatal supplement) at 28 weeks gestation and 9.8% were taking a regular adult multi-vitamin and mineral supplement at 28 weeks gestation (Table 8.7). In addition, 9.8% were taking iron, 11.5% were taking folic acid, and 11.5% were taking calcium supplements. None of the woman who participated in the study smoked and 4 women consumed alcohol during their pregnancy. 46 RESULTS Table 8.3 Participant recruitment results for Study Two n % Letters mailed to participants 504 100.0 Individuals contacted by telephone who were unable 67 13.3 to communicate with interviewer Individuals with telephone numbers not in service 33 6.5 Individuals telephoned, but no answer 230 45.6 Individuals successfully contacted by telephone 174 34.5 Individuals successfully contacted who did not met 23 13.2 eligibility criteria1 Individuals successfully contacted who declined with 63 36.2 no reason given Individuals successfully contacted who declined 18 10.3 because of other commitments Individuals successfully contacted who agreed to 70 40.2 participate Individuals successfully contacted who attended the 60 85.7 first appointment Individuals successfully contacted who attended the 58 82.9 second appointment 1 Eligibility criteria included: registered to deliver their infant at British Columbia's Women's Hospital, 20-40 years of age, singleton pregnancy, no drug use and fewer than 3 alcoholic drinks per week during pregnancy, no tuberculosis, HIV/AIDS, hepatitis, diabetes and no known pre-pregnancy history of high blood cholesterol or triglycerides. 47 RESULTS Table 8.4 Socio-demographic characteristics of women in Study Two n =61 % Race Caucasian 49 80.3 Asian 10 16.4 Other 2 3.3 Age (yr) <25.0 2 3.3 >25<30 19 31.1 >30<35 19 31.1 >35<40 21 34.4 Education Completed High School 9 14.8 College/Technical School 14 23.0 Undergraduate Degree 31 50.8 Graduate Degree 7 11.5 Gross Annual Family Income < $20,000 7 11.5 > $20,000 < $50,000 15 24.6 > $50,000 39 63.9 Number of Individuals Dependent on Family Income  1 1 4 6.6 2 32 52.5 3 21 34.4 >4 4 6.5 1 Number of individuals residing in the household who were dependent on the family income, including the participant, spouse, children, any extended family or other persons 48 RESULTS Table 8.5 Characteristics of women in Study Two1 Height (cm), n=61 164.2 ± 0 . 9 (146.7-177.1) Pre-pregnancy weight (kg), n=61 63.3 ± 3 . 0 (40.0-90.0) Weight at 28 weeks gestation (kg), n=60 71.8 ± 2 . 9 (50.9-94.5) Weight at 35 weeks gestation (kg), n=57 75.9 ± 3 . 0 (54.5-100.0) Pregnancy weight gain (kg), n=57 12.3 ± 1.3 (-0.08-22.7) 1 Values reported are means ± S E (range) and were self-reported 49 RESULTS Table 8.6 Birth outcomes of infants born to women in Study Two1 n % of participants Previous Pregnancies, n=61 0 27 44.3 1 17 27.9 2 11 18.0 >3 6 9.8 Previous Live Births, n=61 0 37 60.7 1 19 31.1 2 5 3.3 Length of gestation (wk) n=46 <38 2 4.3 > 38 < 42 44 95.7 >42 0 0.0 Mean (range) 40 (36-42) Birthweight (g), n=46 <2500 1 2.2 > 2500 < 3000 6 13.0 > 3000 < 3500 13 28.3 > 3500 < 4000 15 32.6 >4000 11 23.9 Mean (range) 3523 (2035-4810) Infant Length (cm), n=44 > 45 < 50 6 13.6 > 50 < 55 32 72.7 >55.0 6 13.6 Mean (range) 52 (46-57) Head Circumference (cm), n=45 > 33 < 35 18 40.0 > 35 < 37 24 53.3 > 37 < 39 3 6.7 Mean (range) 35 (31-39) Gender, n=46 Male 23 50.0 Female 23 50.0 1 Collected, where" recorded, from the participant's medical chart record 50 RESULTS Table 8.7 Diet and health characteristics of participants in Study Two n =61 % Diet Lacto-ovo-vegetarian 4 6.6 Semi-vegetarian1 8 13.1 Other2 1 1.6 No diet restrictions 46 75.4 Vitamin/Mineral Supplements 3 Prenatal 49 80.3 Folic acid 7 11.5 Calcium 7 11.5 Iron 6 9.8 Multi-mineral/vitamin 6 9.8 B vitamins 4 6.6 Vitamin E 3 4.9 Smokers 0 0 Alcohol Intake/Week 4 0 drinks 57 93.4 > 0 < 1 drink 4 6.6 > 1 drink 0 0 2 Wheat and dairy free diet 3 Women were allowed to check more than one supplement 4 Alcohol consumption during pregnancy, 1 drink = 1 bottle of beer/cider, 4 ounces wine or 1 ounce spirits 8.2 Estimated Energy and Dietary Fat Intakes The mean daily energy intake in the second trimester was 2691.8 (range 1251-5775) kcal/d and in the third trimester was 2404.8 (range 1246-3697) kcal/d (Table 8.8). The average daily energy intake in the third trimester was about 300 kcal/d lower than in the second trimester. The mean daily fat intake was 85.8 and 73.9 g/d in the second and third trimester, respectively, contributing less than 30% of energy in both trimesters. Total fat 51 RESULTS intake ranged from 37.5-200.8 g/d in the second trimester and from 22.4-124.3 g/d in the third trimester (Table 8.8). Thirty-eight and 35 women consumed between 50 and 100 g/d of total fat in the second and third trimester, respectively (Table 8.9). The mean daily dietary TFA intake was 3.8 and 3.4 g/d in the second and third trimester, respectively, contributing approximately 1% of total energy in both trimesters. The TFA intake ranged from 0.7-11.3 g/d in the second trimester and from range 0.9-9.6 g/d in the third trimester. Twenty-seven and 31 women consumed between 0 and 3 g/d of TFA in the second and third trimester, respectively. The mean daily polyunsaturated fatty acid intake was 13.6 and 12.2 g/d in the second and third trimester, respectively, contributing about 4.5% of energy in both trimesters. The polyunsaturated fatty acid intake ranged from 5.8-40.0 g/d in the second trimester and from 2.9-23.3 g/person/d in the third trimester. Thirty-one and 33 women consumed between 10 and 20 g/d polyunsaturated fatty acids in the second and third trimester, respectively. The mean daily monounsaturated fatty acid intake was 31.5 and 26.4 g/d in the second and third trimester, respectively, contributing about 10% of energy in both trimesters. The monounsaturated fatty acid intake ranged from 13.9-78 g/d in the second trimester and from 3.7-53.3 g/d in the third trimester. Thirty-eight and 40 women consumed between 20 and 40 g/d monounsaturated fatty acids in the second and third trimester, respectively. The mean daily saturated fatty acid intake in the first and second trimester was 29.7 and 26.3 g, respectively, contributing 9.8% of energy in both trimesters. The saturated fatty acid intake ranged from 11.0-67.8 g/d in the second trimester and from 52 RESULTS 7.3-51.4 g/d in the third trimester. Thirty-four and 31 women consumed between 20 and 40 g/d saturated fatty acids in the second and third trimester, respectively. The total energy intake and total fat intakes of individual women at 28 weeks gestation were significantly related to the total energy and total fat intakes at 35 weeks gestation. The correlation coefficient for energy was 0.63, total fat was 0.69, TFA was 0.54, polyunsaturated fatty acids was 0.61, monounsaturated fatty acids was 0.64 and saturated fatty acids was 0.7 (Table 8.8). The trans fatty acid intake (g/d) was significantly related to the total fat intake (g/d) in the second trimester, r = 0.49, p<0.05 and in the third trimester, r = 0.57, p<0.05 (Figure 8.1). Similarly, TFA and total fat intake as a percent of total energy, were also significantly related at both 28 and 35 weeks gestation, at r = 0.10, p<0.05 and r = 0.26, p<0.05, respectively (Figure 8.2). 53 RESULTS 28 weeks 35 weeks Fat intake (g) Fat intake (g) Figure 8.1 Scatter plots of estimated dietary trans fatty acid and total fat intakes (g/d) of women at 28 and 35 weeks gestation, n=57 Fat Intake (%en) Total fat (% en) Figure 8.2 Scatter plots of estimated dietary trans fatty acid and total fat intakes as percent total energy (%en) of women at 28 and 35 weeks gestation, n=571 1 The percent total energy of total fat and TFA were calculated by multiplying the total fat and TFA intake (g) by 9kcal/g and dividing by total energy (kcal) 54 RESULTS Table 8.8 Estimated dietary trans, polyunsaturated, monounsaturated and saturated fatty acids, total fat and energy intakes as determined by a food frequency questionnaire at 28 and 35 weeks gestation1 Gestation Week 28 35 r n=60 n=57 Energy (kcal/d) 2691.8 ± 1 0 1 . 6 2404.8 + 82.6 0.63 (1250.7-5774.5) (1245.6-3696.9)3 O.001 Total Fat (g/d) 85.8 ± 4.3 (37.5-200.8) 73.9 ± 3.2 (22.4-124.3)3 0.69 <0.001 % Energy from total 28.3 ± 0.7 (18.5-42.4) 27.5 ± 0.7 (16.1-42.4) °/cfat Trans fatty acids (g/d) 3.8 + 0.3(0.7-11.3) 3.4 + 0.3(0.9-9.6) 0.542 O.001 % Energy from trans 1.3 + 0.1(0.3-2.8) 1.3 + 0.1(0.3-3.2) °A fatty acids Polyunsaturated fatty acids 13.6 ± 0 . 9 (5.8-40.4) 12.2 ± 0.6 (2.9-23.3)3 0.61 (g/d) O.001 % Energy from 4.5 ± 0.2 (2.4-8.1) 4.6 ± 0 . 2 (2.0-11.9) polyunsaturated fatty acids Monounsaturated fatty acids 31.5 ± 1.8 (13.9-78.0) 26.4 ± 1.3 (3.7-53.3)3 0.64 (g/d) <0.001 %Energyfrom 10.4 ± 0.4(6.5-22.4) 9.8 ± 0 . 3 (2.7-17.6) monounsaturated fatty acids Saturated fatty acids (g/d) 29.7 ± 1.6 (11.0-67.8) 26.3 ± 1.3 (7.3-51.4)3 0.75 <0.001 % Energy from 9.8 ± 0 . 3 (5.3-14.8) 9.8 ± 0 . 3 (4.5-14.6) saturated fatty acids 1 Values are reported as mean ± SE (range) 2 Pearson correlation coefficients of intakes at 28 and 35 weeks 3 Significantly different from dietary intakes at 28 weeks, p < 0.05 55 RESULTS Table 8.9 Distribution of women by quartiles of estimated trans, polyunsaturated, monounsaturated and saturated fatty acids and total fat intakes(g/d) as determined by a food frequency questionnaire Quartiles 1 2 3 4 Total fat (g/d) 0<50 50 < 100 100< 150 150<200 2 n d trimester1 5 38 12 5 3 r d trimester2 11 35 11 0 Trans fatty acids (g/d) 0<3 3 < 6 6 <9 9 < 12 2 n d trimester 27 23 7 3 3 r d trimester 31 19 6 1 Polyunsaturated fatty 0<10 10<20 20<30 30<40 acids (g/d) 8 1 2 n d trimester 20 31 3 r d trimester 19 33 5 0 Monounsaturated 0<20 20<40 40<60 60<80 fatty acids (g/d) 2 n d trimester 10 38 9 3 3 r d trimester 14 40 3 0 Saturated fatty acids 0<20 20<40 40<60 60<80 (9/d) 2 n trimester 16 34 9 1 3 r d trimester 17 31 9 0 1 n=60 at 28 weeks gestation 2 n=57 at 35 weeks gestation 56 RESULTS 8.3 Dietary Sources and Contribution of Trans Fatty Acids by Food Groups Baked foods were the major source of TFA in the women's diets, contributing over 30% of total dietary TFA in the second and third trimester (Figures 8.3 and 8.4). Most of the women consumed baked foods in the second (n=58/60) and third trimester (n=57/57) providing a mean 1.3 and 1.1 g of total dietary TFA, respectively (Table 8.10). The top five ranked foods, classified by food group, were all baked foods (Figures 8.5 and 8.6). Snack foods and breads each contributed 11% of total dietary TFA in the second trimester and 9 and 10%, respectively, of the total dietary TFA in the third trimester. Snack foods were consumed by more women in the second trimester (n=58/60) than in the third trimester (n=49/57), and provided an average of 0.4 g of dietary TFA in both trimesters of those women who consumed snack foods. Fast foods contributed approximately 11 and 13% of total dietary TFA in the second and third trimester, respectively. Forty-five women consumed fast foods in the second trimester, which contributed an average of 0.5 g of dietary TFA to the diet of those women who consumed fast foods. In the third trimester 46 women consumed fast foods also contributing an average of 0.5 g of dietary TFA. Less significant contributors to the total dietary TFA intake in the second and third trimester were margarines and shortenings (7 and 9%, respectively), dairy products (6 and 7%, respectively), breakfast cereals (5 and 6%, respectively), pasta and other grains such as stuffing and bread crumbs (4 and 6%, respectively), meats (3%), oils and sauces (3%), peanut butter (3 and 2%, respectively) and chocolate and candies (2 and 0%, respectively). Margarines and shortenings contributed 0.5 and 0.6 g dietary TFA to the diet of those eating these fats in the second (n=32/60) and third trimester (n=29/57), respectively. Of note, 28 women did not use margarines or shortenings as an added 57 RESULTS fat, e.g. as a spread on bread or with vegetables, in either the second or third trimester. Breads contributed 0.4 g of dietary TFA to the diet of women who consumed bread in both the second (n=58/60) and third trimester (n=51/57). Cereals, including waffles and pancakes, provided an average of 0.3 g of dietary TFA to the diet of women who consumed these foods in the second (n=33/60) and third trimester (n=36/57). Pasta and other grains such as bread crumbs and stuffing contributed an average of 0.3 and 0.4 g of dietary TFA to the diet of women who consumed these foods in the second (n=32/60) and third trimester (n=27/57), respectively. The majority of women consumed dairy products, contributing an average of 0.2 g of dietary TFA in the second (n=59/60), and third trimester (n=56/57). Twenty-seven and 25 women consumed peanut butter, contributing 0.2 g of dietary TFA in both second and third trimester, respectively. Meats, e.g. beef, contributed an average of 0.2 g of dietary TFA in both second (n=43/60) and third trimester (n=36/57). Most women consumed oils and sauces such as mayonnaise and salad dressings in the second (n=52/60) and third trimester (n=50/57). Oils and sauces contributed an average of 0.2 and 0.1g of dietary TFA, to the diet of women who consumed oils and sauces in the second and third trimester, respectively. Battered fish contributed 0.2 and 0.1 g of dietary TFA to the diet of a small number of women in the second (n=8/60) and third trimester (n=10/57), respectively. Fruits and vegetables, such as fruit leather and frozen potato products, contributed an average of 0.2 g of dietary TFA to the diet of women who consumed these products in the second (n=17/60) and third trimester (n=10/57), respectively. Chocolates and candy contributed about 0.1 g of dietary TFA to the diet of women who consumed chocolate and candy in the second trimester (n=31/60) and less than 0.1 g in the third trimester (n=16/57). Canned and packaged soups contributed 0.1 g of dietary TFA in 58 RESULTS both the second (n=11/60) and third trimester (n=6/57) but again were eaten by relatively few of the women. Pasta/Grains Meat Figure 8.3 Estimated contribution of trans fatty acids by food groups to the total trans fatty acid intake of women in their second trimester, n=601 1 The mean percent contribution of each food group to the total TFA intake was calculated for all 60 women 59 RESULTS Pasta/Grains Meat Figure 8.4 Estimated contribution of trans fatty acids by food groups to the total trans fatty acids women in their third trimester, n=571 1 The mean percent contribution of each food group to the total TFA intake was calculated using all 57 women 60 RESULTS Table 8.10 Quantity of trans fatty acids consumed by food group in the second and third trimester1 Food Group 2 n d Trimester 3 r a Trimester Baked Foods 2 Fast Foods Margarine Snack Foods 3 Breads Cereal/Waffles/Pancakes Pasta and other Grain Products4 Dairy Products Peanut Butter Meat Fish Oil/Sauces/Dressings Fruits and Vegetables 5 Chocolate/Candy/lcing Soup (9) 1.34 ± 0.02 (0.05-6.43) 1.09 ± 0.11 (0.01 -3.84) n=58 n=57 0.54 ± 0.01 (0.02-6.66) 0.55 ± 0.08 (0.01-4.06) n=45 n=46 0.50 ± 0.01 (0.01-2.46) 0.60 ± 0.17 (0.02-3.64) n=32 n=29 0.44 ± 0 . 0 1 (0.01-2.79) 0.35 ± 0.04 (0.01-1.11) n=56 n=49 0.44 ± 0 . 0 1 (0.02-2.41) 0.36 ± 0.05 (0.01-1.40) n=58 n=51 0.32 ± 0 . 0 1 (0.01-1.32) 0.30 ± 0 . 0 6 (0.01-1.21) n=33 n=36 0.30 ± 0.01 (0.01-5.13) 0.41 ± 0.17 (0.01-4.06) n=32 n=27 0.24 ± 0.00 (0.02-0.75) 0.22 ± 0.02 (0.01 -0.66) n=59 n=56 0.23 ± 0.00 (0.01-1.43) 0.15 ± 0.03 (0.01-0.49) n=27 n=25 0.17 ± 0.00 (0.01-0.79) 0.16 ± 0.02 (0.03-0.55) n=43 n=36 0.15 ± 0.00 (0.06-0.37) 0.09 ± 0.01 (0.03-0.19) n=8 n=10 0.15 ± 0.00 (0.01-1.11) 0.12 ± 0.04 (0.01-1.47) n=52 n=50 0.14 ± 0.00 (0.01-0.75) 0.16 ± 0.06 (0.01-0.55) n=17 n=10 0.13 ± 0.00 (0.01-0.96) 0.04 ± 0.01 (0.01-0.13) n=31 n=16 0.10 ± 0.00 (0.01-0.19) 0.10 ± 0.02 (0.01-0.17) n=11 n=6 1 Values are expressed as mean ± SE (range) for women consuming the food group and were obtained from food frequency data 2 Includes cookies, cakes, pies, muffins, scones, croissants, pastries 3 Includes potato, corn and taco chips, popcorn, crackers, cereal, granola bars ' Includes pasta, breadcrumbs, croutons, taco shells 'includes fruit leather and frozen potato products 61 RESULTS mm baked foods cereals dairy fast foods grains WmW margarine oils/sauces snack foods nuts fruits/veg Ranking Figure 8.5 Top five individual foods classified by food groups that contributed to trans fatty acid intake of women at 28 weeks gestation by rank order n=601 1 The top five foods that provided the highest amount of dietary TFA (g), to each woman's total TFA intake, were ranked 1 to 5 and were classified by food group. The frequency represents the number of women who consumed a food containing TFA, categorized by food group. 62 J RESULTS ~ ~ Baked Foods Cereals Dairy Fast Foods Grains Margarine Nuts Oils/sauces WVft Snack Foods Fruit/veg Ranking Figure 8.6 Top five individual foods classified by food groups which contributed to trans fatty acid intake of women at 35 weeks gestation by rank order, n=571 1 The top five foods that provided the highest amount of dietary TFA (g), to each woman's total TFA intake, were ranked 1 to 5 and were classified by food group. The frequency represents the number of women who consumed a food containing TFA, categorized by food group. 63 • • RESULTS 8.4 Maternal Plasma Fatty Acids 8.4.1 Triglyceride The mean total trans fatty acids in maternal plasma TG was 3.98 (range 1.26-7.90)% of total fatty acids (Table 8.11). Trans 18:1 and 18:2 were the predominant trans isomers and represented a mean of 2.07 (range 0.65-5.06)% and 1.91 (range 0.40-3.27)% of plasma T G total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 20.11 (range 13.26-33.64)% of total fatty acids in maternal plasma TG. Cis 18:2n-6 was the predominant polyunsaturated fatty acid in maternal plasma T G , and represented a mean of 13.65 (range 7.75-26.16)% of total fatty acids. Cis 18:3n-3 was present at a mean of 1.21 (range 0.42-3.75)% of total fatty acids and the remaining cis polyunsaturated fatty acids, 18:3n-6, 20:2n-6, 20:3n-6, 20:3n-9, 20:4n-6, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and CLA were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean 42.89 (range 33.85-48.54)% of total fatty acids in maternal plasma TG. The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1 and represented a mean of 35.61 (range 26.97-43.90)% and 4.28 (range 1.48-10.07)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1, each represented less than 1.0% of total fatty acids in maternal plasma TG. Saturated fatty acids represented a mean of 37.00.(range 26.00-47.60)% of total fatty acids in maternal plasma TG. Palmitic acid (16:0) was the predominant saturated fatty acid in TG, and represented a mean of 29.51 (range 19.92-38.07)% of total fatty acids followed by 18:0 and 14:0 with a mean of 4.12 (2.53-9.25)% and 2.77 (range 0.96-4.62)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 20:0, 22:0 and 24:0, were each present at less than 1.0% of total fatty acids. 64 : RESULTS 8.4.2 Phospholipid The mean total trans fatty acids in maternal plasma PL was 2.37 (range 1.13-4.47)% of total fatty acids (Table 8.11). Trans 18:1 and 18:2 were the predominant trans isomers present, with a mean of 1.37 (range 0.58-3.01)% and 1.00 (range 0.35-1.69)% of total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 42.99 (range 38.76-47.31)% of total fatty acids in maternal plasma PL. Cis 18:2n-6 was the predominant polyunsaturated fatty acid in maternal plasma PL, and represented a mean of 20.81 (range 13.59-27.24)% of total fatty acids. Cis 20:3n-6, 20:4n-6 and 22:6n-3 were present at a mean of 3.78 (range 2.35-4.98)%, 8.73 (range 5.55-11.76)%, and 5.04 (range 2.84-8.11)% of total fatty acids, respectively. The remaining cis polyunsaturated fatty acids, 18:3n-3, 18:3n-6, 20:2n-6, 20:3n-9, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3 and CLA were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean of 15.21 (range 11.89-18.67)% of total fatty acids in maternal plasma PL. The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1 and represented a mean of 12.28 (range 9.10-14.53)% and 1.05 (range 0.58-2.41)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1,.each represented less than 1.0% of total fatty acids in maternal plasma PL. Saturated fatty acids represented a mean of 41.84 (range 38.90-41.84)% of total fatty acids in maternal plasma PL. Palmitic acid (16:0) was the predominant saturated fatty acid in PL and represented a mean of 30.61 (range 27.94-30.61)%' of total fatty acids. Stearic acid (18:0) represented a mean of 10.40 (range 9.37-11.81)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 14:0, 20:0, 22:0 and 24:0 were each present at less than 1.0% of total fatty acids. 65 RESULTS 8.4.3 Cholesteryl ester The mean total trans fatty acids in maternal plasma C E was 1.57 (range 0.64-3.70)% of total fatty acids (Table 8.11). Trans 18:1 and 18:2 were the predominant trans isomers identified with a mean 0.64 (range 0.18-1.71)% and 0.92 (range 0.32-2.60)% of total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 53.27 (range 44.34-66.05)% of total fatty acids in maternal plasma C E . Cis 18:2n-6 was the predominant polyunsaturated fatty acid in maternal plasma CE, and represented a mean of 42.44 (range 33.19-56.73)% of total fatty acids. Cis 20:4n-6 and 18:3n3 were present at a mean of 5.31 (range 2.48-8.37)% and 1.02 (range 0.05-2.62) % of total fatty acids, respectively. The remaining cis polyunsaturated fatty acids, 18:3n-6, 20:2n-6, 20:3n-6, 20:3n-9, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and CLA were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean of 27.89 (range 19.34-36.59)% of total fatty acids in maternal plasma C E . The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1 and represented a mean of 22.24 (range 16.66-27.14)% and 4.41 (range 2.03-11.25)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1 each accounted for less than 1.0% of total fatty acids in maternal plasma CE. Saturated fatty acids represented a mean of 18.85 (range 12.97-28.82)% of total fatty acids in maternal plasma CE. Palmitic acid (16:0) was the predominant saturated fatty acid in C E and represented a mean of 13.79 (range 10.62-16.77)% of total fatty acids followed by 18:0 and 14:0 with a mean 3.29 (range 0.87-9.70)% and 1.15 (range 0.04-1.71)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 20:0, 22:0 and 24:0, were each present at less than 1.0% of total fatty acids. 66 RESULTS Table 8.11 Fatty acid composition of maternal plasma triglyceride (TG), phospholipid (PL) and cholesteryl ester (CE) at 35 weeks gestation (g/100g)1 Fatty Acid TG PL CE 14:0 2 . 7 7 ± 0 . 1 2 (0.96-4.62) 0.39 ±0 .01 (0.18-0.64) 1.15 ± 0 . 0 4 (0.04-1.71) 16:0 29.51 ± 0 . 5 1 (19.92-38.07) 30.61 +0.15 (27.94-30.61) 13.79 ± 0 . 2 0 (10.62-16.77) 18:0 4 . 1 2 ± 0 . 1 4 (2.53-9.25) 10.40 ± 0 . 0 8 (9.37-11.81) 3.29 ± 0.33 (0.87-9.70) 20:0 0.15 ± 0.01 (0.03-0.32) 0.09 + 0.00 (0.06-0.14) 0.15 ± 0 . 0 2 (0.02-0.54) 22:0 0.12 ± 0 . 0 1 (0.05-0.30) 0.23 ±0 .01 (0.08-0.49) 0.17 ± 0 . 0 2 (0.02-0.59) cis 16:1 4.28 + 0.18 (1.48-10.07) 1.05 + 0.16 (0.58-2.41) 4.41 +0.21 (2.03-11.25) cis 18:1 35.61 ± 0 . 4 8 (26.97-43.90) 12.28 ± 0 . 1 5 (9.10-14.53) 22.24 ± 0 . 4 5 (16.66-27.14) cis 20:1 0.58 ± 0 . 0 2 (0.34-1.08) 0.20 ±0 .01 (0.12-0.36) 0.29 ± 0 . 0 3 (0.01-1.35) cis 22:1 0.16 + 0.01 (0.06-0.44) 0.09 ± 0 . 0 1 (0.02-0.28) 0.18 ± 0 . 0 2 (0.02-0.81) cis 18:2n6 13.65 ± 0 . 4 9 (7.75-26.16) 20.81 +0.40 (13.59-27.24) 42.44 ± 0 . 6 8 (33.19-56.73) cis 18:3n6 0.14 ± 0 . 0 1 (0.04-0.37) 0.06 ± 0 . 0 0 (0.00-0.12) 0.51 ± 0 . 0 3 (0.17-1.27) cis 18:3n3 1.21 ± 0 . 0 8 (0.42-3.75) 0.36 ± 0 . 0 2 (0.17-0.75) 1.02 ± 0.05 (0.05-2.62) cis 20:2n6 0.29 ± 0 . 0 1 (0.14-0.50) 0.49 ±0 .01 (0.30-0.72) 0.09 ± 0 . 0 1 (0.01-0.24) cis 20:3n6 0.25 ± 0 . 0 1 (0.16-0.44) 3.78 + 0.07 (2.35-4.98) 0.77 ±0 .01 (0.49-1.03) cis 20:3n9 0.12 ± 0 . 0 1 (0.03-0.29) 0.17 ±0 .01 (0.06-0.51) 0.10 ± 0 . 0 1 (0.03-0.27) cis 20:4n6 0.88 ± 0 . 0 4 (0.47-2.12) 8.73 + 0.17 (5:55-11.76) 5.31 ± 0 . 1 4 (2.48-8.37) cis 20:5n3 0.10 ± 0 . 0 1 (0.02-0.42) 0.52 ± 0 . 0 3 (0.11-1.64) 0.58 ± 0 . 0 5 (0.13-1.64) cis 22:4n6 0,13 ± 0 . 0 1 (0.06-0.26) 0.39 ±0 .01 (0.18-0.75) 0.04 ±0 .01 (0.00-0.61) cis 22:5n6 0.16 ± 0 . 0 1 (0.04-0.38) 0.57 ± 0 . 0 3 (0.19-1.27) 0.06 ± 0 . 0 1 (0.00-0.17) c/s 22:5n3 0 . 1 3 ± 0 . 0 1 (0.04-0.37) 0.71 ± 0 . 0 2 (0.24-1.11) 0.07 + 0.02 (0.00-0.86) c/s 22:6n3 0.53 ± 0 . 0 4 (0.23-1.40) 5.04 ± 0 . 1 7 (2.84-8.11) 0.69 ± 0 . 0 3 (0.05-1.37) CLA 2 0.59 ± 0 . 0 3 (0.23-1.41) 0.32 ±0 .01 (0.10-0.58) 0.61 ± 0 . 0 6 (0.12-1.82) trans 18:13 2.07 ± 0 . 1 2 (0.65-5.06) 1.37 ± 0 . 0 7 (0.58-3.01) 0.64 ± 0 . 0 6 (0.18-1.71) frans 18:23 1.91 ± 0 . 0 9 (0.40-3.27) 1.00 ± 0 . 0 4 (0.35-1.69) 0.92 ± 0.04 (0.32-2.60) Total frans3 3.98 ± 0 . 2 1 (1.26-7.90) 2.37 ± 0 . 1 0 (1.13-4.47) 1.57 ± 0 . 1 0 (0.64-3.70) Saturates4 37.00 ± 0 . 6 4 (26.00-47.60) 4 1 . 8 4 ± 0 . 1 4 (38.90-41.84) 18.85 ± 0 . 5 6 (12.97-28.82) MUFA 5 42.89 ± 0 . 4 3 (33.85-48.54) 15.21 ± 0 . 2 3 (11.89-18.67) 27.89 ± 0 . 5 2 (19.34-36.59) PUFA 6 20.11 ± 0 . 5 2 (13.26-33.64) 42.99 ± 0 . 2 3 (38.76-47.31) 53.27 ± 0.63 (44.34-66.05) 1 Data presented are means ± SE (range), n=58 2 CLA = conjugated linoleic acids ' Includes all trans and cis-trans geometric and positional isomers Sum of all saturates, including 12:0 and 24:0 ' Sum of all cis monounsaturates, including 14:1 and 24:1 ' Sum of all cis polyunsaturates 67 RESULTS 8.5 Relations Between Trans Fatty Acids and Cis n-6 and n-3 Fatty Acids in Maternal Plasma The maternal plasma T G percent TFA showed a significant inverse relation with cis 18:2n-6 (p<0.05) and cis 18:3n-3 (p<0.05) (Figure 8.7 and Table 8.12). There was no significant relation between the percent TFA in the maternal plasma T G and cis 20:4n-6 and 22:6n-3. The maternal plasma PL percent TFA showed no significant relation to the percent cis 18:2n-6, 18:3n-3, 20:4n-6 or 22:6n-3. The maternal plasma C E percent TFA, however, showed a significant inverse relation with cis 18:2n-6 (p<0.01), cis 20:4n-6 (p<0.05), and cis 22:6n-3 (p<0.05) and a positive relation with cis 18:3n-3 (p<0.01) (Figure 8.9). 68 R E S U L T S Table 8.12 Relations between trans fatty acids and selected cis n-6 and n-3 fatty acids in maternal plasma at 35 weeks gestation and in newborn cord plasma at birth Triglyceride Cis 18:2n-6 Cis 18:3n-3 cis 20:4n-6 cis 22:6n-3 Maternal plasma n=58 -0.531 -0.411 -0.08(n.s.) : -0.17 (n.s.) (r) Cord Plasma n=70 -0.431 -0.12 (n.s.) -0.06 (n.s.) -0.361 Phospholipid cis 18:2n-6 cis 18:3n-3 cis 20:4n-6 cis 22:6n-3 -0.19 (n.s.) -0.10 (n.s.) -0.03 (n.s.) -0.11 (n.s.) -0.05 (n.s.) 0.14 (n.s.) 0.06 (n.s.) -0.11 (n.s.) Cholesteryl ester cis 18:2n-6 cis 18:3n-3 cis 20:4n-6 cis 22:6n-3 -0.681 0.361 -0.283 -0.323 -0.351 0.07 (n.s.) -0.27 3 -0.12 (n.s.) 1 p<0.01 2 Not statistically significant 3 p<0.05 69 RESULTS 30 -i in T J 25 -o 20 -15 -total 10 --s 5 -0 -0 1 2 3 4 5 6 7 8 9 Trans fatty acids (% total fatty acids) 4 -i acids 3 ->. •2 2 -total 1 -0 -_ cis 18:3n-3 \ r = -0.41 p.< 0.01 ~i—i—i—i 0 1 2 3 4 5 6 7 8 9 Trans fatty acids (% total fatty acids) cis 20:4n-6 3 i 0 1 2 3 4 5 6 7 8 9 Trans fatty acids (% total fatty acids) •g o (0 >. CO ~ 1 cis 22:6n-3 0 1 2 3 4 5 6 7 8 9 Trans fatty acids (% total fatty acids) Figure 8.7 Scatter plots of selected cis n-6, n-3 and trans fatty acids in maternal plasma triglyceride at 35 weeks gestation, n=58 70 RESULTS 28 co 26 E 24 I 2 2 4 | 20 18 H 14 H 12 cis 18:2n-6 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) CO •g o CD CO -I—' o 0.8 0.7 0.6 H 0.5 0.4 0.3 0.2 0.1 cis 18:3n-3 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) 13 12 -11 -10 9 -8 -7 6 5 cis 20:4n-6 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) co 8 "D o CO ro o 7 6H 5 4 3 2 c/s 22:6n-3 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) Figure 8.8 Scatter plots of selected cis n-6, n-3 and trans fatty acids in maternal plasma phospholipid at 35 weeks gestation, n=581 1 No statistically significant relations were found. 71 RESULTS 60 in 55 ro 50 f 45 1 40 o £ 3 5 - 1 30 r = -0.68 p<0.01 #• ft. 0 1 2 3 4 Trans fatty acids (% total fatty acids) CO I c CO 00 4 3 2H 1 0 c/s 18:3n-3 r = 0.36 p<0.01 0 1 2 3 4 Trans fatty acids (% total fatty acids) in _J o ro >. -*-« o c/s 20:4n-6 0 1 2 3 4 Trans fatty acids (% total fatty acids) 3 -| in T J 2 -aci >, .2 1 -"ro 0 -"5 -1 -c/s 22:6n-3 r = -0.32 p<0.05 0 1 2 3 4 Trans fatty acids (% total fatty acids) Figure 8.9 Scatter plots of selected c/s n-6, n-3 and trans fatty acids in maternal plasma cholesteryl ester at 35 weeks gestation, n=58 72 RESULTS 8.6 Cord Plasma Fatty Acids 8.6.1 Triglyceride The mean total trans fatty acids in cord plasma T G was 2.87 (range 0.64-12.79)% of total fatty acids (Table 8.13). Trans 18:1 and 18:2 were the predominant trans isomers, and represented a mean of 1.63 (range 0.06-9.61)% and 1.23 (range 0.12-3.18)% of total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 22.62 (range 8.39-31.34)% of total fatty acids in cord plasma TG. Cis 18:2n-6 was the predominant polyunsaturated fatty acid in cord plasma TG, and represented a mean of 10.09 (range 2.93-17.00)% of total fatty acids. Cis 20:4n-6 and 22:6n-3 were present at a mean 3.58 (range 1.04-7.26)% and 2.77 (range 0.49-5.91)% of total fatty acids. The remaining cis polyunsaturated fatty acids 18:3n-3, 18:3n-6, 20:2n-6, 20:3n-6, 20:3n-9, 20:5n-3, 22:4n-6, 22:5n-6 and 22:5n-3 were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean of 41.58 (range 33.63-61.98)% of total fatty acids in cord plasma TG. The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1, and represented a mean of 33.23 (range 25.34-54.64)% and 5.56 (range 2.46-9.29)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1, each represented less than 1.0% of total fatty acids in cord plasma TG. Saturated fatty acids represented a mean of 35.80 (range 29.63-45.96)% of total fatty acids in cord plasma TG. Palmitic acid (16:0) was the predominant saturated fatty acid in T G and represented a mean of 25.73 (range 18.70-31.30)% of total fatty acids followed by 18:0 and 14:0 with a mean 7.00 (3.95-12.74)% and 1.69 (range 0.60-3.88)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 20:0, 22:0 and 24:0, were each present at less than 1.0% of total fatty acids. 73 RESULTS 8.6.2 Phospholipid The mean total trans fatty acids in cord plasma PL was 0.67 (range 0.11-1.33)% of total fatty acids (Table 8.13). Trans 18:1 and 18:2 were the predominant trans isomers present with a mean of 0.40 (range 0.04-0.97)% and 0.27 (range 0.03-0.52)% of total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 42.51 (range 38.22-45.52)% of total fatty acids in cord plasma PL. Cis 20:4n-6 was the predominant polyunsaturated fatty acid in cord plasma PL, and represented a mean of 17.71 (range 13.79-23.04)% of total fatty acids. Cis 22:6n-3, 18:2n-6 and 20:3n-6 were present at a mean 7.65 (range 4.35-11.67)%, 7.52 (range 5.04-10.66)%, and 5.72 (range 2.55-9.96)% of total fatty acids, respectively. The remaining cis polyunsaturated fatty acids, 18:3n-3, 18:3n-6, 20:2n-6, 20:3n-9, 20:5n-3, 22:4n-6, 22:5n-6 and 22:5n-3, were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean of 12.62 (range 9.99-17.38)% of total fatty acids in cord plasma PL. The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1, and represented a mean of 11.19 (range 9.20-15.49)% and 1.01 (range 0.64-1.70)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1, each represented less than 1.0% of total fatty acids in cord plasma PL. Saturated fatty acids represented a mean of 44.68 (range 42.86-46.18)% of total fatty acids in cord plasma PL. Palmitic acid (16:0) was the predominant saturated fatty acid in PL and represented a mean of 28.82 (range 26.10-31.00)% of total fatty acids followed by 18:0 with a mean 14.94 (range 13.04-16.80)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 14:0, 20:0, 22:0 and 24:0, were each present at less than 1.0% of total fatty acids. 74 RESULTS 8.6.3 Cholesteryl ester The mean total trans fatty acids in cord plasma C E was 2.04 (range 0.72-4.30)% of total fatty acids (Table 8.11). Trans 18:1 and 18:2 were the predominant trans isomers present, with a mean of 1.00 (range 0.11-2.94)% and 1.04 (range 0.13-3.10)% of total fatty acids, respectively. Polyunsaturated fatty acids represented a mean of 33.19 (range 19.21-42.35)% of total fatty acids in maternal plasma C E . Cis 18:2n-6 and 20:4n-6 were the predominant polyunsaturated fatty acid in maternal plasma C E , and represented a mean of 15.60 (range 8.97-22.72)% and 11.79 (range 2.05-17.40)% of total fatty acids, respectively. Cis 22:6n-3 and 20:3n-6 were present at a mean 1.44 (range 0.55-3.14) and 1.34 (range 0.40-2.51)% of total fatty acids, respectively. The remaining cis polyunsaturated fatty acids, 18:3n-6, 18:3n-3, 20:2n-6, 20:3n-9, 20:5n-3, 22:4n-6, 22:5n-6 and 22:5n-3 were each present at less than 1.0% of total fatty acids. Monounsaturated fatty acids represented a mean of 38.39 (range 29.58-56.03)% of total fatty acids in cord plasma CE. The predominant monounsaturated fatty acids were the cis isomers of 18:1 and 16:1, and represented a mean of 30.00 (range 22.30-48.90)% and 6.94 (range 2.71-11.16)% of total fatty acids, respectively. The remaining monounsaturated fatty acids, 14:1, 20:1, 22:1 and 24:1, each represented less than 1.0% of total fatty acids in cord plasma CE. Saturated fatty acids represented a mean of 28.41 (range 24.52-35.05)% of total fatty acids in cord plasma C E . Palmitic acid (16:0) was the predominant saturated fatty acid in CE and represented a mean of 20.66 (range 16.55-25.49)% of total fatty acids followed by 18:0 and 14:0 with a mean 5.26 (range 2.71-8.89)% and 1.06 (range 0.51-3.38)% of total fatty acids, respectively. The remaining saturated fatty acids, 12:0, 20:0, 22:0 and 24:0, were each present at less than 1.0% of total fatty acids. 75 RESULTS 8.7 Relations Between Cord Plasma Trans Fatty Acids and C/s n-6 and n-3 Fatty Acids The cord plasma T G percent TFA showed a significant inverse correlation to the percent cis 18:2n-6 (p<0.01) and cis 22:6n-3 (p<0.01) (Table 8.12 and Figure 8.10). The cord plasma PL percent TFA showed no significant relation to the percent cis 18:2n-6, 18:3n-3, 20:4n-6 or22:6n-3. The cord plasma C E percent TFA, however, showed a significant inverse relation to the percent cis 18:2n-6 (p<0.01) and cis 20:4n-6 (p<0.05). (Figure 8.11). 76 RESULTS Table 8.13 Fatty acid composition in cord plasma triglyceride (TG), phospholipid (PL) and cholesteryl ester (CE) of term gestation infants at birth (g/100g)1 Fatty Acid TG PL C E 12:0 0.22 ± 0 . 0 3 (0.00-0.92) 0.02 ± 0.00 (0.00-0.08) 0.32 ± 0 . 0 3 (0.00-1.19) 14:0 1.69 ± 0 . 0 7 (0.60-3.88) 0.24 ±0 .01 (0.16-0.51) 1.06 ± 0 . 0 5 (0.51-3.38) 16:0 25.73 ± 0 . 3 1 (18.70-31.30) 28.82 + 0.12 (26.10-31.00) 20.66 ± 0 . 1 7 (16.55-25.49) 18:0 7.00 ± 0 . 2 2 (3.95-12.74) 14.94 ± 0 . 0 9 (13.04-16.80) 5.26 ± 0 . 1 5 (2.71-8.89) 20:0 0.36 ± 0 . 0 3 (0.11-1.22) 0.16 ± 0 . 0 0 (0.10-0.30) 0.34 ± 0.04 (0.08-2.50) 22:0 0.53 ± 0 . 0 3 (0.12-1.22) 0.30 ± 0 . 0 1 (0.08-0.57) 0.52 + 0.03 (0.03-1.48) 24:0 0.28 ± 0.01 (0.00-0.68) 0.19 + 0.01 (0.07-0.29) 0.25 ± 0 . 0 2 (0.03-0.78) c/s 16:1 5.56 ± 0 . 1 7 (2.46-9.29) 1.01 ± 0 . 0 2 (0.64-1.70) 6.94 ± 0 . 1 9 (2.71-11.16) cis 18:1 33.23 ± 0.74 (25.34-54.64) 11.19 ± 0 . 1 5 (9.20-15.49) 30.00 ± 0 . 5 6 (22.30-48.90) cis 20:1 0.34 ± 0 . 0 3 (0.02-1.06) 0.07 ± 0 . 0 0 (0.02-0.13) 0.08 ± 0 . 0 2 (0.00-0.70) cis 22:1-n9 0.83 ± 0 . 1 0 (0.00-3.54) 0.08 ± 0 . 0 1 (0.00-0.26) 0.37 ± 0 . 0 5 (0.00-2.07) cis 18:2n6 10.09 ± 0 . 3 5 (2.93-17.00) 7.52 ± 0 . 1 5 (5!04-10.66) 15.60 ± 0 . 3 5 (8.97-22.72) cis 18:3n6 0.37 ± 0 . 0 2 (0.13-0.87) 0.07 ± 0.00 (0.02-0.20) 0.61 ± 0 . 0 3 (0.08-1.27) cis 18:3n3 0.48 ± 0 . 0 2 (0.16-0.96) 0.05 ± 0 . 0 0 (0.02-0.18) 0.15 ± 0 . 0 1 (0.00-0.48) cis 20:2n6 0.30 ± 0 . 0 2 (0.14-0.91) 0.38 ± 0.01 (0.24-0.54) 0.10 ± 0 . 0 1 (0.00-0.49) cis 20:3n6 0.86 ± 0 . 0 3 (0.28-1.44) 5.72 ± 0 . 1 3 (2.55-9.96) 1.34 ± 0 . 0 4 (0.40-2.51) cis 20:3n9 0.55 ± 0 . 0 2 (0.21-0.99) 0.60 ± 0 . 0 4 (0.11-1.97) 0.28 ± 0.02 (0.04-0.88) cis 20:4n6 3.58 ± 0 . 1 6 (1.04-7.26) 17.71 ± 0 . 2 4 (13.79-23.04) 11.79 ± 0 . 3 7 (2.05-17.40) cis 20:5n3 0.30 ± 0 . 0 2 (0.03-1.33) 0.38 ± 0 . 0 2 (0.06-1.47) 0.33 ± 0 . 0 3 (0.09-1.46) cis 22:4n6 0.62 ± 0 . 0 3 (0.16-1.36) 0.74 ± 0 . 0 2 (0.47-1.16) 0 : 0 3 ± 0 . 0 1 (0.00-0.57) cis 22:5n6 0.93 ± 0 . 0 4 (0.36-1.64) 0.86 ± 0 . 0 4 (0.26-1.68) 0.13 ± 0 . 0 1 (0.00-0.90) c/s 22:5n3 0.52 ± 0 . 0 3 (0.18-1.37) 0.64 ± 0 . 0 3 (0.20-1.38) 0.36 ± 0 . 0 7 (0.00-3.11) cis 22:6n3 2.77 ± 0 . 1 5 (0.49-5.91) 7.65 ± 0 . 2 0 (4.35-11.67) 1.44 ± 0 . 0 7 (0.55-3.14) Trans 18:12 1.63 ± 0 . 1 5 (0.06-9.61) 0.40 ± 0.02 (0.04-0.97) 1.00 ± 0 . 0 8 (0.11-2.94) Trans 18:22 1.23 ± 0 . 0 7 (0.12-3.18) 0.27 ± 0.01 (0.03-0.52) 1.04 ± 0 . 0 5 (0.13-3.10) Total trans2 2.87 ± 0 . 1 9 (0.64-12.79) 0.67 ± 0 . 0 3 (0.11-1.33) 2.04 ±0 .11 (0.72-4.30) Saturates3 35.80 ± 0.40 (29.63-45.96) 44.68 ± 0 . 1 1 (42.86-46.18) 28.41 ± 0 . 3 0 (24.52-35.05) MUFA 4 41.58 ± 0 . 7 1 (33.63-61.98) 12.62 ± 0 . 1 8 (9.99-17.38) 38.39 ± 0 . 6 0 (29.58-56.03) PUFA 5 22.62 ± 0 . 6 1 (8.39-31.34) 42.51 ± 0 . 1 9 (38.22-45.52) 33.19 ± 0 . 6 1 (19.21-42.35) 1 Data are presented as means ± S.E. (range), n=70 2 includes all trans and cis-trans geometric and positional isomers 3 sum of all saturates 77 RESULTS - a 'o CD If 10 CD o c/s 18:2n-6 • V -1 1— 2 4 6 8 10 12 14 Trans fatty acids (% total fatty acids) 1.0 ] 0.9 -| •g 0.8 '8 0.7 0 6 ~ .2 0.5 -JS 0.4 2 0.3 # 0.2 0.1 0 2 4 6 8 10 12 14 Trans fatty acids (% total fatty acids) m •o o CD >* CD -t—' O 8 -i 7 -6 -5 4 3H 2 1 c/s 20:4n-6 0 2 4 6 8 10 12 14 Trans fatty acids (% total fatty acids) T3 O CD CD O c/s 22:6n-3 r = -0.36 p<0.01 0 2 4 6 8 10 12 14 Trans fatty acids (% total fatty acids) Figure 8.10 Scatter plots of selected cis n-6, n-3 fatty and trans fatty acids in cord plasma triglyceride at birth, n=70 78 RESULTS in T> O CD >, t i & ro —^< o 11 10 9 8 -7 -6 -5 -4 -3 2H 1 c/s 18:2n-6 0 1 2 3 Trans fatty acids (% total fatty acids) in o ro >. ro o 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 c/s 18:3n-3 V%1 I . I I I 0 1 2 3 Trans fatty acids (% total fatty acids) 21 0 J 1 1 1 1 2 -I 1 1 1 0 1 2 3 0 1 2 3 Trans fatty acids (% total fatty acids) Trans fatty acids (% total fatty acids) Figure 8.11 Scatter plots of selected cis n-6, n-3 and trans fatty acids in cord plasma phospholipid at birth, n=701 1 No statistically significant relations were found. 79 RESULTS c/s 18:2n-6 24 -, 22 -in 20 -XI 18 -o CO 16 ->1 14 -12 -10 -"co 8 -"o 6 -%o 4 -2 -0 -r = -0.35 p<0.01 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) 0.6 -, in 0.5 -TJ o 0.4 -to >» 0.3 -JD «•— 0.2 -"<5 t> +-< 0.1 -0.0 -c/s 18:3n-3 _ • • • • 0 1 2 3 4 5 Trans fatty acids (% total fatty acids) Figure 8.12 Scatter plots of selected cis n-6, n-3 and trans fatty acids in cord plasma cholesteryl ester at birth, n=70 80 RESULTS 8.8 Relations Between Selected Fatty Acids in Maternal and Cord Plasma A significant, positive relation was present between the level of cis 20:4n-6 in maternal and cord plasma T G (r = 0.35) and between the level of cis 22:6n-3 in maternal and cord plasma T G (r = 0.50) (Table 8.14 and Figure 8.12). Significant associations were also present between the percent of each of cis 18:2n-6 (r = 0.51), 20:4n-6 (r = 0.48) and 22:6n-3 (r = 0.48) in maternal and cord plasma PL as well as between total trans isomers in maternal and cord plasma PL (r = 0.53) (Figure 8.13). A significant association was also present between the levels of cis 18:2n-6 (r = 0.36) and 18:3n-3 (r = 0.33) in maternal and cord plasma in C E (Figure 8.14). No other statistically significant relations were present between maternal and cord plasma 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 in TG, PL or C E . Table 8.14 Relations between trans fatty acids, cis n-6 and n-3 fatty acids in maternal plasma at 35 weeks gestation and cord plasma lipids at birth1 Triglyceride n=46 Phospholipid n=46 Cholesteryl ester n=46 18:2n-6 0.04 0.513 0.362 18:3n-3 0.22 -0.23 0.332 20:4n-6 0.352 0.483 0.21 22:6n-3 0.503 0.483 0.26 total trans 0.07 0.533 -0.02 isomers 1 Values given are Pearson correlations (r) 2p<0.05 3p<0.01 81 RESULTS co TJ 30 O ra 25 ra 20 -"ra *-> o 15 -+-> 10 -"in c 5 -cu ro 0 2 0 cis 18:2n-6 • • • • • - i — i — i — i — i — i — i — i — i 2 4 6 8 10 12 14 16 18 Cord (% total fatty acids) 4 i co TJ O ra £ 3 ra "ro o 2 m 1 cis 20:4n-6 r = 0.35 p<0.05 0 1 2 3 4 5 6 7 Cord (% total fatty acids) £ 3 ro o 2 ro ' c 03 ro o cis 18:3n-3 i i i i — i i i i i i 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Cord (% total fatty acids) CO TJ ro o to 0 cis 22:6n-3 r = 0.50 p<0.01 • 5 V 0 1 2 3 4 5 6 7 Cord (% total fatty acids) .co g trans fatty acids o ro 8H t 7 6H a 5 2 4 * 3 ro 2 I 1 ro o . • ' • • I f * 0 1 2 3 4 5 Cord (% total fatty acids) 1 not statistically significant Figure 8.13 Scatter plots of selected fatty acids in triglyceride in maternal plasma at 35 weeks gestation and cord plasma at birth, n=46 82 RESULTS in XJ o CO CO o 28 -i 26 24 22 20 H 18 16 14 12 10 8 6 4 V cis 18:2n-6 r = 0:51 p<0.01 1 2 3 4 5 6 7 8 9 10 Cord (% total fatty acids) o ro CO o 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 cis 18:3n-3 "!! 0.00 0.05 0.10 0.15 Cord (% total fatty acids) 0.20 13 _ o 12 -ro 11 -10 -ro 9 -8 _ ro -t-» 7 -o •»-» 6 -VO 5 _ 0s 4 -"ro 3 -c 2 -CD 1 -ro 0 2 0 cis 20:4n-6 cis 22:6n-3 • ft r = 0.48 p<0.01 - i 1 1 r — i i 1 1 1 1 1 2 4 6 8 10 12 14 16 18 20 22 Cord (% total fatty acids) "ro H—1 o 9 -i 8 7-\ 6 :1 3 2 1 0 r = 0.48 p<0.01 - i 1 r—r-0 1 2 3 4 5 6 7 8 9 10 11 12 Cord (% total fatty acids) -a 5 o ro ro o 4 3 ^ 2 C 1 _ CD 0 frans fatty acids r = 0.53 p<0.01 1 2 Cord (% total fatty acids) Figure 8.14 Scatter plots of selected fatty acids in phospholipid in maternal plasma at 35 weeks gestation and cord plasma at birth, n=46 83 RESULTS (0 TJ 60 -i o 55 -ro 50 -45 -40 -'o total 35 -30 -25 -20 -"ro 15 -E 10 -CD 5 -ro 0 -cis 18:2n-6 r = 0.36 p<0.05 0 2 4 6 8 10 12 14 16 18 20 22 24 Cord (% total fatty acids) ° R ro ° f 6 ro 5 5 4 £ 3 ro 2 E 1 * n ro 0 c/s 20:4n-6 2 4 6 8 10 12 14 16 18 Cord (% total fatty acids) o ro c/s 18:3n-3 r = 0.33 p<0.05 0.0 0.1 0.2 0.3 0.4 0.5 Cord (% total fatty acids) •g 3 o ro 45 2 3 £ ^ 1 "ro E o ro 0 2 c/s 22:6n-3 1 2 3 Cord (% total fatty acids) in TJ 4 -, aci fatty 3 -total 2 -1 -temal ro 5 0 -0 frans fatty acids •••• • 1 2 3 4 Cord (% total fatty acids) Figure 8.15 Scatter plots of selected fatty acids in cholesteryl ester in maternal plasma at 35 weeks gestation and cord plasma at birth n=46 84 . . RESULTS 8.9 Relations Between Trans Fatty Acid Intake and Level of Trans Fatty Acids in Maternal Plasma Lipids 8.9.1 Triglyceride A significant association was present between the dietary TFA intake and the level of TFA in maternal plasma T G at 28 weeks gestation (r = 0.40) and at 35 weeks gestation (r = 0.31) (Table 8.15). When these relations were adjusted for total energy intake, the relations were still significant, r = 0.45 and 0.33 at 28 and 35 weeks, respectively. 8.9.2 Phospholipid A significant association was present between the dietary TFA intake and the level of TFA in maternal plasma PL at both 28 weeks gestation (r = 0.36) and at 35 weeks gestation (r = 0.35). When these relations were adjusted for total energy intake, the relations were still significant, r = 0.40 and 0.39 at 28 and 35 weeks, respectively. 8.9.3 Cholesteryl ester As for the plasma triglyceride and phospholipid, a significant association was present between the dietary TFA intake and level of TFA in maternal plasma C E at 28 weeks gestation, r = 0.26. However, when the intake of TFA was adjusted for total energy intake, the relation between TFA intake and cord plasma C E TFA was statistically significant (r = 0.34, p<0.05). 8.10 Relations Between Trans Fatty Acid Intake and Level of Trans Fatty Acids in Cord Plasma Lipids A significant association was present between the dietary TFA intake at 28 weeks gestation and level of TFA in cord plasma PL (r = 0.31) both without and with 85 RESULTS adjustment for total energy intake (r = 0.38). No significant association was present between the dietary intake of TFA at 35 weeks gestation and level of TFA in cord plasma PL. However, when the intake of TFA was adjusted for total energy a statistically significant relation (r = 0.36, p<0.05) was present. There were no other statistically signification relations present between dietary intake of TFA without or with adjustment for energy intake, at either 28 or 35 weeks gestation and the cord plasma TG, PL or C E level of TFA. Table 8.15 Relations between trans fatty acid (TFA) intake at 28 and 35 weeks gestation and trans fatty acids in maternal plasma at 35 weeks gestation and cord plasma at birth1 ' Plasma lipid TFA Intake (g) TFA Intake (g) adjusted for • energy 28 weeks 35 weeks 28 weeks 35 weeks Triglyceride Maternal 0.392 0.313 0.452 0.333 Cord 0.08 0.02 0.14 0.05 Phospholipid Maternal 0.362 0.352 0.392 0.392 Cord 0.313 0.21 0.383 0.363 Cholesteryl ester Maternal 0.263 0.24 0.26 0.343 Cord 0.08 0.03 0.20 0.06 1 Pearson correlation coefficients, r 2p<0.01 3 p<0.05 86 RESULTS 8.11 Dietary Trans Fatty Acid Intake and Birth Outcomes There were no statistically significant relations between the maternal dietary TFA intakes and infant birthweight, length, head circumference or length of gestation. 8.12 Relations Between Cord Plasma Lipid Fatty Acids and Birth Outcomes 8.12.1 Triglyceride A significant relation was present between the length of gestation and cis 20:4n-6 (r = 0.40, p<0.01) and the total trans isomers (r = -0.28, p<0.05) in cord plasma T G (Table 8.16). A significant inverse relation was also present between the isomers of CLA in cord plasma T G and the length of gestation (r = -0.44, p<0.01) (Figure 8.16), between cis 20:4n-6 and infant birthweight (r = 0.37, p<0.01) (Figure 8.17), and between cis 20:4n-6 (r = 0.52, p<0.01) and cis 20:5n-3 (r = 0.37, p<0.05) and infant length (Figure 8.18). No significant relations were found between infant head circumference and the cord plasma T G levels of TFA, CLA, cis n-6 or n-3 fatty acids (Figure 8.19). 87 RESULTS Table 8.16 Relations between selected fatty acids in cord plasma triglyceride at birth and birth outcomes Birth Outcome Fatty acid n=46 1 n=70 2 Length of gestation (wk) 18:2n-6 0.27 0.23 20:4n-6 0.503 0.403 18:3n-3 0.13 0.13 20:5n-3 0.27 -0.20 22:6n-3 0.23 0.09 20:5n-3/20:4n-6 0.00 -0.06 Total trans -0.19 -0.284 C L A 5 -0.443 — Birth weight (g) 18:2n-6 -0.03 -0.07 20:4n-6 0.403 0.373 18:3n-3 0.12 0.09 20:5n-3 0.22 0.17 22:6n-3 0.10 -0.03 20:5n-3/20:4n-6 0.04 -0.03 Total trans -0.03 0.06 CLA -0.21 — Infant length (cm) 18:2n-6 0.07 — 20:4n-6 0.523 — 18:3n-3 0.25 — 20:5n-3 0.374 — 22:6n-3 0.27 — 20:5n-3/20:4n-6 0.16 — Total trans -0.03 CLA -0.24 — Head circumference (cm) 18:2n-6 -.042 — 20:4n-6 .181 — 18:3n-3 .062 — 20:5n-3 .040 — 22:6n-3 .068 — 20:5n-3/20:4n-6 -.094 Total trans -.034 — CLA -.183 — 1 Cord plasma from Study Two 2 Cord plasma from Study One and Two 3p<0.01 4 p<0.05 5 Conjugated linoleic acid 88 RESULTS 0 18 £ 1 6 f 14 SO 12 — 10 CD 1 8 ^ 6 oo ^ 4 o 2 "S o c/s 18:2n-6 8 I s Hf. t 35 36 37 38 39 40 41 42 43 Length of gestation (wk) cn "g o co .to CO 66 w Q •a o O i.o 1 0.9 -| 0.8 0.7 0.6 0.5 0.4 0.3 H 0.2 0.1 0.0 c/s 18:3n-3 35 36 37 38 39 40 41 42 43 Length of gestation (wk) TD 8 i 7 6H 5 4 3 2 1 0 c/s 20:4n-6 35 36 37 38 39 40 41 42 43 Length of gestation (wk) o 7 n  CO CO CO (Si CNI o o 5 -4 -3 2 -1 -0 c/s 22:6n-3 —i ; 1 1 1 1—;—i 1 35 36 37 38 39 40 41 42 43 Lengthof gestation (wk) CO fatty aci 14 -i 12 --9 10 -CO T3 8 -O CO 6 -CO 4 -trans 2 -0 -1— o O trans fatty acids 35 36 37 38 39 40 41 42 43 Length of gestation (wk) «? 3 o CO "o o O 2 H c/s 20:5n-3 i d * 35 36 37 38 39 40 41 42 Length of gestation (wk) 43 Figure 8.16 Scatter plots of length of gestation and selected fatty acids in cord plasma triglyceride at birth, n=70 89 RESULTS TJ o co 49 <? c CN GO CO G TJ O O 18 16 14 -12 -10 8 6H 4 2 0 c;s 18:2 n-6 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) TJ CO 49 ro <_ CO 66 co G XI o O 1.0 -, 0.9 0.8 -I 0.7 -I 0.6 0.5 0.4 0.3 H 0.2 0.1 0.0 c/s 18:3n-3 • Aw 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) tn T J o co >* .CO CD c •*t o CN .<o 0 •a o O tn ~o o co >^  t: 42 tn T J o co c T J i o O 8 7 6 5 4 3 -2 -1 c/s 20:4n-6 ( r = 0.37 • • * * p<0.05 • 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 14 -, 12 10 8 6 4 2H trans fatty acids — i 1 1 1 1 1 1 1500 2000 2500 3000 3500 4000 4500 5000 tn T J o CD (Si CN 7 6H 5 4 3 2 1 E 0 c/s 22:6n-3 T J O CO >. .CO CO c in o CM G T J 1 o o 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 3 c/s 20:5n-3 i _ i . — i — i . 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) Birthweight (g) Figure 8.17 Scatter plots of infant birthweight and selected fatty acids in cord plasma triglyceride at birth, n=70 90 RESULTS CO TD O CO > . 40 "co o o O 18 16 14 • 12 10 8H 6 4 2 H 0 44 cis 18:2n-6 • v . > — i — 1 — 1 — i — 1 — 1 — i 46 48 50 52 54 56 58 TD O CO > , CO o o O 1.0 0.9 0.8 -I 0.7-j 0.6 0.5 0.4 H 0.3 0.2 0.1 0.0 c/s 18:3n-3 * A •* PS 44 46 48 50 52 54 56 58 Infant length (cm) . Infant length (cm) <o TD O CO > . CO o TJ i o O c/s 20:4n-6 r = 0.52 p<0.05 1 r 1 1 1 1 1 44 46 48 50 52 54 56 58 Infant length (cm) ,—. 7 -j CO TD O 6 -CO 5 -4 -CO -4—1 0 3 --*-« 2 -• — 1 -O O 0 -c/s 22:6n-3 • • * * 44 46 48 50 52 54 56 58 Infant length (cm) (spi 6 -j aci 5 ->* *s •B 4 -"S 3 -(% tol 2 -Cord 1 -0 -frans fatty acids • • • 44 46 48 50 52 54 56 58 Infant length (cm) cn TD CJ CO > . CO o o o 1.0 0.9 -I 0.8 -| 0.7 0.6 0.5 0.4 0.3 A 0.2 -I 0.1 0.0 c/s 20:5n-3 r = 0.37 p<0.01 — i — 54 44 46 48 50 52 Infant length (cm) 56 58 Figure 8.18 Scatter plots of infant length and selected fatty acids in cord plasma triglyceride at birth, n=44 91 RESULTS ^ ^ 18 i in -a 16 -o to 14 ->. a 42 12 -To 10 -o 8 -3- 6 -XI 4 -b O 2 -c/s 18:2n-6 • • • ! 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 1.0 -j XJ 0.9 -o to 0.8 ->. 3=s 0.7 -49 0.6 -to 0.5 -o 0.4 -0.3 -T J 0.2 -i o O 0.1 -c/s 18:3n-3 — i — i — i — i — i — i — i — i 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 7 -i in XI 6 -o to >. 5 -a a 4 -CO ~o 3 --s 2 -XI 1 -b o 0 -c/s 20:4n-6 , . . . t..h — i — i — i — i — i — i — i — i 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) in x> o co >» V 45 CO o XI o O c/s 22:6n-3 * . 1 * — i — i — i — i — i — i — i — i — i 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) to XI o CO >, ti 49 CO -4-« o XI o O 6 i 5 -4 3 2 1 0 trans fatty acids • IT . i -30 31 32 33 34 35 36 37 38 39 Head circumference (cm) ids) 1.0-i 0.9 -CJ to 0.8 -fatty 0.7 -0.6 -to 0.5 -0.4 ->S 0.3 -Cord (< 0.2 -0.1 -0.0 -c/s 20:5n-3 i . i r 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) Figure 8.19 Scatter plots of infant head circumference and selected fatty acids in cord plasma triglyceride at birth, n=451 1 No statistically significant relations were found. 92 _ _ _ RESULTS 8.12.2 Phospholipid A significant inverse relation was found between the cord plasma PL cis 18:2n-6 and length of gestation (r = -0.25, p<0.05) (Figure 8.20), infant birthweight (r = -0.39, p<0.01) (Figure 8.21) and infant length at birth (r = - 0.38, p<0.05) (Figure 8.22 and Table 8.17). No significant relations were found between the infant head circumference and the cord plasma PL level of TFA, CLA, cis n-6 or n-3 fatty acids (Figure 8.23). 8.12.3.Cholesteryl ester A significant relation was found between the length of gestation and the cord plasma C E cis 20:4n-6 (r = 0.33, p<0.01) and total trans isomers (r = -0.25, p<0.05) (Table 8.18 and Figure 8.24). A significant relation was also found between the cord plasma C E cis 20:4n-6 and infant birthweight (r = 0.26, p<0.01) (Figure 8.25) and infant length (r = 0.43, p<0.01), as well as between cis 22:6n-3 and infant length (r = 0.31, p<0.05) (Figure 8.26). A significant inverse relation was found between the cord plasma C E isomers of CLA and length of gestation (r = -0.52, p<0.01) infant birthweight (r = -0.35, p<0.05) and infant iength (r = -0.35, p<0.05) (Figure 8.28). No significant relations were present between the infant head circumference and the cord plasma C E level of TFA, CLA, cis n-6 or n-3 fatty acids (Figure 8.27). 93 RESULTS Table 8.17 Relations between selected fatty acids in cord plasma phospholipid at birth and birth outcomes Birth Outcome Fatty acid n=46 1 n=70 r Length of gestation (wk) 18:2n-6 -0.28 -0.25 3 20:4n-6 -0.04 0.03 18:3n-3 -0.15 -0.04 20:5n-3 -0.04 -0.14 22:6n-3 0.14 0.19 20:5n-3/20:4n-6 -0.03 -0.12 Total trans -0.09 -0.16 C L A 4 0.08 Birth weight (g) 18:2n-6 -0.45 5 -0.39 3 . 20:4n-6 0.00 0.02 18:3n-3 -0.12 0.01 20:5n-3 0.07 -0.05 22:6n-3 0.14 0.13 20:5n-3/20:4n-6 0.05 -0.01 Total trans -0.10 -0.02 CLA 0.15 Infant length (cm) 18:2n-6 -0.381 20:4n-6 0.03 18:3n-3 -0.17 20:5n-3 0.16 22:6n-3 0.22 20:5n-3/20:4n-6 0.08 Total trans -0.11 CLA 0.17 Head Circumference (cm) 18:2n-6 -0.17 20:4n-6 -0.03 18:3n-3 0.05 20:5n-3 0.01 22:6n-3 -0.15 20:5n-3/20:4n-6 -0.04 Total trans . -0.08 CLA -0.18 1 Cord plasma from Study Two 2 Cord plasma from Study One and Two 3p<0.05 4 Conjugated linoleic acid 5p<0.01 94 R E S U L T S 11 n 10 9 8 7 6 5 4 H 3 2 1 0 35 c/s 18:2n-6 r = -0.25 p<0.05 36 37 38 39 40 41 42 43 Length of gestation (wk) acids) 0.20 -0.18 -0.16 -49 0.14 -0.12 -CO 0.10 -o ~s 0.08 -0.06 -T l 0.04 -Core 0.02 -0.00 -c/s 18:3n-3 _——-j i i 11 i 35 36 37 38 39 40 41 42 43 Length of gestation (wk) c/s 20:4n-6 c/s 22:6n-3 (sp! 24 -i (sp! 22 -o 20 -tty a 18 -16 -45 14 -CO 12 -o 10 -8 -6 -XJ 4 -O 2 -o 0 -' t i l t 35 36 37 38 39 40 41 42 43 Length of gestation (wk) >—. 14 -i (/> • a o 12 -CO $ 10 -45 8 -43 "o 6 -4 -_ 2 -O O 0 -1 • I. •* 35 36 37 38 39 40 41 42 43 Length of gestation (wk) St 3 •g o 2 H trans fatty acids cis 20:5n-3 • • •• 35 36 37 38 39 40 41 42 43 Length of gestation (wk) * 3 i •g o co >, , ti -.co CO o o O 35 36 37 38 39 40 41 42 43 Length of gestation (wk) Figure 8.20 Scatter plots of length of gestation and selected fatty acids in cord plasma phospholipid at birth, n=70 9 5 RESULTS c/s 18:2n-6 o 1 " O 0 -I 1 1 1 1 1 1 1 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) ~ 0 20 n c / s 1 8 : 3 n " 3 CO ^ 0.18 - • co 0.16 -0.14 -B 0.12 -15 0.10 - • o 0.08- • ^ IE: ' * ' O 0.00 -I 1 1 1 1 1 1 1 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) (spi 24 -, 22 -o 20 -CO 18 -16 -B 14 -CO 12 --I-1 o 10 -8 -3- 6 -"D 4 -i — O 2 -O 0 -c/s 20:4n-6 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 14 </) T3 12 -O CO >, 10 -*3 B 8 -co H—' o 6 -0 s 4 -T D i 2 -o O 0 -c/s 22:6n-3 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 3 i T3 o o 0 frans fatty acids 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) ids) 3 -i aci •B 2 -CO (% tol 1 -Cord 0 -c/s 20:5n-3 i r 1 1 1 1 1 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) Figure 8.21 Scatter plots of infant birthweight and selected fatty acids in cord plasma phospholipid at birth, n=70 9 6 RESULTS ,—. 10 - i 9 -ac 8 ->* 7 -B 6 -"(0 5 -o 4 -3 -— 2 -o 1 -O 0 -c/s 18:2n-6 r = -0.38 p<0.05 - i 1 1 1— — 0 20 ^ 0.18 ro 0.16 >< 0.14 <S 0.12 c/s 18:3n-3 ro 0.10 0.08 £ 0.06 - 0.04 c3 0.02 O 0.00 -1 1 r-44 46 48 50 52 54 56 58 44 46 48 50 52 54 56 58 Infant length (cm) Infant length (cm) 22 20 18 16 14 12 10 8 6 4H 2 0 c/s 20:4n-6 — i 1 1 1 1 1 1 44 46 48 50 52 54 56 58 Infant length (cm) (spi 12 -. 11 -o 10 -ya 9 -8 -B 7 -ro 6 -o 5 --»-< 4 -o - 3 -"O 2 -O 1 -o 0 -c/s 22:6n-3 * • tin"-*: ~i 1 r 44 46 48 50 52 54 56 58 Infant length (cm) T r 3 trans fatty acids 2H 1 H • - • • t _1 • • • • 44 46 48 50 52 54 56 58 Infant length (cm) S 2-° ^ 1.8 ro 1.6 >< 1.4 B 1.2 ro 1.0 o 0.8 £ 0.6 OA o 0.2 O 0.0 c/s 20:5n-3 44 46 48 50 52 54 56 58 Infant length (cm) Figure 8.22 Scatter plots of infant length and selected fatty acids in cord plasma phospholipid at birth, n=44 97 RESULTS ^—^ 10 i in x> 9 -o CO 8 -7 -CO 6 -~s 5 -o 4 -3 -2 -1 -o O 0 -c/s 18:2n-6 r • • i 5 • 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) acids) 0.20 i 0.18 -0.16 -0.14 -0.12 -CD 0.10 -O 0.08 --9 0.06 -0.04 -Core 0.02 -0.00 -c/s 18:3n-3 ~i 1 r-30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 22 20 H 18 16 14 12 10 H 8 6 4 2 0 c/s 20:4n-6 • • • • • V. 30 31 32 33 34 35 36 37 38 Head circumference (cm) 39 (spi 12 - i 11 -o 10 -CO 9 -8 -7 -CO 6 -H—' o 5 -4 -5- 3 -"D 2 -O 1 -o 0 -c/s 22:6n-3 • • • 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 3 1 H trans fatty acids •sf:i:i»# 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) in "O o CO CO 14— "co o o o 2.0 1.8 H 1.6 1.4 1.2 1.0 0.8 H 0.6 0.4 H 0.2 0.0 c/s 20:5n-3 • .11 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) Figure 8.23 Scatter plots of infant head circumference and selected fatty acids in cord plasma phospholipid at birth, n=451 1 No statistically significant relations were found. 98 RESULTS Table 8.18 Relations between selected fatty acids in cord plasma cholesteryl ester at birth and birth outcomes Birth Outcome Fatty acid n=46" n=70 z Length of gestation (wk) 18:2n-6 0.25 0.22 20:4n-6 0!353 0.334 18:3n-3 -0.17 -0.21 20:5n-3 0.05 -0.06 22:6n-3 0.19 0.18 20:5n-3/20:4n-6 -0.14 -0.21 Total trans -0.26 -0.25 3 C L A 5 -0.524 — Birth weight (g) 18:2n-6 -0.09 -0.09 20:4n-6 0.474 0.264 18:3n-3 -0.343 -0.19 20:5n-3 -0.10 -0.15 22:6n-3 0.20 0.18 20:5n-3/20:4n-6 -0.10 -0.19 Total trans -0.22 -0.19 CLA -0.35 3 — Infant length (cm) 18:2n-6 0.01 — 20:4n-6 0.434 • — 18:3n-3 -0.14 — 20:5n-3 0.11 — 22:6n-3 0.313 — 20:5n-3/20:4n-6 0.13 — Total trans -0.13 — CLA -0.35 3 — • Head Circumference (cm) 18:2n-6 -.084 — 20:4n-6 -.021 — 18:3n-3 -.118 — 20:5n-3 -.268 — 22:6n-3 -.073 — 20:5n-3/20:4n-6 -.145 — Total trans -.202 — CLA -.167 — 1 Cord plasma from Study Two 2 Cord plasma from Study One and Two 3p<0.05 4 Conjugated linoleic acid 5p<0.01 99 RESULTS o CO >> 45 CO o o O 24 22 20 18 16 14 12 10 8 6 4 2 0 c/s 18:2n-6 • • •. • — i — i — i — i — i — i — i — i 35 36 37 38 39 40 41 42 43 Length of gestation (wk) acids) 0.6 i 0.5 -$ 45 0.4 -CO 0.3 -(% tol 0.2 -•o 0.1 -o O 0.0 -c/s 18:3n-3 35 36 37 38 39 40 41 42 43 Length of gestation (wk) V) TD O CO 45 CO o "D O o 20 18 16 14 12 10 8 6 4 2 0 c/s 20:4n-6 r= 0.33 p<0.01 c/s 22:6n-3 * # I r.r • • • - i 1 1 1 1 1 r 1 35 36 37 38 39 40 41 42 43 Length of gestation (wk) 4 -i in "D o co >* V 45 CO -4—' o o O 3 2 H 1 I •a • t — i 1 — - 1 1 1 1 1 1 35 36 37 38 39 40 41 42 43 Length of gestation (wk) trans fatty acids c/s 20:5n-3 ids) 5 i o CO 4 -42 3 -CO o -4—' 2 -1 -0 -Cord 35 36 37 38 39 40 41 42 43 Length of gestation (wk) in •g o CO & 45 ro o o O 2.0 1.8 1.6 1.4 1.2 '1.0 0.8 0.6 0.4 0.2 0.0 35 36 37 38 39 40 41 42 43 Length of gestation (wk) • Figure 8.24 Scatter plots of length of gestation and selected fatty acids in cord plasma cholesteryl ester, n=70 100 RESULTS „—_ 24 - i (/) T J 22 -o 20 -ya 18 -16 -14 -to 12 --t-< o 10 -VP 8 -6 -TD 4 -i O 2 -O 0 -c/s 18:2n-6 •• • • •AM '• 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 43 06 ' 1 0 5 4 0.4 .2 "ca 0.3 o S 0.2 "2 0.1 o O 0.0 c/s 18:3n-3 • i • 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) cis 20:4n-6 T 1 1 1 1 1 1 I 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) 4 -i 3'H in TD O CO tJ 15 2 H o £ 1 o O c/s 22:6n-3 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) TD O ro >• .2 CO o o O 5 n 4 3 2 1 H trans fatty acids • % 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) ^ 3 0 1 "O £ 2.5 co V 2.0 .<o 1.5 1.0 0.5 TD O O 0.0 c/s 20:5n-3 1500 2000 2500 3000 3500 4000 4500 5000 Birthweight (g) Figure 8.25 Scatter plots of infant birthweight and selected fatty acids in cord plasma cholesteryl ester, n=70 101 RESULTS m 24 •g 22 ^ 20 ro 18 16 14 12 10 8 6 4 2 0 c/s 18:2n-6 45 TO O "O i o O • • • *_• * 2 44 46 48 50 52 54 56 58 Infant length (cm) » 0 5 •> "O £ 0.4 CO >. e 0.3 42 0.2 o ^ 0.1 -•o 0.0 -o O c/s 18:3n-3 44 46 48 50 52 54 56 58 Infant length (cm) 18 , 16 -| 14 12 10 8 6 4 H 2 0 c/s 20:4n-6 • •• r = 0.43 p<0.01 44 46 48 50 52 54 56 Infant length (cm) 58 » 4 " i "D O .to c/s 22:6n-3 2 H St 1 TJ O O 0 44 46 48 50 52 54 56 58 Infant length (cm) CO T J o CD >s .ro 45 3 co -i—' o •_ o O trans fatty acids • • n _•• • • • 44 46 48 50 52 54 56 58 Infant length (cm) CO •g o co >. a 49 CO o TJ O o 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 c/s 20:5n-3 — i — i — i — i — i — i — i 44 46 48 50 52 54 56 58 Infant length (cm) Figure 8.26 Scatter plots of infant length and selected fatty acids in cord plasma cholesteryl ester at birth, n=44 102 RESULTS 2 4 1 22 A 20 A 18 16 A 14 12 10 11 4 2 0 cis 18:2n-6 ^ c e •• t i l ; 1 : 1 1 — n 1 1 1 1 1 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) (sp 0.5 i ( J CD 0.4 -t » • — 0.3 -~& 0.2 -O -9 0.1 -Cord 0.0 -cis 18:3n-3 •v 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 18 n 16 14 12 10 8 6 u cis 20:4n-6 i • • 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) CO 72 o CD CD O - 9 T J b o 4 -, 3 2 1 0 cis 22:6n-3 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) 5 -i 4 -3 2 -1 -trans f a t t y a c i d s 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) CO T J o CO >. V CO o o O 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 cis 20:5n-3 • •• i — i — i — i — i — i — 30 31 32 33 34 35 36 37 38 39 Head circumference (cm) Figure 8.27 Scatter plots of head circumference and selected fatty acids in cord plasma cholesteryl ester at birth, n=461 1 No statistically significant relations were found. 103 • CO * co . CO o m o i n o i n o i o o i o o l O ^ ^ c o c o ' c s i f ^ ^ T - d d o in o ID o in o if) q m o - 0005 • - OOQfr * * • * - OOOt' • ' - ooge • • < - oooe - 0092 - oooz —1—1—1 - 0091-OLOOLOOLDOinpLOO ixi^^crJcocNoiT-T -bd OlDOlDOlOOlDOlOO i r i ^ ^ c o ' c o c N C v i T - ^ d d i i ' 0 0 ) 0 3 r ^ ( O L O ^ C O C N t - p r d d d d d d d d c i o O C D c O r ^ C D L O ^ T C O C N — O T - ' d d d d d d d d d d \ i—i—i—i—i—i—i i r oo>coi*- tom^rcoeNr -o ^ o o o d o o o o o o r 0009 009fr OOOt' 009e oooe 0092 0002 0091. —i—I—r-•g 'g. "o s: O ^ d o o c i o o o o o o JZ a. (sppe fi^ei o/0) 0 PJ°0 jzt E o o "D CO CO X E JZ in o) c CO CD -*r — 0009 OOQt' 00017 3 OOSC g, CD 000C CO CD "E" CD CO a> o JZ O 5 JZ 1- 0092 § 0002 0091 co CN 2" - I ° » <8 ro . C O co o • £ " ro <° £ CO CD (0 ro E co ra a "S o u < o •a o ra o "53 o c TJ CD ra a) "E o o T J c ra to o k_ 3 (A ra a> E o E o u o 4 -o CO o a i_ a> ra o </) 00 CN 00 0) w 3 O) 104 . DISCUSSION DISCUSSION 9.1 Dietary Trans Fatty Acid Intakes The results of the study have shown dietary TFA intake of pregnant women was about 3-4 g/person/d in both the second and third trimester. The mean (median) intakes of T F A were 3.8 (3.3) and 3.4 (2.9) g/person/d, respectively, representing approximately 1.3% of total energy in both trimesters. The estimates of TFA intake obtained here are lower than reported by Ayyagari et al (1996) for 43 pregnant women in the U.S, with an estimated median of 5 g TFA/person/d. However, despite the lower median intakes of the women in the study here, than that of Ayyagari et al (1996), the range of TFA intakes (range 0.7-11.7 g/person/d) in the study here compare well with those of Ayyagari et al (1996) (range 1.3-14.7 g/person/d). The estimated TFA intake for the pregnant women from Vancouver studied here are, however, similar to information on the TFA intakes of non-pregnant women in the U.S. both as absolute intakes (g/d), and as a percent total energy intake. For example, Garland et al (1998) estimated TFA intakes of 2.8 g/person/d, representing approximately 1.4% of total energy, for 140 non-pregnant nurses (mean age = 52), using two FFQ collected between 1986 and 1987. Similarly, the mean TFA intake in a Boston-area study of 115 non-pregnant women (mean age = 60) was 3.4 g/person/d, representing approximately 1.7% of energy (London 1991). More recently, Allison et al (1999) reported a mean dietary TFA intake of 4.6 g/person/d (10 th percentile = 1.6 g/person/d, 90 t h percentile = 8.0g/person/d) in women aged 20-49 in the U.S. The range of TFA intakes among individuals is wide, with low mean and median values, indicating that TFA intakes are not normally distributed in the population. Therefore, it is difficult to compare mean 105 DISCUSSION dietary TFA intakes here with the study of Allison et al (1999), Garland et al (1998) and London et al (1991), or with the median values reported by Ayyagari et al (1996). One of the reasons why the results reported here differed from the results reported by Allison et al (1999), Garland et al (1998), London et al (1991) and Ayyagari et al (1996) may be due to methodological differences. For example, Allison et al (1999) used 3-day food records to estimate dietary T F A intake whereas Ayyagari et al (1996), Garland et al (1998) and London et al (1991) all used a FFQ to estimate TFA intakes. Food records tend to under-estimate usual dietary intakes while FFQ over-estimate usual dietary intakes (Gibson 1990). Allison et al (1999), however, reported higher TFA intakes using a 3-day food record than dietary TFA intakes found here and by Garland et al (1998) and London et al (1991) who both used a FFQ to estimate dietary TFA intakes. Allison et al (1999) used a different nutrient database to calculate TFA intake than Garland et al (1998) and London et al (1991), which is one possible explanation for the differences in dietary TFA intake. Despite using the same FFQ to estimate dietary intakes, the lower TFA intakes found in non-pregnant women (Garland 1998, London 1991) than that found in pregnant women (Ayyagari 1996) could be explained, at least in part, by increased energy intakes associated with pregnancy. The lower estimated intake of TFA of pregnant women in the study here when compared to the study by Ayyagari et al (1996) could be explained by methodological differences such as the use of a FFQ versus a 3-day food record and the use of different nutrient databases, as well as differences in food supply and in eating habits of different populations. Ayyagari et al (1996), Garland et al (1998) and London et al (1991) all used the Harvard/Willett FFQ to estimate TFA intakes. This questionnaire contains between 116 106 DISCUSSION to 131 food items with standard portion sizes given and nine response categories for frequency of consumption (ranging from never or less than once per month to 6+ times per day). The Harvard/Willett F F Q was designed to assess average nutrient intake over the last year. This questionnaire asks the individual to indicate their consumption of vitamin and mineral supplements, dairy products, fruits, vegetables, eggs, meat, breads, cereals, starches, carbonated and other beverages, sweets, baked goods and miscellaneous foods such as dressings and sauces. There are also questions on type of fat used in baking and as a table spread, consumption of visible fat on meat, fried food consumption, amount of sugar used in beverages, usual oil used in cooking, and usual cold breakfast cereal consumed. At the end of the FFQ, participants were asked to indicate all other foods consumed at least once per week with the usual serving size and number of servings consumed per week. The Harvard/Willett F F Q uses standard portion sizes, collapses various foods into food categories and then assigns an average nutrient value for that category (Willett 1990). In contrast, the F F Q used in the study here was designed to include specific brand names and to provide an estimate of portion sizes using food models. Furthermore, trans fatty acid values for given foods were obtained from a 250-food TFA database (Innis 1999). Innis and Green (1999) caution against collapsing foods into broad food categories to estimate TFA intakes because of the considerable variation within a food category. For example, 6 types of white bread analyzed here were found to vary in TFA content froml .3 -34.9% of total fatty acids (Innis 1999). It is also important to recognize that oils used in the hydrogenation process differ in Canada and U.S. In Canada, canola oil is the most common oil used, whereas in the U.S., soybean oil is more prominent. Canola oil contains predominately 107 • DISCUSSION monounsaturated fatty acids, with approximately 53% cis 18:1 n-9, 21% cis 18:2n-6 and 11% cis 18:3n-3 whereas soybean oil contains approximately 23% cis 18:1 n-9, 54% 18:2n-6 and 8% 18:3n-3 (Laposata 1998). When these oils are hydrogenated, the amount of T F A and the trans isomer content and composition will differ because of the difference in the initial fatty acid profile. When canola oil, a good source of monounsaturated fatty acids, primarily cis 18:1 n-9, is partially hydrogenated, isomers of trans 18:1 may be formed. When soy oil, which contains mostly polyunsaturated fatty acids, specifically cis 18:2n-6, is partially hydrogenated both trans 18:1 and 18:2 isomers may be formed. The amount of trans isomers formed, however, is dependent on the temperature, gas pressure, type and amount of catalyst used and the length of time the hydrogenation process continues. Using different oils for hydrogenation can, therefore, result in a variety of trans isomers and may possibly affect the amount of TFA formed and the amount of TFA consumed. This is an important consideration when comparing TFA intakes from Canada and the U.S. In addition, dietary habits and intakes may differ between the U.S. and Canada and may further explain the differences between the results reported here and those of Ayyagari et al (1996). It is also possible that increased public awareness of the possible role of dietary fat in certain chronic diseases has resulted in changes in dietary fat intakes in the last several years. 9.2 Dietary Fat Intakes: Proportion of Energy From Total, Trans, Polyunsaturated, Monounsaturated and Saturated Fat The mean energy intake of the participants in the present study was 2692 kcal (range 1251-5775 kcal) in the second trimester and 2405 kcal (range 1246-3697 kcal) in 108 DISCUSSION the third trimester. The decrease in mean energy intake from the second to the third trimester is consistent with previous studies that have estimated energy intake throughout pregnancy. Beal (1971) and Haste et al (1990) both reported a decrease in energy intake of pregnant women in the U.S. in their third trimester compared to energy intakes in their second trimester of pregnancy. The total fat intake of women in the second and third trimesters of pregnancy, in this study, ranged from 38-201 and 22-124 g/person/day, respectively. The mean fat intake was 86 g in the second trimester and 74 g in the third trimester. Similar results were reported by Al et al (1996) in a study of the fat intake of 176 Dutch women using a food-frequency questionnaire in the second and third trimesters. Al et al (1996) found fat intakes of 88 and 87 g/person/day in the second and third trimester, respectively. Studies from the southwest region of England and Spain have also reported mean fat intakes of 70 and 101 g/person/day in the second and third trimester, respectively (Ortega 1996, Rogers 1998). The study in England by Rogers and Emmett (1998) used a food frequency questionnaire with standard portion sizes to estimate nutrient intakes of 11,923 women. Ortega et al (1996) used a 5-day food record to obtain diet information on 292 Spanish women. Rogers and Emmett (1998) reported a mean dietary fat intake of women in their third trimester (32 weeks gestation) of 70.4 g, similar to the mean fat intake of 74 g/person/d in the third trimester found in the present study. The percent energy from fat, however, reported by Rogers and Emmett (1998) was 34.4%, whereas in the study here, the percent energy from fat was less than 30% in both the second and third trimesters. The higher fat intakes in England may be explained by differences in food choices and diet between England and Vancouver, but may involve differences in the methods used to estimate fat intake. In the study here, a 109 DISCUSSION quantitative FFQ was designed to not only measure the frequency of food consumption, but also to measure typical portion sizes. Rogers and Emmett (1998) also used a FFQ, but rather than measuring portion size, standard portion sizes were used. This possibly may have over-estimated the mean fat intake. In contrast to Rogers and Emmett (1998), the mean fat intake reported by Ortega et al (1996) of 101 g/d for women in Spain, representing about 40% of daily energy from fat, was substantially higher than the mean fat intake found in the study here or by Rogers and Emmett (1998) for women in England. In addition to the cultural and food supply differences, the use of a five-day food record in the study by Ortega et al (1996) could possibly have provided an over-estimation of the true mean fat intake. However, dietary records usually under-estimate rather than over-estimate dietary intakes. In the study here, fat accounted for about 28% of total energy intake both in the second and third trimester. Health and Welfare Canada (Committee 1990) and the American Heart Association (Krauss 1996) have recommended a decrease in total fat intake to 30% of total energy. Despite a decrease in the mean fat intakes in the third trimester, the mean percentage of dietary energy from fat was the same in the second and third trimester. These results indicate a proportional decrease in the total energy and fat intakes from the second to third trimester. A decrease in fat intake, in late pregnancy, related to a proportional reduction in the total energy intake, has been reported by other investigators (Beal 1971, Haste 1990). The daily intakes of saturated, polyunsaturated and monounsaturated fat in this study were 30, 14 and 32g/d in the second trimester, and 26, 12 and 26g/d in the third trimester in this study. The proportion of energy as saturated, monounsaturated and polyunsaturated fat was constant at 9%, 5% and 10% in both trimesters, respectively. The American Heart 110 DISCUSSION Association has also recommended that saturated fatty acid intake should not exceed 10% of total energy intake and to increase polyunsaturated fatty acids to no more than 10% of total energy (Krauss 1996). The women in the study reported here met current recommendations not only for total fat but also for saturated and polyunsaturated fatty acid intakes. The lack of change in the percent of energy as saturated, polyunsaturated and monounsaturated fat from the second to third trimester suggests a decrease in total fat intake rather than a change in the type of fat consumed. Consistent with this interpretation, the energy and nutrient intakes of individual women in the second trimester show a strong, significant correlation to the intakes in the third trimester, r = 0.54 for TFA, r = 0.61 for polyunsaturated fat, r = 0.64 for monounsaturated fat and r = 0.75 for saturated fat. These statistically significant relations give further evidence that the energy distribution of fat from macronutrients and the food sources of fat of the women in this study were fairly consistent from the second to third trimester. The high correlation coefficients further suggest a high degree of reproducibility of the questionnaire. Relations between dietary fat intakes of pregnant women in the second and third trimester by Al et al (1996) have been reported to be similar with an r = 0.61, 0.58, 0.62 and 0.66 for total fat, saturated fat, monounsaturated fat and polyunsaturated fat, respectively. 9.3 Dietary Sources of Trans Fatty Acids In recent years health professionals have advised the public to decrease total fat intake and to choose fats and oils containing polyunsaturated and monounsaturated fat rather than saturated fat, in order to lessen their risk for heart disease. Saturated fats 111 DISCUSSION such as butter and lard or tropical (palm/coconut) vegetable oils can be replaced with vegetable oil margarines and shortenings, or in some foods and cooking, with liquid vegetable oils. Most solid and semi-solid margarines and vegetable shortenings, however, contain significant amounts of trans fatty acids. Therefore, a switch from butter or other saturated animal and vegetable fats to margarines and shortenings may decrease the proportion of saturated fat from one's diet while increasing the proportion of TFA. When decreasing total fat intake, consumers often choose products perceived as low fat with the intent of increasing carbohydrates, and thus decreasing fat intake. However, many products, for example baked foods, contain hydrogenated and/or partially hydrogenated fat, therefore TFA. Although products such as breads contain relatively small amounts of hydrogenated fat these foods are consumed frequently. As a result, they can contribute significant amounts of TFA to an individual's total TFA intake. The five most important sources of TFA in the diet of the women in the study here were baked foods, fast foods, breads, snack foods and margarines (Figures 5.3 and 5.4). These foods contributed over 70% of the estimate total TFA intake. In the study here, baked foods was the most frequent food category for each of the first, second, third, fourth and fifth-ranked contributors to the average TFA intake of the participants in the present study. This result clearly shows that a variety of baked foods containing hydrogenated fats were being consumed regularly. Further, these results show that although margarines may contain the highest amounts of TFA, margarines represents a relatively low proportion of the daily fat intake. Consequently, margarines are not the main contributors of an individual's TFA intake. Because margarines are a visible fat, a low contribution of margarines to the total fat intake would be expected, 112 ; DISCUSSION particularly if individuals are attempting to decrease total fat intake in an attempt to meet the dietary recommendations of 30% energy from fat. Similarly, in the study by Garland et al (1998) the five foods most predictive of the dietary TFA intake were store bought cookies (r=0.46), pastry (r=0.46), margarine (r=0.41), french fries (r=0.36) and crackers (r=0.24). Together these foods accounted for a cumulative r of 0.89. When correlated with a biochemical marker, fat aspirate TFA content, the five most predictive foods were: brownies (r=0.32), pastry (r=0.28), margarine (r=0.26), chocolate (r=0.22) and frozen potato products (r=0.20). Most of the women in the present study (>95%) consumed baked foods which provided a mean of about 1.2 g TFA/person/d for those women who ate these foods. Data compiled by the US Department of Agriculture found that cakes and related baked goods provided only 0.3 g of TFA/person/day . The latter data, however, were estimated between 1989 and 1991, using three-day food records of individuals. One acknowledged problem of food records is that they are known to under-estimate actual food intakes and are not representative of the variety of foods consumed by individuals (Gibson 1990). The present study, in contrast, used a FFQ to estimate usual dietary intakes covering a one-month period in 1998. Using a FFQ, rather than a food record, may over-estimate rather than under-estimate usual dietary intakes because of the time period it spans and the number of foods the FFQ contains. A wide variety of foods can be collected using a FFQ in comparison to a three-day food record because over 30 days it is more likely that an individual will consume a wider variety of foods than over 3 days (Gibson 1990). 113 DISCUSSION 9.4 Maternal Plasma Fatty Acids 9.4.1 Relations Among Selected Fatty Acids in Maternal Plasma The total trans isomers were present in maternal plasma TG, PL and C E at a mean of 4.0 (range 1.3-7.9), 2.4 (range! 1-4.5) and 1.6 (range 0.6-3.7) % of total fatty acids, respectively. No published data are available on the plasma TFA levels of women during pregnancy. Several studies, however, have measured plasma TFA in women immediately following delivery of their infant. Berghaus et al (1998) measured maternal plasma fatty acids in 41 German women after delivery. All births were between 38 and 42 weeks gestation and the median age of the women was 30 years. The median and interquartile range of TFA in TG, PL, C E and free fatty acids were 0.83 (0.63), 0.42 (0.32), and 0.28 (0.38), respectively. A limitation of this study is that a 50 m column was used in the gas chromatograph for separation and identification of fatty acids. For complete separation of fatty acid isomers, particularly the separation of cis and trans isomers, a 100 m column is recommended (Ratnayake 1995). In a preliminary report, Ayyagari et al (1996) noted median values for trans 18:1 isomers in maternal T G and PL of 2.57 (range 1.0-7.8) and 1.44 (range 0.4-2.6) % of total fatty acids, respectively. Preliminary data from France (Billeaud 1998) estimated plasma C E TFA of 0.38% of total fatty acids in pregnant women. A significant inverse relation was found between TFA and 18:2n-6 and 18:3n-3 in maternal plasma T G in the study here. This relation may suggest that participants who consumed higher amounts of TFA consumed less 18:2n-6 and 18:3n-3 than participants who consumed lower amounts of TFA. In addition, dietary sources of TFA are relatively poor sources of 18:2n-6 and 18:3n-3 and, therefore, as the intake of TFA increases, the intake of 18:2n-6 and 18:3n-3 may decrease. The plasma C E T F A was inversely 114 DISCUSSION related to plasma CE18:2n-6, 20:4n-6 and 22:6n-3. These relations may be a result of an effect of TFA on cholesterol metabolism, specifically on LCAT or C E T P activity. The TFA intake in the second and third trimester was significantly and positively associated with maternal plasma T G , PL and C E TFA. These significant relations between dietary intake and plasma TFA were increased when the values for TFA intake were adjusted for total energy intake. It was anticipated that TFA intake estimated in the third trimester would relate to the plasma level of TFA because the blood sample was taken at the same time that the third trimester FFQ was administered. Previous studies have reported positive relations between TFA intake and blood levels of TFA in TG, PL and C E (Mensink 1995, Seppanen-Laakso 1996, Zock 1997). Mensink and Hornstra (1995) studied the effect of replacing the usual sources of fat in the Dutch diet (animal and hydrogenated fat) with palm oil. All 40 participants consumed a control diet, containing usual Dutch fat sources, for three weeks. The participants were then randomly divided into two groups, one group receiving the control diet and the other group receiving the palm oil diet for six weeks. This was followed by a washout period of three weeks, and then the dietary regimen was crossed-over. The proportion of trans 18:1 n-9 in serum T G and in platelet PL decreased during the consumption of the palm oil diet and increased when the control diet, containing hydrogenated oil was consumed. The proportion of trans 18:1 n-9 in the diet was positively related to the level of TFA in serum T G (r = 0.41, p<0.05) after the first three weeks of consuming the control diet. The authors concluded that the level of TFA in serum T G reflects the dietary intake of TFA over the previous three weeks. The level of elaidic and frans-vaccenic acids in plasma PL have also been considered as indicators of dietary intake oitrans 18:1 isomers (Seppanen-Laakso 115 DISCUSSION 1996). A study of 46 Finnish men and women examined the changes in plasma PL fatty acids when consumers who ate margarine were switched to butter and vice versa for three to six weeks. When butter consumers switched to margarine they consumed on average 3.7 g TFA/day, representing 4.3% of total fat and 1.7% of total energy. After three weeks of consuming margarine, the individuals who usually consumed butter had similar TFA levels to the individuals who usually consumed margarine. These findings show that the levels of PL elaidic and frans-vaccenic acids are reliable indicators for 18:1 trans fatty acid intake. Two cross-over studies by Zock et al (1997) have measured the effects of modifying dietary fat content on plasma fatty acids. In the first study, 25 men and 34 women consumed a diet rich in monounsaturated fat, TFA, or saturated fat for three weeks each in random order. In the second study, 26 men and 30 women consumed a diet in which 18:2n-6 was replaced with trans 18:1 or stearic acid. Blood was sampled on the 21 s t day of each diet period. The results of these studies showed that the level of TFA in C E increased when participants consumed a TFA-rich diet and decreased when the TFA-rich diet was replaced with a diet rich in monounsaturated fat or saturated fat. In addition to this finding, the authors concluded that the proportions of TFA increased to approximately the same level in all subjects, but an associated decrease in 18:2n-6 was not seen in all individuals when a diet rich in 18:2n-6 was replaced with TFA. The studies by Zock et al (1997), Seppanen-Laakso et al (1996) and Mensink and Homstra (1995) confirm that when a diet containing TFA is consumed, TFA are present in plasma or serum TG, PL and C E . Because TFA are not synthesized in vivo, the presence of TFA in plasma lipid can be used as evidence of dietary TFA intake. The findings of Zock et al (1997), Seppanen-Laakso et al (1996) and Mensink and 116 DISCUSSION Hornstra (1995) suggest that plasma TFA reflect the last three weeks of dietary intake. Because of this, it seemed probable that the TFA levels in the 35 week blood sample taken in the study here may not have correlated with the FFQ data on TFA intake at 26 weeks gestation. Positive associations, however, were found between TFA intake during the second trimester and plasma TFA measured in the third trimester. This can reasonably be explained by the similar intake of TFA in the second and third trimester. 9.4.2. Relations Between Selected Fatty Acids in Maternal Plasma and Dietary Trans Fatty Acid Intake The correlation between dietary TFA intake and plasma PL T F A at 28 weeks (r = 0.36, n=60) and at 35 weeks (r = 0.35, n=58) are similar to the preliminary findings of Ayyagari et al (1996). Ayyagari et al (1996) reported a positive relation between dietary TFA intake and PL trans 18:1 isomers (r = 0.4), with no correlation between PL total TFA {trans 18:1 and 18:2) and dietary TFA intake. 9.5 Cord Plasma Fatty Acids 9.5.1 Relations Among Selected Fatty Acids in Cord Plasma The results of this study show the presence of TFA in the plasma of infants at birth, and demonstrate considerable variability in the plasma T F A levels among infants. The range of TFA in cord plasma was, for T G 0.6-12.8; for PL 0.1-1.3; and for C E 0.7-4.3% total fatty acids. TFA were present in highest levels in the TG , with mean levels of 2.6% fatty acids, when compared to PL (0.7%) and C E (2.0%). The trans 18:1 isomers represented 1.6% of total fatty acids in the TG. The trans 18:1 isomers represented a mean of 0.4 and 1.0% fatty acids in plasma PL and C E , respectively. Similarly, a 117 ; ; DISCUSSION preliminary report by Ayyagari et al (1996), has noted that trans 18:1 isomers represented 1.2% and 0.5% fatty acids in cord blood T G and PL, respectively, of newborn term infants in the U.S. Studies in Germany, however, have noted mean levels of about 1.6, 1.1 and 2.0% TFA in plasma TG, PL and C E , respectively, of 4-day old premature infants (Koletzko 1992). The study reported here found no statistically significant relation between TFA and cis 18:2n-6, 18:3n-3, 20:4n-6 or 22:6n-3 in cord plasma PL, or cis 18:2n-6, 18:3n-3 and 20:4n-6 in T G or cis 18:2n-6, 18:3n-3 and 22:6n-3 in C E . An inverse relation was found between TFA and cis 22:6n-3 in TG , and between TFA and cis 20:4n-6 in CE . Similarly, Koletkzo (1992) found no inverse relations between T F A and 18:2n-6, 18:3n-3, 20:4n-6 or 22:6n-3 in cord plasma PL of premature infants, but did report a significant inverse relation between TFA and cis 22:6n-3 in TG , and between TFA and cis 20:4n-6 in C E . The inverse association between TFA and cis 22:6n-3, but not cis 18:3n-3 in T G suggests that the association of high TFA with low cis 22:6n-3 is not explained by low intakes of cis 18:3n-3. However, cord plasma cis 18:3n-3 levels are very low in all infants less than 1.0% fatty acids therefore a significant relation would be difficult to find. Some studies have suggested TFA may inhibit desaturation of 18:2n-6 and 18:3n-3 (Anderson 1975, Beyers 1991, Cook 1990, Rosenthal 1984). Further, Carlson et al (1997) suggested that TFA could interfere with the final steps in the formation of 22:6n-3 , which occurs in the peroxisome (Sprecher 1995). The inverse relation between T F A and 22:6n-3 in cord blood in T G in the study reported here is consistent with this suggestion. However, differences in maternal intakes of 18:3n-3 or 22:6n-3 among women with high versus low dietary TFA intakes, or other dietary, genetic, or environmental factors could also explain these relations. In view of the current interest 118 ; : DISCUSSION in the importance of 22:6n-3 in fetal and infant growth and development these findings suggest the need for further investigation. However, no inverse relations were found here between TFA and 22:6n-3 in cord plasma PL or C E , although the cord plasma C E TFA was inversely related to 20:4n-6. If TFA have the ability to inhibit the desaturase enzymes, a decrease in 22:6n-3 and in 20:4n-6 would be expected in all lipid classes. Phospholipids (PL) are critical components of the cell membrane structure and, therefore, in cell growth, it would be expected that PL fatty acids such as 20:4n-6 and 22:6n-3 would be positively related with growth. No associations between PL fatty acids including 22:6n-3 and 20:4n-6, and birth outcomes were present in cord plasma. It is possible, however, that the sample size in this study was too small to detect significant effects. Further, all but 2 newborns were born at term, between 37-42 weeks gestation. The study reported also shows TFA are incorporated into C E , with isomers of 18:2 representing 50% of all trans fatty acids in maternal and cord plasma C E . This pattern appears consistent with the preferential use of 18:2n-6 for acylation of plasma unesterified cholesterol via the lecithin:cholesteryl acyl transferase (LCAT) reaction in humans. Whether or not this has any significance to lipoprotein metabolism, particularly in relation to the increase in LDL and Lp(a), that may accompany diets high in TFA (Sundram 1997), may be worth further consideration. Several studies have shown fetal plasma PL and T G are relatively enriched in 20:4n-6 and 22:6n-3 when compared to maternal plasma PL and T G (Hoving 1994, Koletzko 1990). This could reflect preferential uptake and/or release of 20:4n-6 and 22:6n-3 to the fetus by the placenta. Alternatively, the high levels of 20:4n-6 and 22:6n-3 in fetal plasma PL could reflect selection or synthesis from 18:2n-6 and 18:3n-3, 119 DISCUSSION retention and preferential acylation of 20:4n-6 and 22:6n-3 by the fetal liver during PL and T G synthesis. 9.5.2 Relations Between Selected Fatty Acids in Cord Plasma and Maternal Dietary Trans Fatty Acid Intake The levels of TFA in the newborn cord plasma PL were positively associated with the maternal intake of TFA in both the second and third trimester. Ayyagari et al (1996) also reported a positive relation between maternal TFA intake and TFA in cord plasma PL. Ayyagari et al (1996), however, correlated dietary TFA with 18:1 trans isomers in cord plasma, rather than with the total trans isomers as was done in the present study. No other studies have reported a relation between maternal TFA intake and cord plasma TFA. Phospholipids are critical components of membranes, thus, high levels of TFA in PL could possibly alter the function of developing membranes. Whether the developing fetus of women with high intakes of TFA is adversely affected with respect to normal growth and development is not known. In contrast to the PL, the present study found no relation between the maternal TFA intake and TFA in cord plasma C E or TG. The lack of relation between maternal TFA intake and cord plasma C E may be explained by the low concentration of cholesterol and C E in the fetal plasma. Cord plasma T G fatty acids reflect synthesized and secreted by the fetal liver. In contrast to postnatal life, the fetus does not obtain fatty acids from the gut but rather the fetus receives unesterified (free) fatty acids through the placental vein with de novo synthesis of fatty acids also contributing to the fetal TF pool. The hepatic fatty acids can then be oxidized for energy, or packaged into T G for secretion to plasma. Rodriguez et al (1998) reported significant A 5 and A6 120 . ; DISCUSSION desaturase activity in 16 fetal liver samples from legally approved abortions conducted between 17 and 35 weeks gestation. These enzymes are required to produce 20:4n-6 and 22:6n-3 from 18:2n-6 and 18:3n-3, respectively. This suggests that the fetal liver is capable of synthesizing long chain n-6 and n-3 fatty acids. The fetus is in an anabolic state and favoring the production of adipose tissue, muscle, and skeletal growth. Fatty acids from the placenta may be used for triglyceride synthesis and accumulation in adipose, and for oxidation for energy by skeletal muscle. Most information suggests placental transfer of fatty acids is low relative to the de novo synthesis of fatty acids from glucose in the fetal liver. This may explain the lack of a relation between maternal TFA intake and TFA in cord plasma TG. The dietary TFA intake of the women between 35 weeks gestation and parturition, when the cord blood sample was collected, might also have differed. It is also possible that changes in the T G pool may occur towards the end of pregnancy due to decrease intakes, and/or during labor due to the potential of a fasting state. 9.6 Relations Between Selected Fatty Acids in Maternal and Cord Plasma The mean (median) percent T F A in maternal plasma T G was 4.0% (4.2%), about 30% higher than in cord plasma T G of 2.9% (2.6%). Similarly, the mean (median) percent T F A in maternal plasma PL was 2.4% (2.3%), approximately 1.5 times higher than in cord plasma PL of 0.7% (0.6%). The median percent TFA in maternal and cord plasma T G and PL at 0.8% (maternal TG), 0.5% (cord TG), 0.4% (maternal PL) and 0.2% (cord PL) in the study by Berghaus et al (1998) were lower than the TFA values found in the study here. In contrast the mean (median) percent TFA in the cord plasma was higher than in the maternal plasma; C E TFA 2.0% (1.8%) and 1.6% (1.3%). for 121 DISCUSSION total maternal and cord plasma, respectively. Similarly, Berghaus et al (1998) reported a higher percentage of TFA in cord plasma C E (0.8%) than in maternal C E (0.3%). Koletzko and Muller (1990) also measured in maternal and cord plasma TFA but used total plasma lipid (TG, PL and C E fractions) rather than separated lipid classes. The mean percent of TFA in the maternal plasma total lipid was 1.99% of total fatty acids, about 18% higher than the 1.66% TFA in cord plasma total lipid. The results of our study and those of Berghaus et al (1998) and Koletzko and Muller (1990) clearly indicates that TFA cross the placenta to the fetus. In maternal plasma T G and PL, the trans 18:1 isomers were the predominant trans isomers representing over 50% of the trans isomers. In cord plasma C E , however, trans 18:2 isomers represented 50% of total TFA; in maternal plasma C E trans 18:2 represented about 60% of total TFA. Whether the accumulation of TFA in plasma C E , specifically trans 18:2 isomers, has any adverse effect on cholesterol metabolism in the fetus is not known. The levels of essential fatty acids, 18:2n-6 and 18:3n-3 were lower in the newborn cord plasma TG, PL and C E than in maternal blood at 35 weeks of gestation in the study here (Table 8.11 and 8.13). In contrast, the level of 20:4n-6 and 22:6n-3 values in newborn cord plasma TG, PL and C E were higher than in the maternal blood at 35 weeks of gestation. Higher proportions of 20:4n-6 and 22:6n-3 and lower 18:2n-6 in newborn than maternal blood has been documented in many previous studies including studies in the U.S. (Reece 1997), Germany (Berghaus 1998, Koletzko 1992), the Netherlands (Hoving 1994), Britain (Hendrickse 1985) and Japan (Young 1997). The higher level of 20:4n-6 and 22:6n-3 in the fetus than the mother is consistent with the higher need of the fetus for the long chain polyunsaturated n-6 and n-3 fatty acids for growth and development. 122 DISCUSSION 9.7 Relations Between Cord Plasma Fatty Acids and Birth Outcomes The study here found a significant, positive relation between T G and C E 20:4n-6 and length of gestation, birthweight and infant length. Arachidonic acid (20:4n-6) is found in phospholipids throughout the body and is required in growth and development. Hoving et al (1994) also reported a gestational age-dependent increase in 20:4n-6 in a study of 503 cord blood T G and C E samples collected from spontaneous deliveries in the Caribbean. However, Rodriguez et al (1998) reported that the amount of 20:4n-6 in 16 fetal liver microsomal PL remained relatively stable, regardless of length of gestation, while 22:6n-3 increased and was positively correlated with length of gestation and fetal weight. Because liver PL were measured in the study by Rodriguez et al (1998), rather than plasma PL, it is difficult to compare the results from Rodriguez et al (1998) with the results of Hoving (1994) and the study here. The studies here found a significant positive relation between 22:6n-3 and infant length at birth, van Houwelingen et al (1995) also found this relation in a study of 86 umbilical plasma samples obtained by trans-abdominal puncture during ongoing pregnancies. It is reasonable to expect a significant positive relation between 22:6n-3 and infant length because 22:6n-3 was positively related to length of gestation. The longer the gestation, the more time the fetus has to grow in both weight and length. Koletzko (1992) reported an inverse relation between TFA and birthweight in cord plasma PL and C E of 29 premature infants. This relation was not present in our study with term infants; however, there was an inverse association between T G and C E TFA and length of gestation of the 70 newborns studied here. 123 DISCUSSION The study here also found inverse relations between cord plasma PL 18:2n-6 and length of gestation, birthweight and infant length. Possibly, the inverse relation between 18:2n-6 and each of length of gestation, birthweight and infant length is explained by increased utilization of 18:2n-6 for 20:4n-6 synthesis with progressing gestation. The study here provides the first data on conjugated linoleic acid (CLA) in cord plasma lipids. The T G and C E CLA were inversely related to the length of gestation and C E CLA was inversely related to birthweight and infant length. Conjugated linoleic acid has been found to inhibit tumor growth during the post-initiation phase of mammary carcinogenesis Conjugated linoleic acid has been suggested to have anticarcinogenic properties because of its antioxidant properties or alterations in eicosanoid metabolism, however, the mechanism by which CLA inhibits tumor growth is not known (Cunningham 1997, Ip 1997). Conjugated linoleic acid is found in dairy products and in the meat of ruminant animals. There are many other fatty acids other than CLA, and also other nutrients common to these foods that may explain the inverse relation between CLA and birth outcome measures. However, further work on the relation of CLA to growth would seem important. 9.8 Conclusions and Future Directions The major food source of TFA for the women in the study here was baked foods, which provided approximately one third of the mean total TFA intake of 3.8 and 3.4 g/person/d, respectively. Despite relatively low intakes of TFA, with mean intakes representing about 1% of total dietary energy, maternal TFA intakes adjusted for energy at 35 weeks gestation were related to the maternal plasma levels of TFA in C E (r = 124 DISCUSSION 0.34) and in T G (r = 0.33). In contrast, cord plasma C E and T G levels of TFA showed no significant relation to the maternal TFA intake. The level of TFA in cord plasma PL, however, was significantly related to the maternal TFA intakes adjusted for energy at 28 weeks (r = 0.38) and 35 weeks of gestation (r = 0.36). Because PL are important in all cell membranes, high maternal TFA intakes, could possibly have an effect on fetal membrane function and therefore on normal growth and development, however, all but 2 infants studied here were born term (37-42 weeks) and were of appropriate birthweights for gestational age. Inverse relations between the level of TFA and 22:6n-3 in cord plasma TG, and between the level of TFA and 20:4n-6 in C E were found . Whether or not variability in 22:6n-3 and 20:4n-6 states at birth has any physiological significance to infant growth and development is not known. Many factors such as race, parity, maternal height, pre-pregnancy weight, weight gain during pregnancy and activity level are associated with birth outcome measures. In order to control for these environmental and genetic effects, studies with a much larger sample size than used in the study here are needed to determine whether the amounts of TFA in the food supply have any effect on birth outcome. Significant inverse associations were found, however, between the levels of CLA in cord plasma C E and length of gestation, birthweight and length, and between CLA in cord plasma T G and length of gestation. These statistical associations require further investigation to further elucidate whether CLA has a direct effect on fetal growth or pregnancy duration, or whether another fatty acid or nutrient present in dairy products, or some other lifestyle factor associated with high dairy product intake, is responsible for the relations. 125 DISCUSSION The findings from the present study do not substantiate the hypothesis that dietary TFA have adverse effects on normal fetal growth and development. However, it is possible that the amounts of TFA consumed by the women in this study were too low for adverse effects to be evident, or that the generally high socio-economic and nutritional status of the women studied here compensated for any potential adverse effects. The results of this study, therefore, should not be generalized to all pregnant Canadian women, particularly those who may be in the highest quartile of TFA intake. Of note, only one mother in the study here had a TFA intake at 35 weeks gestation >9g/d, equivalent to about 3-4% of dietary energy. With respect to TFA intake, the eating habits of the women in the study here may not reflect usual eating habits of other socio-economic or ethnic groups, or of other women in other regions of Canada. For example, many of the women in the study here consumed a diet with a variety of fresh fruits and vegetables and included fish and seafood on a regular basis. Many of the women studied here also indicated that they consumed only organic fruits, vegetables and grains. The results of this research suggest the need to consider the value of ingredient labeling on foods such as bakery items in grocery stores and in coffee shops with respect to fat content and composition. It is evident from the results of the study here that most of the dietary TFA intake was found in invisible fats and often in foods that the women in the study here may not have considered as contributing to fat or TFA intake. In addition to ingredient labeling, consumers need to be aware that many processed and prepared foods such as pie shells, bakery items, prepared pasta meals and granola bars contain TFA. It is important to educate the consumer to identify food sources of TFA on ingredient labels, by looking for ingredients such as partially hydrogenated fats 126 DISCUSSION and oils and vegetable shortenings on packaged foods. It is also important that the public understands the importance of n-6 and n-3 fatty acids in normal growth and development, particularly in children and pregnant women, and that foods containing TFA are often poor dietary sources of n-6 and n-3 fatty acids. Future research should address whether diets containing TFA have lower n-6 and n-3 fatty acid levels than diets devoid of TFA. To understand whether TFA are likely to inhibit the A 5 and A 6-desaturation of 18:2n-6 and 18:3n-3 at usual TFA intakes, future studies will need to determine dietary n-6 and n-3 fatty acid intakes in addition to estimating dietary TFA intakes. A large, prospective study with pregnant women will be needed to determine whether TFA, CLA or other n-6 and n-3 fatty acids influence birth outcomes. This is necessary to control for all other known nutritional, environmental and genetic factors that contribute to birth outcome. Studies are also needed to understand the mechanism(s) that allow fatty acids to be transported to the fetus through placental transport. 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Am J Epidemiol 145, no. 12:1114-22. 136 APPENDIX 1 APPENDIX 1 Informed consent: Study One 137 A P P E N D I X 2 APPENDIX 2 Research study questionnaire: Study One 140 APPENDIX 2 ID No. Initials Fatty Acids in Infant Cord Blood Research Study Questionnaire Mother's age Smoker Gestational age (wk) Yes No Level of Education (highest level achieved) <Secondary Infant Gender Secondary M Post-Secondary F Community/Technical Undergraduate Infant Birthweight (g) Graduate Doctorate Gross Family Income $0< $10,000 $10,000 < $20,000 $20,000 < $35,000 $35,000 < $50,000 _ _ $50,000 < $75,000 $75,000 or < Nutrition 1. How often do you eat the following foods? <1x/wk 1-2x/wk 3-4x/wk >4x/wk - fried fast foods (french fries, onion ring, fish) - commercial baked goods (muffins, cookies, donuts, crackers, cakes) 2. Do you regularly (weekly) do your own baking? Yes No. If yes, what type of fat do you use in baking? a) butter d) vegetable oil b) lard e) stick margarine c) shortening f) tub margarine . 141 APPENDIX 2 3. What type of fat do you use as a table spread (i.e. on breads, buns, vegetables, pasta)? 4. Do you follow any particular diet (e.g. vegetarian)? 5. Do you purposefully avoid any foods (allergies, intolerances, dislikes)? If yes, what foods? 142 APPENDIX 3 APPENDIX 3 Recruitment letter: Study Two 143 APPENDIX 4 APPENDIX 4 Informed consent: Study Two 145 APPENDIX 5 APPENDIX 5 Socio-demographic and health questionnaire: Study two 149 APPENDIX 5 Fatty Acids in Maternal Diet, Maternal Blood and Infant Cord Blood Socio-Demographic and Health Questionnaire Date: ID _________ Some of these questions are personal but very important to our study. Please answer questions as accurately as possible. Remember, your answers will be kept strictly confidential. Your name is not attached to any of these sheets. Socio-Demographic Information 1. DateofBirth ( ) ( ) ( ) 2. Country of birth 3. Mother's Ethnicity • White • First Nations • Black • Chinese • East Indian • Other Asian (please specifiy) • Other (please specify) 150 APPENDIX 5 4. Father's Ethnicity • White • First Nations • Black • Chinese • East Indian • Other Asian (please specify) • Other (please specify) 5. What is the highest level of education you have completed? • Less than high school • High school • Community College/Technical school • Undergraduate degree • Graduate degree 6. What is your usual occupation? • 7. What is your gross family income? • less than $20,000 • $20,000 - $50,000 • >$50,000 8. How many people are dependent on this income? 151 APPENDIX 5 Health Information The next set of questions ask about your personal health and lifestyle habits. These questions are very important to our clinical study. I would again like to remind you that all the information given is strictly confidential. 1. What is your height? feet inches 2. What was your pre-pregnancy weight? lbs 3. What is your present weight? lbs 4. What week of pregnancy are you at? . weeks 5. Is this your first pregnancy? • Yes • No If no, how many pregnancies have you had? • How many live births have you had? Of these live births, how many were male? Of these live births, were there any multiple pregnancies, i.e. twins? • YesD No 6. Have you smoked cigarettes during your pregnancy? • No • Yes If yes, what is the average number of cigarettes you smoke per week? 7. Have you consumed alcoholic beverages during your pregnancy? • No . • Yes If yes, what is the average number of drinks per week? ' (1 drink =1.5 ounces of spirits, whiskey, etc.; 12 oz beer; 5 oz wine) 152 APPENDIX 5 8. During your pregnancy have you taken any vitamin and/or mineral supplement? • No • Yes If yes: 1. supplement name how often do you take them? when did you start taking them? 2. supplement name how often do you take them?_ when did you start taking them? (if more than two supplements were used, please use back side of page) 9. During your pregnancy have you taking any medications? (includes current use). • No • Yes If yes: 1. medication dose condition time period 2. medication dose condition time period 3. medication dose condition time period (if more than three medications were taken, please continue on the back side) 153 . • , APPENDIX 5 10. During your pregnancy did you follow any particular diet? • No • Yes If yes what diet did you follow: • Lacto-ovo vegetarian (eats all milk and milk products and eggs) • Semi-vegetarian (eats all milk and milk products, eggs, poultry and fish) • Vegan (avoids ALL animal products) • Other (please specify) Thank you for taking the time to complete this questionnaire. Your cooperation is greatly appreciated! 154 A P P E N D I X 6 APPENDIX 6 Food frequency questionnaire: Study Two 155 APPENDIX 6 FOOD FREQUENCY QUESTIONNAIRE (TO BE COMPLETED BY NUTRITIONIST) Subject Code Date Week of Gestation Date of Follow-up Visit Day Month Year Day of week 156 APPENDIX 6 I T E M N A M E B R A N D / H O M E M A D E A M O U N T D A I R Y P R O D U C T S 1) Milk (drinking) - cow (homo, 2%,1%, skim) goat, rice, soy, chocolate, hot choc (used in cereal) (used in tea/coffee) (used in cooking/ baking) 2) Cheese (hard) -cheddar, mozarella (soft) - brie/camembert, cottage, edam 3) Processed Slices 4) Cheese Spreads - Cheez Whiz -Country Crock • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade C O D E : F R E Q U E N C Y _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _• oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _Qoz/g • cup/ml • piece _Doz/g • cup/ml • piece • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml 0 piece • brand • homemade _Doz/g • cup/ml • piece _doz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week D Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 157 APPENDIX 6 I T E M N A M E B R A N D / H O M E M A D E A M O U N T C O D E : F R E Q U E N C Y 5) Yogurt 6) Puddings V) Ice cream/ Frozen Yogurt 8) Milkshakes/ Yogurt Shake 9) - fried, scrambled - poached, deviled, boiled - omelettes, quiches 10) Other Dairy Products - eggnog, Caresse fresh cheese T A B L E / C O O K I N G F A T 1) Margarine (for spreading) • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade _Doz/g • cup/ml • piece _ • oz/g , • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece • oz/g 0 cup/ml • piece _Doz/g • cup/ml • piece • brand • homemade _Ooz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 158 A P P E N D I X 6 C O D E : I T E M N A M E B R A N D / H O M E M A D E A M O U N T (used in baking) • brand • homemade F R E Q U E N C Y _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 2) Shortening • brand • homemade J3oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 3 ) Cooking Oils • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 4) Salad dressings • brand • homemade _Ooz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 5) Mayonnaise Miracle Whip • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 6) Peanut Butter - tahini, nut butters • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 7) Coffee Whiteners (non dairy liquid, powder) • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 8) Whipped Toppings - Cool Whip, NutriWhip - whipping cream • brand • homemade • brand • homemade _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month 159 APPENDIX 6 C O D E : I T E M N A M E B R A N D / H O M E M A D E A M O U N T F R E Q U E N C Y 9) Other Table/ Cooking Fats - cereal cream, cream cheese, sour cream • brand • homemade _rjoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month B R E A D S / C E R E A L S A N D B A K E D G O O D S 1) Bread (including pita/bagels) - corn, rye, sourdough - chapati, roti • brand • homemade • brand • homemade JHoz/g • cup/ml • piece JHoz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 2) Buns/Rolls (including hamburger/hotdog buns) • brand • homemade JHoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade JD'oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 3) Breadsticks/Croutons • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 4) Cereals • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade 160 _Doz/g • cup/ml • piece • Day • Week • Month APPENDIX 6 CODE: ITEM NAME BRAND/HOMEMADE AMOUNT FREQUENCY • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 5) Wheat germ (used in/on foods) • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 6) V) Pancakes/Waffles Muffins • brand • homemade • brand • homemade _Doz/g • cup/ml • piece _doz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week ' • Month 8) English Muffin • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 9) Scones/Tea Biscuits • brand • homemade • brand • homemade _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month 10) Poptarts/Toaster Pastries_ • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 11) Donuts/Fritters • brand • homemade • brand • homemade _ • oz/g • cup/ml • piecE _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month 12) Danish/pastries • brand • homemade _Qoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _doz/g • cup/ml • piece • Day • Week • Month 161 APPENDIX 6 I T E M N A M E B R A N D / H O M E M A D E A M O U N T C O D E : F R E Q U E N C Y 13) Croissants 14) Cakes/Squares - brownies, cake rolls, cheesecake 15) Pies/Tarts 16) Cookies 17) Other Breads/Cereals and Baked Goods -cinnamon buns, wagon wheels fruit crisps, stuffing S N A C K I T E M S 1) Chocolate Bars/ Chocolates/ Candies 2) Frostings/Icings • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand D homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade _rjoz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _doz/g • cup/ml D piece _Doz/g • cup/ml • piece JDoz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _Qoz/g • cup/ml • piece JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece • brand • homemade JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 162 APPENDIX 6 C O D E : I T E M N A M E B R A N D / H O M E M A D E A M O U N T F R E Q U E N C Y 3) Potato Chips • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 4) Corn/Taco Chips • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 5) Other Chips - apple chips - banana chips • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 6) Buttered Popcorn - microwave, air - movie • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade _Qoz/g • cup/ml • piece • Day • Week • Month 7) Party snacks - Nuts & Bolts, pretzel - Crunch N' Munch • brand • homemade JDoz/g Q cup/ml • piece • Day • Week • Month • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month 8) Crackers • brand • homemade _Doz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece .•Day • Week • Month 9) Granola/Cereal Bars • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 10) Nuts/Seeds - alone or in salads, baking • brand • homemade • brand • homemade • brand • homemade _Doz/g • cup/ml • piece _ • oz/g • • cup/ml • piece _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month 163 , APPENDIX 6 C O D E : I T E M N A M E B R A N D / H O M E M A D E A M O U N T F R E Q U E N C Y 11) Other snack items • oz/g • Day - Cheezies, Bugles • brand • cup/ml • Week • homemade • piece • Month Qoz/g DDay • brand • cup/ml • Week • homemade • piece • Month • oz/e • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month C O M B I N A T I O N F O O D S / M E A L S (Purchased and Home-made) Prepared Sauces • oz/g • Day - Cheese, Hollandaise • brand • cup/ml • Week Pesto, Bernaise • homemade • piece • Month • oz/e • Day • brand • cup/ml • Week • homemade • piece • Month Prepared Pasta/Rice • oz/g • Day Dishes (bottled, canned, boxed) • brand • cup/ml • Week - KD, Lipton's • homemade Q piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month Rice • oz/g • Day (other than mentioned in 2) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month Pasta • oz/g • Day (other than mentioned in 2) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month Seasoning/Coating Mixes • oz/g • Day - Shake n'Bake • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 164 APPENDIX 6 I T E M N A M E B R A N D / H O M E M A D E A M O U N T C O D E : F R E Q U E N C Y 6) Frozen Dinners V) Fries - trench, New York 8) Perogies - potato, cheese 9) Tacos/Burritos Quesidias 10) Pizza (including rolls/pops) 11) Wieners/Frankfurters - beef, turkey, veggie, tofu, other 12) Sausages - Polish, farmer, breakfast 13) Luncheon Meats/Spreads_ - in sandwiches/subs • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade _doz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g D cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece _Doz/g • cup/ml • piece JDoz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece • brand • homemade _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 165 APPENDIX 6 CODE: I T E M N A M E BRAND/HOMEMADE AMOUNT FREQUENCY 14) Meat Pies • oz/e • Day Shepards Pie • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 15) Hamburgers • oz/g • Day -beef, veggie,soy • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 16) Fish sticks/ • oz/g • Day pieces • brand • cup/ml • Week frozen fast food • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 17) Fish • oz/g • Day (BakedVSteamed/BBQ etc) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 18) Chicken/Fish • oz/g • Day Burgers • brand 0 cup/ml • Week D homemade • piece • Month • oz/g • Day Q brand • cup/ml • Week • homemade • piece • Month 19) Fried Chicken • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 20) Chicken Nueeets • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 21) Chicken Wines • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 166 A P P E N D I X 6 CODE: ITEM NAME BRAND/HOMEMADE AMOUNT FREQUENCY 22) Poultry • oz/g • Day (Baked/Roast/BBQ) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 23) Beef • oz/g • Day (Baked/Roast/BBQ) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 24) Pork • oz/g • Day (Baked/Roast/BBQ) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 25) Lamb • oz/g • Day (Baked/Roast/BBQ) • brand • cup/ml • Week • homemade • piece • Month 26) Other meat • oz/g • Day -game, venison, rabbit • brand • cup/ml • Week - lobster, mussels • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 27) Shrimp • oz/g • Day (fried/boiled) • brand • cup/ml • Week (cooked, canned) • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 28) Other seafood • oz/g • Day -scallops, clams • brand • cup/ml • Week -lobster, mussels • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 29) Legumes, dry peas. • oz/g • Day beans, lentils (canned,cooked) • brand • cup/ml • Week • homemade • piece • Month • oz/g • Day • brand • cup/ml • Week • homemade • piece • Month 1 6 7 APPENDIX 6 CODE: ITEM NAME BRAND/HOMEMADE AMOUNT FREQUENCY 30) Tofu Doz/g rjDay • brand • cup/ml • Week • homemade • piece • Mon SOUPS • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month Cream Soups 2) Vegetable/Noodle Soups include cup of soups 3) Instant Noodle Soups - Ichiban, Chow Mein 4) Other Soups - borscht, miso VEGETABLES rCanned. fresh, frozen) Green/yellow beans Peas 2) Broccoli, Brussel sprouts_ Cabbage • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade _ • oz/g • cup/ml • piece JDoz/g • cup/ml • piece _Qoz/g • cup/ml • piece JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece JDoz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece. • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day 0 Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 168 APPENDIX 6 CODE: 3) I T E M N A M E Lettuce, Kale, Spinach BRAND/HOMEMADE AMOUNT 4) Squash, Corn 5) Potatoes - other than fries 6) Vegetable juices V) Other Vegetables FRUITS (Canned, fresh, frozen) 1) Apples/applesauce 2) Oranges, Grapefruits Lemons, Limes 3) Pear, Plums, Peaches Nectarines • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade FREQUENCY _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g • cup/ml • piece JDoz/g • cup/ml • piece _Doz/g • cup/ml • piece _ • oz/g • cup/ml • piece _Doz/g • cup/ml • piece _Cloz/g • cup/ml • piece _ • oz/g • cup/ml • piece • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece _oz/g • cup/ml • piec _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month 169 APPENDIX 6 CODE: I T E M N A M E BRAND/HOMEMADE AMOUNT • brand • homemade FREQUENCY • oz/g • cup/ml • piece • Day • Week • Month 4) Raisins, prunes, Dried fruit • brand • homemade _Qoz/g • cup/ml • piece • Day • Week • Month 5) Melons, Lychees • brand • homemade • brand • homemade _ • oz/g • cup/ml • piece _ • oz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 6) Bananas • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 7 ) Berries • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade _ • oz/g • cup/ml • piece • Day • Week • Month 8) Fruit cocktail/ Fruit salads • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 9) Fruit Juices • brand • homemade JD oz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month 10) Other Fruits • brand • homemade JD oz/g • cup/ml • piece • Day • Week • Month • brand • homemade JDoz/g • cup/ml • piece • Day • Week • Month • brand • homemade JD oz/g • cup/ml • piece • Day • Week • Month 170 APPENDIX 6 ITEM N A M E BRAND/HOMEMADE AMOUNT CODE: FREQUENCY BEVERAGES rother than milk and juices) 1) Soft Drinks (regular) 2) Drink Mixes (Iced teas, Kool-Aids) 3) Other Beverages • brand • homemade • brand • homemade D brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade • brand • homemade JDoz/g • cup/ml • piece JDoz/g • cup/ml • piece JDoz/g • cup/ml • piece JD oz/g • cup/ml • piece JDoz/g • cup/ml • piece JD oz/g • cup/ml • piece JD oz/g • cup/ml • piece JD oz/g • cup/ml • piece JD oz/g • cup/ml • piece JDoz/g • cup/ml • piece JDoz/g • cup/ml • piece • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week • Month • Day • Week •Month 171 

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