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The oxidation of fatty acids and other substrates in healthy men fed butterfat versus beef tallow MacDougall, Diane E. 1995

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T H E OXIDATION OF F A T T Y ACIDS A N D OTHER SUBSTRATES IN H E A L T H Y M E N FED B U T T E R F A T VERSUS B E E F T A L L O W by Diane E . MacDougall B. Sc. (Nutrition) St. Francis Xavier University, 1989  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES S C H O O L OF F A M I L Y A N D NUTRITIONAL SCIENCES DIVISION OF H U M A N NUTRITION We accept this thesis as conforming totijy?^quired standard  T H E UNIVERSITY OF BRITISH C O L U M B I A June 1995 ©  Diane E. MacDougall, 1995  In  presenting  degree freely  this  at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  scholarly purposes may be her  representatives.  permission.  of  WvJvwcuA  K\<jdhT\4\oO  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  for  an advanced  Library shall make  it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  Department  requirements  British Columbia, I agree that the  available for reference and study. I further  copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT To assess the influence of dietary fat composition on postprandial oxidation of dietary myristic acid (MA), palmitic acid (PA) and other substrates, healthy males (n=8) consumed prepared solid food diets containing 22% of energy as butter or tallow. Using a randomized cross-over design, subjects were prefed 11 day test diets which were identical in nutrient content except for the specific fat treatment. The diets provided 40%, 45%, and 15% of energy as fat, carbohydrate and protein, respectively, and were fed at a level equivalent to calculated energy requirements. On day 8 and day 11 of each diet cycle, a randomly assigned bolus of [1- C]MA or 13  [1- C]PA (20 mg/kg body weight) was ingested with the breakfast meal. Hourly 13  breath samples were collected for 9 hours thereafter and C 0 enrichment was 1 3  2  determined using isotope ratio mass spectrometry. Continuous respiratory gas exchange was also monitored and carbohydrate oxidation, fat oxidation and energy expenditure were determined. Treatment fat did not influence the fractional oxidation of dietary [1- C]MA (% dose/9 hours; x±SEM; 7.1±1.0% and 8.6±0.9% after the 13  butter and tallow meal, respectively) or [1- C]PA (3.3±0.7% and 3.0±0.9% after the 13  butter and tallow meal, respectively) (P<0.01, M A versus PA). M A contents of the butter and tallow meals were 4.6 g and 2.4 g, respectively, while the PA contents were 13.6 g and 11.2 g, respectively. While net dietary M A oxidation, calculated as the product of percent oxidation * meal fatty acid content, was greater (p<0.05) after the butter (329±45 mg) compared to tallow (212±25 mg) breakfast, no difference in net oxidation of dietary PA was observed between butter (441 ±99 mg) and tallow (348±95  ii  mg) meals. Overall, dietary M A or PA accounted for less than 1% of fat oxidized for 9 hours postprandial. Cumulative postprandial energy expenditure (kcal/9 hours; 7 8 3 ± 2 6 kcal and 781±31 kcal after the butter and tallow meal, respectively), cumulative postprandial fat oxidation (g/9 hours; 4 6 ± 3 g and 4 4 ± 4 g after the butter and tallow meal, respectively), and cumulative postprandial carbohydrate oxidation (g/9 hours; 85±8 g and 89±7 g after the butter and tallow meal, respectively) did not differ due to fat treatment.  These findings suggest that the percent oxidation of M A  and P A within a meal is independent of the blend of fatty acids consumed. However, it is markedly enhanced with decreasing chain length. Conversely, net oxidation of fatty acids contained within a meal is proportional to the mixture of fatty acids contained therein.  iii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E O F CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F FIGURES  viii  ACKNOWLEDGEMENTS  ix  1.  INTRODUCTION  1  2.  L I T E R A T U R E REVIEW  4  2.1  2.2  Structure-Dependent Differences in the Intermediary Metabolism of Fatty Acids 2.1.1 Digestion and Absorption 2.1.2 Transport 2.1.3 Hepatic Extra-mitochondrial Metabolism 2.1.4 Mitochondrial Beta-oxidation and Subsequent Utilization 2.1.5 Other Factors Affecting Fat Utilization 2.1.5.1 Facultative Thermogenesis 2.1.5.2 Long and Short Term Prefeeding The Influence of Dietary Fat Composition on Substrate Utilization and Energy Expenditure 2.2.1 Fat Oxidation 2.2.1.1 Dietary Fatty Acid Oxidation Based on C and C - Bolus Tracer Methodologies 2.2.1.2 Postprandial Net Fat Oxidation Based on Respiratory Gas Exchange Analysis 2.2.2 Energy Balance 2.2.2.1 Thermogenic Response to Dietary Fat Based on Respiratory Gas Exchange Analysis 2.2.2.2 Energy Balance Due to Long Term Consumption of Different Dietary Fats  4 4 5 7 9 11 11 13 15 16  1 3  1 4  3.  16 19 20 20 22  METHODOLOGY  25  3.1 Study Subjects 3.2 Study Diets 3.3 Study Protocol  25 25 26 iv  T A B L E OF CONTENTS 3.4 Analytical Procedures and Data Analysis 3.4.1 Respiratory Gas Exchange Measurement 3.4.2 Respiratory Gas Exchange Analysis 3.4.3 Collection of C 0 Breath Samples 3.4.4 Purification of C 0 Breath Samples 3.4.5 Mass Spectrometry 3.4.6 Gas Liquid Chromatography 3.4.7 Determination of Breakfast Meal Fatty Acid Content and Oxidation 3.5 Statistics 2  2  4. RESULTS  29 29 29 30 30 31 33 34 34 35  4.1 Study Subjects 4.2 Fatty Acid Analysis of Treatment Fats and Test Breakfast Meals 4.3 Fractional Recovery of Butter and Tallow Breakfast Meal [l- C]Fatty Acids 4.3.1 Fractional Recovery of Breakfast Meal [l- C]Myristic Acid 4.3.2 Fractional Recovery of Breakfast Meal [l- C]Palmitic Acid 4.3.3 Fractional Recovery of Breakfast Meal [l- C]Myristic versus [l- C]Palmitic Acid 4.4 Energy Expenditure and Substrate Oxidation Prior to Consumption of the Test Breakfast Meal and for Nine Hours Postprandial 4.4.1 Energy Expenditure 4.4.2 Carbohydrate Oxidation 4.4.3 Fat Oxidation 4.5 Dietary Fatty Acid Metabolism Relative to Whole Body Fat Metabolism 4.5.1 Content and Net Oxidation of Breakfast Meal Myristic and Palmitic Acids 4.5.1.1 Breakfast Meal Fatty Acid Oxidation Relative to Fat Intake 4.5.1.2 Breakfast Meal Fatty Acid Oxidation Relative to Postprandial Fat Oxidation 13  35 35 39  13  39  13  43  13  13  v  44  44 46 46 50 52 52 52 55  T A B L E OF CONTENTS 5. DISCUSSION  57  5.1 5.2 5.3  Suitability of the Present Protocol Suitability of the Present Isotopic Model Suitability of the Present Protocol to Determine Energy Expenditure and Substrate Utilization in Healthy Humans 5.4 Comparative Dietary Fatty Acid Oxidation in Healthy Humans Consuming Different Dietary Fats 5.5 Comparative Substrate Utilization in Healthy Humans Consuming Different Dietary Fats 5.6 The Contribution of Dietary Fat to Postprandial Fat Oxidation  57 58 60 62 66 68  6. CONCLUSIONS  70  BIBLIOGRAPHY  71  Appendix 3-1  Subject Consent Form  79  Appendix 3-2  Food/Nutrient Composition Analysis of Meals  81  Appendix 4-1  Hourly [l- C]Fatty Acid Recovery For Butterfat and Beef Tallow Treatments  93  Postprandial Energy Expenditure for Butterfat and Beef Tallow Treatments  94  Postprandial Carbohydrate Oxidation for Butterfat and Beef Tallow Treatments  95  Postprandial Fat Oxidation for Butterfat and Beef Tallow Treatments  96  Cumulative Fractional Oxidation of C-Fatty Acids for Individual Subjects  97  Cumulative Substrate Utilization and Energy Expenditure for Individual Subjects After the Butter Meal  98  Cumulative Substrate Utilization and Energy Expenditure for Individual Subjects After the Tallow Meal  99  Appendix 4-2  Appendix 4-3  Appendix 4-4  Appendix 4-5  Appendix 4-6  Appendix 4-7  13  13  vi  LIST O F T A B L E S Table 2-1  Postprandial Thermogenesis in Healthy Humans Consuming Medium Versus Long Chain Triglycerides  21  Long Term Energy Balance in Rats Consuming Medium Versus Long Chain Triglycerides  23  Table 4-1  Subject Parameters  36  Table 4-2  Weight Change Over Eleven Day Diet Cycles  37  Table 4-3  Fatty Acid Analysis of Treatment Fats and Test Breakfast Meals  Table 2-2  Table 4-4  Cumulative Oxidation, Peak Oxidation and Hour of Peak Oxidation for [l- C]Fatty Acid  38  13  Table 4-5  Table 4-6  Table 4-7  Table 4-8  Table 5-1  41  Energy Expenditure and Substrate Utilization Prior to Consumption of the Test Meal and for Nine Hours Postprandial  45  Content and Net Oxidation of Breakfast Meal Myristic and Palmitic Acids  53  Breakfast Meal Fatty Acid Oxidation Relative to Fat Intake  54  Breakfast Meal Fatty Acid Oxidation Relative to Postprandial Fat Oxidation  56  Comparative Fatty Acid Oxidation Between Studies  64  vii  LIST O F F I G U R E S Figure 3-1  Study Protocol  Figure 3-2  Carbon Dioxide Purification Apparatus  Figure 4-1  Hourly [l- C]Fatty Acid Oxidation Over Nine Hours Postprandial  Figure 4-2  Cumulative [l- C]Fatty Acid Oxidation Over Nine Hours Postprandial  Figure 4-3  Bi-hourly Energy Expenditure Over Nine Hours Postprandial  Figure 4-4  Bi-hourly Carbohydrate Oxidation Over Nine Hours Postprandial  Figure 4-5  Bi-hourly Fat Oxidation Over Nine Hours Postprandial  13  13  viii  ACKNOWLEDGMENTS It is my pleasure to gratefully acknowledge many individuals for their contribution to this thesis project. First, I would like to thank Dr. Peter Jones for his constant support, enthusiasm and expertise. I would also like to thank Dr. David Kitts for his constant involvement and wise counsel in this project. The support and input of Drs. Linda McCarger, Joseph Leichter, and P. Terry Phang are also gratefully acknowledged. This thesis project could not have been completed with out the assistance of Lynn Blake in the kitchen, and Tim Bailey and Janet Vogt in the laboratory. I sincerely appreciate the patience and cooperation of the eight individuals who volunteered as subjects in this project. The financial support of the Dairy Bureau of Canada is also very much appreciated. Finally, I would like to thank my family and friends who, in a very patient and understanding way, encouraged me to finish.  ix  1.  INTRODUCTION The preferential partitioning of dietary short and medium chain fatty acids for  oxidation has been observed in numerous studies. Geliebter et al. (1983) showed that rats fed medium chain fatty acids gained 20% less weight than those fed equivalent amounts of long chain fatty acids. Chain-length dependent differences in fatty acid oxidation have also been observed in studies examining respiratory gas exchange response to diet fat type where consumption of polyunsaturated (Jones et al., 1988; 1992) and shorter chain (Seaton et al., 1986; Hill et al., 1989; Scalfi et al., 1991) fatty acids increased oxidation and/or energy expenditure compared with saturated fats. Tracer studies in animals have similarly demonstrated that dietary medium chain fatty acids are preferentially oxidized to labelled carbon dioxide compared with long chain fatty acids (Johnson et al., 1990; Leyton et al., 1987). Correspondingly, the storage of labelled dietary fatty acids in muscle, adipose and other tissues is reduced with shorter chain fatty acids (Johnson et al., 1990; Leyton et al., 1987). In humans, fractional oxidation of dietary C - or C-octanoate has been shown to exceed that of palmitate 1 3  14  (Sabatin et al., 1987; Watkins et al., 1982) and oleate (Paust et al., 1990; Metges et al., 1991; Watkins et al., 1982) when administered in fat emulsions, lending further support to the view that shorter chain fatty acids are preferentially directed toward oxidation versus storage uses. Although the dependency of oxidation of dietary fatty acids on acyl chain length has been described, how changes in dietary fat composition influence the partitioning of individual fatty acids for energy remains poorly understood in healthy  1  humans. Whether changes in percentage (i.e., fractional) and absolute (i.e., net) oxidation of dietary fatty acids occur with dietary shifts in medium chain fatty acid content has not been established. Increases in net dietary fatty acid oxidation rates could result from either higher fractional oxidation, or unchanged fractional oxidation but increased dietary pool size. The extent to which fat composition and corresponding fatty acid pool size influences dietary fatty acid oxidation is presently unknown. Further, it is unknown whether oxidative effects result from immediate discrimination of meal fats within pathways leading to oxidation, or alternatively, whether mobilization of tissue fatty acid stores is responsible. To better understand chain length-dependent discrimination of fatty acid utilization in healthy humans, the objectives of this study were as follows: (1) To compare postprandial oxidation of dietary M A and PA in healthy volunteers fed test diets containing either primarily butter or beef tallow. For this analysis  1 3  C 0 enrichment in hourly breath samples following the 2  ingestion of [1- C]MA or PA was measured for 9 hours using isotope ratio mass 13  spectrometry. (2) To compare resting, total and diet-induced energy expenditure and substrate utilization in healthy humans consuming butter or tallow diets for 11 days. Energy expenditure, fat oxidation and carbohydrate oxidation were quantitated prior to consumption of butter or tallow breakfast meals and for 9 hours postprandial using respiratory gas exchange data.  2  (3) To examine the relative contribution of dietary M A and PA to total fat oxidation following consumption of butter or tallow test meals. This required the integration of dietary [l- C]fatty acid oxidation and total fat 13  oxidation results. It was hypothesized that consumption of butter, which contains a higher concentration of medium chain fatty acids than most other commonly consumed fats, may shift the partitioning of dietary fatty acids toward oxidation rather than storage. The null hypotheses under investigation in the current study were as follows: (HI) Fatty acid chain length and fat composition do not influence postprandial dietary fatty acid oxidation. (H2) Dietary fat composition does not influence postprandial energy expenditure or substrate utilization. (H3) Dietary fatty acid chain length and dietary fat composition do not influence the contribution of dietary fatty acid to total postprandial fat oxidation.  3  2.  LITERATURE REVIEW  2.1  STRUCTURE-DEPENDENT DIFFERENCES IN THE INTERMEDIARY METABOLISM OF FATTY ACIDS The molecular size and water solubility of structurally different fatty acids have  been shown to influence metabolic handling during digestion, absorption and chyloportal partitioning. Further, structurally different fatty acids show unequal affinity toward various catabolic and anabolic pathways. As detailed below, structure dependent differences in fatty acid intermediary metabolism may affect the partitioning of dietary fat toward oxidation versus storage.  2.1.1  DIGESTION AND ABSORPTION The hydrolysis of dietary triglyceride is initiated in the stomach by gastric and  lingual lipase (Levy et al., 1981). At this stage of digestion, small quantities of medium chain fatty acids, unlike long chain fatty acids, may undergo direct absorption into venous circulation (Vallot et al., 1985; Levy et al., 1984). Digestion of the majority of fats occurs in the intestinal lumen. In conditions of fat malabsorption, medium chain triglycerides are able to access the enterocyte independent of bile salts (Watkins et al., 1982) and pancreatic lipase (Blomstrand et al., 1968). Similarly, in normal conditions, medium chain triglycerides are hydrolysed and absorbed to a greater degree than long chain triglycerides (Bach et al., 1982; Caspary, 1992). For example the level of infant fatty acid absorption declined with  4  increased triglyceride-fatty acid chain length from 99% for caprylic acid and capric acid, to 95.7% for lauric acid, and 62.7% for oleic acid, as a percentage of triglyceride-fatty acid present in infant formula (Jensen et al., 1986). Fatty acid absorption is also influenced by level of saturation (DeSchrijver et al., 1991b), as illustrated by findings in healthy humans consuming mixed meals where dietary triglyceride-stearic acid was absorbed less efficiently than either -oleic or -linoleic acids (Jones et al., 1985). Although differences exist in the absorption of triglyceride-fatty acids, overall this physiological process is very efficient (Caspary, 1992). In males, the absorption of oleic acid (99%), palmitic acid (96-97%) and stearic acid (90-94%) was found to be high, regardless of whether triglyceride-fatty acids were components of butter, beef tallow or cocoa butter (Denke et al., 1991). Leyton et al. (1987) found that recovery of individually labelled dietary fatty acids in the urine and feces was low (1-3%). Finally, oral versus parenteral administration of C-triglyceride had no influence on 13  cumulative postprandial fatty acid oxidation (Metges et al., 1991). Overall, these studies suggest that the role of dietary fat composition in fat utilization is minimally influenced by digestion.  2.1.2  TRANSPORT The chylo-portal partitioning of fatty acid is dependent on its quantity,  composition and affinity for cystolic fatty acid binding protein (Bernard et al., 1990). Medium chain fatty acids show a low affinity for fatty acid binding protein, thus few  5  are esterified for chylomicron incorporation (Ockner et al., 1972). Studies conducted in vivo show that the carbon chain length as well as the degree of saturation influence the extent to which fatty acids are transported via the portal vein. Following digestion and absorption saturated long chain fatty acids are transported primarily via the thoracic duct by chylomicrons. In contrast, higher concentrations of medium chain and unsaturated fatty acids travel directly to the liver by preferential absorption and release into the portal blood. For example, lauric, myristic, palmitic, stearic, oleic and linolenic acids, individually infused into the rat duodenum, bypassed the lymphatic pathway at percentages of 72, 58, 41, 28, 58 and 68, respectively (McDonald et al., 1980). In a similar study, 49%, 7.8%, 6.4% and 10.6% of capric, oleic, linoleic and arachidonic acid, respectively, were recovered from portal circulation when individually infused in rat intestinal mucosa (Bernard et al., 1990). Of the few exogenous fatty acids which were oxidized in the intestine, medium chain and unsaturated fatty acids were oxidized more rapidly than saturated fatty acids (Bernard et al., 1990). The positive relationship between increasing fatty acid chain length and incorporation into chyle is also seen in humans. Swift et al. (1990) reported that chylomicron triglyceride transport from a long chain triglyceride diet was approximately five times greater than that from a medium chain triglyceride diet. Metges and Wolfram (1991) observed increased C 0 peak time for trioleate when 1 3  2  administered orally versus parenterally, thus demonstrating the extra time required for fatty acid absorption and transport across the thoracic duct. Thus, it can be concluded  6  from these studies that medium chain fatty acids are absorbed principally via the portal vein and oxidized to a greater extent in the liver (Bach et al., 1982; Metges and Wolfram, 1991). Similarly, portal transport accounts for a significant portion of unsaturated fatty acid metabolism (Bernard et al., 1990; McDonald et al., 1980)  2.1.3  HEPATIC EXTRA-MITOCHONDRIAL METABOLISM Characteristic structure-dependent differences in the metabolism of fatty acids  have been demonstrated following uptake by hepatic tissue. In the liver, as in the intestinal mucosa, fatty acid binding protein was most effective in the promotion of long chain fatty acid re-esterification (Ockner et al., 1972). Consequendy, few medium chain fatty acids are recovered in triglyceride (Christensin et al., 1991), phospholipid or cholesterol ester fractions (Mascioli et al., 1989), and low concentrations of medium chain fatty acids are deposited in various tissues (Johnson et al., 1990; Leyton et al., 1987). The effect of fatty acid saturation on hepatic esterification, secretion and subsequent utilization is less definitive. Lai et al. (1991) reported elevated hepatic triglyceride secretion and plasma triglyceride levels in response to saturated versus polyunsaturated fatty acid consumption, though this was dependent on the specific fatty acid consumed. For example, ingestion of butterfat or beef tallow increased triglyceride secretion by 20% and 30-60%, respectively, relative to corn oil (Lai et al., 1991). Further, depressed lipoprotein lipase activity in the heart and soleus muscle, accompanied by reduced fatty acid oxidation and serum triglyceride  7  level in rats fed beef tallow compared to unsaturated fat, has been reported (Shimomura et al., 1990). In the liver, fatty acid not directed toward esterification may follow various catabolic pathways including omega-oxidation, peroxisomal beta-oxidation or mitochondrial beta-oxidation. The contribution of omega-oxidation to fatty acid metabolism is controversial. Lauric and capric acids show a high affinity toward this metabolic pathway (Christensen et al., 1991). This is consistent with observed dicarboxylic aciduria following the administration of medium chain triglycerides in children and animals (Whyte et al., 1986; Bohles et al., 1987), as omega hydroxylated products produce water soluble dicarboxylic acids which can be excreted in the urine. Dias et al. (1990) investigated energy loss due to dicarboxylic aciduria in normal adults consuming a medium chain triglyceride diet for 3 days and reported that less than 1% of ingested energy was excreted as urinary dicarboxylic and keto acids due to medium chain triglyceride consumption. The level of urinary dicarboxylic acid excretion in this study was similar to previous studies and may have reflected demands in excess of beta-oxidation capacity. Thus, differential urinary excretion of medium chain versus long chain fatty acid would minimally influence dietary fat utilization. An alternate form of fatty acid oxidation is peroxisomal oxidation. Unlike mitochondrial beta-oxidation, peroxisomal beta-oxidation produces H 0 and is not 2  2  coupled with respiration (Ganning et al., 1988). Peroxisomal oxidation has been found to increase with greater cytosolic fatty acid concentration, potentially due to saturation  8  of the mitochondrial transport system (Reubsaet et al., 1988; Berge et al., 1988; Handler et al., 1988). With elevated cytosolic fatty acid concentration, it was estimated that as much as 25% of beta-oxidation occurred in the peroxisome of the perfused rat liver (Handler et al., 1988). Further, the relative affinity of individual fatty acids toward peroxisomal oxidation is dependent on acyl-CoA concentrations (Reubsaet et al., 1988). Although peroxisomal oxidation has been classically associated with both complete and partial oxidation of very long chain fatty acids (Ganning et al., 1988; Handler et al., 1988), at high fatty acid concentrations, caprylic, capilic and lauric acids produced the greatest peroxisomal activity in rat liver extract (Reubsaet et al., 1988) and perfused rat liver (Handler et al., 1988), relative to fatty acids of increasing or decreasing chain length. Thus, increased thermogenic and oxidative effects associated with medium chain triglyceride consumption may result from their increased affinity toward this energetically inefficient pathway.  2.1.4  MITOCHONDRIAL BETA-OXIDATION AND SUBSEQUENT UTILIZATION The final and most favourable pathway of fat catabolism is mitochondrial beta-  oxidation. To cross the mitochondrial membrane, long chain fatty acids require the assistance of carnitine palmityl transferase 1 and 11. Consequently, the concentration of long chain fatty acids crossing the mitochondrial membrane is limited (Bach et al., 1982). Conversely, medium chain fatty acids are able to penetrate the mitochondria free of this transferase system (McCarry and Foster, 1971a; 1971b; Freidman et al.,  9  1990). For example, Christensen et al. (1989) reported that most lauric acid gained independent access to the mitochondria, although carnitine-dependent mitochondrial oxidation did occur to a minor extent. This is consistent with elevated hepatic acetyl-CoA levels observed with medium chain triglyceride consumption (Bach et al., 1976). The oxidation of C-fatty acid to C 0 in isolated hepatocytes has consistently 14  14  2  been shown to decrease with increasing chain length (Pegorier et al., 1988; Crozier et al., 1988; Christensen et al., 1991; Christensen et al., 1989). However, oxidative capacity is limited (McCary et al., 1971b) and unlike low levels of acetyl-CoA, which are preferentially directed toward the production of C0 , higher levels of acetyl-CoA 2  also stimulate ketone body formation (Berry et al., 1983). The ketogenic effects of medium chain triglyceride ingestion are well documented in animals and humans (Crozier et al., 1987; Dias et al., 1990; Bach et al., 1976). Relative to saturated fatty acids, polyunsaturated fatty acids have also been associated with elevated ketone body formation (Beyne and Katan, 1985) and mitochondrial fatty acid oxidation (Bjorntorp, 1968). Acetyl-CoA, derived from fatty acid, particularly medium chain fatty acid, may also contribute to fatty acid elongation (Bach et al., 1982). Christensen et al. (1989) reported significant conversion of labelled lauric acid to palmitic and lesser amounts of myristic, palmitoleic, stearic and oleic acids in isolated hepatocytes derived from rats re-fed fructose. This is complemented by findings in the human where fasting plasma triglyceride-PA concentration was two fold higher with 6 day consumption of a  10  medium versus long chain triglyceride diet, although the PA content of the medium chain triglyceride diet was 5 fold less than that of the long chain triglyceride diet. Relative increases in fasting plasma triglyceride-stearic and -oleic acid concentrations were also associated with the medium chain triglyceride diet treatment, suggesting that both chain elongation and desaturation had occurred (Hill et al., 1990). Alternatively, acetyl-CoA derived from fatty acid may be resynthesized into fatty acid and esterified (Hill et al., 1989; Mascioli et al., 1989). In fact, increased carnitine transport of acyl and acetyl groups out of the mitochondria has been observed with infusion of medium chain versus long chain fatty acids in the human (Rossle et al., 1990). The theoretical thermogenic cost of octanoate metabolism is 3.3% if directed toward immediate oxidation, 6.7% if directed toward ketone formation and 32.3% if directed toward de novo fatty acid synthesis (Hill et al., 1989). This suggests that the potential metabolic pathways of fat metabolism are not equivalent in terms of energy efficiency.  Consequently, increased affinity of medium chain versus long chain fatty  acids toward pathways of ketone formation and de novo fatty acid synthesis may result in an elevated thermogenic response to medium chain fatty acid consumption.  2.1.5  OTHER FACTORS AFFECTING FAT UTILIZATION  2.1.5.1  F A C U L T A T I V E THERMOGENESIS Facultative thermogenesis refers to heat production in excess of obligatory  demands, occurring in response to environmental change, and resulting in no net  11  increase in substrate concentration. Facultative thermogenesis includes uncoupling or reduced coupling of oxidation to phosphorylation (Mercer et al., 1987), substrate cycling (Wolfe et a l , 1990) and reversed electron transport (Berry et al., 1983), all of which may be dependent on dietary fat composition. Non-shivering heat production results from uncoupling of oxidative phosphorylation in brown adipose tissue. This process, associated with cold adapted and cafeteria-fed rodents, is controversial in humans. Mercer et al. (1987) reported that in rats, dietary corn oil increased stimulation of brown adipose tissue relative to that resulting from the consumption of beef tallow. It was suggested that excess stimulation of brown adipose tissue was mediated through polyunsaturated fat consumption which potentially stimulated the sympathetic nervous system or altered membrane function (Mercer et al., 1987). However, rats overconsuming medium chain versus long chain triglyceride diets for 6 weeks, following recovery from interscapular brown adipose tissue (IBAT) removal, gained significandy less weight and contained 25% less dissectible fat, confirming the thermogenic effect of medium chain triglyceride overfeeding even in the absence of IBAT. Dietary fat metabolism may also stimulate substrate cycling. Substrate cycling is the simultaneous activation of opposing reactions, which together consume energy, produce heat, and result in no net product formation. Crozier (1988) proposed that the cycling of carbon between beta-oxidation and lipogenesis may be a mechanism utilized to cope with the large influx of acetyl-CoA precursors associated with medium chain triglyceride consumption.  12  Reverse electron transport has been observed in isolated liver cells from starved (Berry et al., 1983) and cafeteria fed (Berry et al., 1985) rats and is associated with the reduction of acetoacetate to 3-hydroxybutyrate during the flavin-linked step of fatty acid oxidation. Specifically, reducing equivalents are channelled from mitochondrial flavin to mitochondrial nicotinamide adenine dinucleotide (NAD) to cytoplasmic N A D back to mitochondrial flavin. Reverse electron transport is associated with greater heat production, suggesting that hepatic fat metabolism may play a role in altered fat thermogenesis (Berry et al., 1985). This process, which is stimulated by the betaoxidation of fatty acids to acetyl-CoA, maintains mitochondrial N A D in a greater reduced state and allows for rapid disposal of high concentrations of fatty acid, probably toward ketogenesis. The authors suggest that increased substrate flow to the liver may stimulate this process (Berry et al., 1985). Given a large hepatic influx of medium chain fatty acid (Bach et al., 1982), increased rates of beta-oxidation (Bach et al., 1976) and elevated levels of beta-hydroxybutyrate (Seaton et al., 1986; Hill et al., 1990; Rossle et al., 1990; Wiley et al., 1973) resulting from medium chain versus long chain fatty acid administration, this process may contribute to enhanced thermogenesis associated with the consumption of medium chain triglyceride.  2.1.5.2  L O N G AND SHORT T E R M PREFEEDING In addition to features already mentioned, the intermediary metabolism of fatty  acids may be independently influenced by long and short term prefeeding of treatment diets (Pegorier et al., 1988; Crozier, 1988; 1987; Hill et al., 1989). Crozier (1988)  13  found that hepatocytes derived from rats consuming medium chain versus long chain triglyceride or low fat diets utilized 2 to 4 times more oleic and caprylic acids. Elevated fat utilization in hepatocytes derived from medium chain triglyceride fed rats was associated with concurrent activation of rapid and extensive fatty acid catabolism, expressed as C 0 production, and anabolism, in the form of ketogenesis, lipogenesis 2  and glucose production. Pegorier et al. (1988) suggested that decreased sensstivity of carnitine palmityl transferase 1 to malonyl-CoA inhibition may be responsible for enhanced fatty acid metabolism associated with long term medium chain triglyceride consumption. In whole body studies blood ketone body concentrations were reported to decline two fold (Crozier et al., 1987), or to levels similar to that of other fat treatments (Chanez et al., 1991), with progressive adaptation to a medium chain triglyceride diet. This adaptive response may involve decreased ketone production and/or increased utilization. In humans, postprandial thermogenesis following consumption of a medium chain triglyceride test meal increased significandy from day 1 to day 6 of a medium chain triglyceride diet while postprandial thermogenesis due to long chain triglyceride feeding remained the same over 6 days (Hill et al., 1989). Based on comparative fasting plasma lipid levels, elevated postprandial thermogenesis on day 1 of the medium chain triglyceride diet may have resulted from increased ketone body formation. The further rise in postprandial thermogenesis by day 6 of the medium chain triglyceride diet may have been due to a shift toward increased de novo fatty acid synthesis (Hill et al., 1990). These studies suggest that medium chain  14  triglyceride pre-feeding may actually enhance structure dependent differences in fatty acid metabolism. It is apparent that structure-dependent differences in the intermediary metabolism of fatty acids do exist. These differences result in unequal affinity of fatty acids toward various metabolic pathways. Ease of absorption (Jensen et al., 1986), preferential transport in the portal blood (Bernard et al., 1990; Swift et al., 1990), a low affinity toward esterification (Ockner et al., 1972), independent penetration of the mitochondrial membrane (McCarry and Foster, 1971a; 1971b; Freidman et al., 1990) and a greater affinity toward energetically inefficient pathways of fat metabolism such as ketogenesis (Crozier et al., 1987; Dias et al., 1990; Bach et al., 1976), fatty acid elongation (Bach et al., 1982; Christensen et al., 1989), fatty acid synthesis (Rossle et al., 1990) and facultative thermogenesis (Crozier, 1988) are associated with fatty acids of shorter chain length. Similarly, reports indicate that unsaturated fatty acids show increased absorption (DeSchrijver et al., 1991b; Jones et al., 1985), portal transport (McDonald et al., 1980) and ease of oxidation (Bjorntorp, 1968) relative to saturated fatty acids.  2.2.  T H EINFLUENCE O F DIETARY F A T COMPOSITION O N SUBSTRATE UTILIZATION A N DE N E R G Y  EXPENDITURE  The influence of dietary fat composition on substrate utilization and energy expenditure has been examined using a variety of methodologies including various isotope tracer techniques, short term respiratory gas exchange measurement and long  15  term whole body substrate/energy balance methods. Determination of C 0 enrichment 2  following an oral bolus of labelled fatty acid or triglyceride provides an accurate estimation of dietary fatty acid oxidation, while short term respiratory gas exchange and long term substrate/energy balance reflects the overall effect of dietary fat composition on substrate utilization (dietary and endogenous) and energy expenditure.  2.2.1  FAT OXIDATION  2.2.1.1  DIETARY FATTY ACID OXIDATION BASED ON C- AND C13  14  BOLUS TRACER METHODOLOGIES Structure dependent differences have been found to influence dietary fatty acid oxidation, based on the findings of C - and C - bolus tracer methodologies in the rat. 1 3  1 4  For example, administration of intravenous lipid emulsions produced a more complete oxidation of C-medium chain (90% of administered dose) versus C-long chain (45% 14  14  of administered dose) triglyceride over 24 hours (Johnson et al., 1990). Level of fatty acid saturation also influenced dietary fatty acid oxidation where dietary [l- C]linoleate, compared to [l- C]palmitate, was preferentially oxidized to 14  14  respiratory C 0 (Cenedella et al., 1969). In fact, for only one hour, from 5 to 6 1 4  2  hours post isotope administration, approximately 11% of [l- C]palmitate and 21% of 14  [l- C]linoleate were recovered as respiratory C 0 . Leyton et al. (1987) analyzed 14  1 4  2  respiratory C 0 evolution following oral dosing of C-fatty acid and reported that I 4  14  2  after 24 hours the oxidation of dietary saturated fatty acids decreased with increasing  16  chain length as 63%, 40%, 32% and 25% of lauric, myristic, palmitic and stearic acid, respectively, were recovered in respiratory C 0 . Using the same model it was shown 2  that rates of oleic and linolenic acid oxidation were similar to that observed for lauric acid (Leyton et al., 1987). Fatty acid chain length appears to affect not only the quantity of dietary fat oxidized, but the rate in which oxidation occurs. In studies where healthy children, ranging in age from 3 months to 17 years, were administered an oral bolus of C 1 3  triolein, C - P A or C-trioctanoin, the appearance of C 0 from C-trioctanoin 13  13  1 3  13  2  reached maximum levels 2 to 4 hours after administration (Watkins et al., 1982), C 1 3  triolein oxidation peaked between 4 and 6 hours. Labelled PA appeared as  1 3  slowly, increasing gradually over a 6 hour period. Cumulative excretion of  1 3  C 0 very 2  C0  2  for  6 hours was 27.6% for trioctanoin, 11.3% for triolein and 6.6% for PA. Similar results were reported in new born infants (Paust et al., 1990). The method of administering lipids for the purpose of determining differential oxidation has a large impact on the extent of fatty acid oxidation over time. A concern of particular relevance is the effect of parenteral versus oral administration on subsequent oxidation. For example, cumulative and peak  1 3  C0  2  oxidation of octanoate  were independent of oral versus parenteral administration in humans (Schwabe et al., 1968; Metges et al., 1991). In contrast, peak C 0 breath enrichment of C-oleate 1 3  13  2  was faster when administered parenterally, although cumulative oxidation remained unaffected by administration route (Metges et al., 1991). Interpretation of these results require consideration that orally administered long chain triglyceride and parenterally  17  administered long and medium chain triglyceride are acted upon by lipase in all tissue. In contrast medium chain triglyceride administered orally, is primarily removed from the plasma by hepatic lipase before entering the liver (Johnson and Cotter, 1986). This suggests that extra-hepatic tissue is as effective as hepatic tissue at rapidly oxidizing medium chain fatty acid. Another factor which has been shown to affect the extent of dietary fatty acid oxidation is the diet fat/carbohydrate ratio. Oxidation of C-medium chain fatty acid 13  appears to be inhibited by simultaneous administration of carbohydrate (Paust et al., 1990). Thirty five percent of a C-palmitate bolus was recovered over 8 hours in the 14  rat. This dropped considerably to 7% when the C-palmitate bolus was accompanied 14  by a high carbohydrate meal (Toorop et al., 1979). The influence of meal composition on the fractional oxidation of dietary fat warrants further attention as it impacts on the comparability of fat utilization studies and their application to a normal feeding situation. The effect of fat composition on its subsequent utilization has also been demonstrated during exercise where the oxidation of dietary C-medium chain 13  triglyceride exceeded that of dietary C-long chain triglyceride (Satabin et al., 1987) 13  and equalled (Massicotte et al., 1992) or fell short of dietary C-glucose (Satabin et 13  al., 1987). Massicotte et al. (1992) reported that 54% of C-trioctanoate, ingested 1 13  hour pre-exercise, was oxidized 2 hours after exercise initiation. Satabin et al. (1987) observed an oxidation rate of 45% for [l- C]octanoate under similar experimental 14  18  conditions. During the latter study, the corresponding oxidation of [l- C]palmitate 14  was 9% of adrninistered dose.  2.2.1.2  POSTPRANDIAL NET FAT OXIDATION BASED ON RESPmATORY GAS EXCHANGE ANALYSIS Complimentary, though less precise findings of differential fat utilization have  been observed in humans using respiratory gas exchange data. The intravenous administration of a medium chain triglyceride emulsion produced a significant increase in medium chain triglyceride oxidation over 10 hours, while the oxidation of long chain triglyceride from its corresponding emulsion remained similar to basal levels (Mascioli et al., 1991). In this study, the rise in energy expenditure observed with the medium chain triglyceride treatment could be entirely accounted for by enhanced fat oxidation (Mascioli et al., 1991). Similarly, a diet high in polyunsaturated fat resulted in enhanced postprandial diet-induced fat oxidation, relative to a similar saturated fat diet (Jones et al., 1988; 1992). In contrast, Flatt et al. (1985b) investigated postprandial fat oxidation over 9 hours and found no significant difference due to fat composition. In general, there is sufficient data to indicate that the whole body utilization of dietary fat differs on the basis of composition. Dietary medium chain and unsaturated fatty acids show a greater affinity toward immediate oxidation, while longer chain fatty acids are more likely directed toward cell structure and storage.  19  2.2.2  ENERGY BALANCE Dietary fat composition may effectively alter not only fat oxidation, but the  oxidation of other macronutrients. For example, compared to a similar long chain fatty acid administration, the administration of medium chain fatty acid maintained whole body protein balance (Yamazaki et al., 1972; Hill et al., 1989; Mascoili et al., 1991), while increasing fat oxidation (Metges et al., 1991; Mascioli et al., 1991) and maintaining (Flatt et al., 1985b; Mascioli et al., 1991) or actually increasing (Sato et al., 1992) carbohydrate oxidation, possibly through increased response to insulin (Yost et al., 1989). Unsaturated fats have also been associated with increased fat oxidation over basal levels (Jones et al., 1988; 1992) with subsequent reduction (Jones et al., 1988) or maintenance (Jones et al., 1992) of carbohydrate oxidation over basal levels, compared to saturated fat. The body is most efficient at oxidizing carbohydrate (Flatt et al., 1977) and storing fat (Flatt et al., 1985a). Medium chain and unsaturated fatty acids seem to disrupt the efficient orchestration of macromolecular metabolism.  2.2.2.1  T H E R M O G E N I C RESPONSE T O D I E T A R Y F A T B A S E D O N RESPIRATORY GAS E X C H A N G E ANALYSIS With the exception of Flatt et al. (1985b) researchers have consistently  observed enhanced thermogenic response to medium chain versus long chain fatty acid meal consumption, regardless of varied caloric and fat intake ( T A B L E  2-1).  Integration of the above findings from several studies involving consumption of test meals in healthy humans (Flatt et al., 1985b; Seaton et al., 1986; Hill et al., 1989;  20  T A B L E 2-1  POSTPRANDIAL THERMOGENESIS IN H E A L T H Y HUMANS CONSUMING MEDIUM VERSUS L O N G CHAIN TRIGLYCERIDES  Treatment fat (g)  Energy content (kcal)  44  1000  day 1 - 80±8 vs 58±8 kcal* day 6 - 120±13 vs 6 6 ± 1 0 kcal*  Hill et al., 1989  45  400  53 vs 17 kcal*  Seaton et al., 1986  30  1300  88±2 vs 59±7 kcal*  Scalfi et al., 1991  40  858  105±13 vs 96±11 kcal  Flatt et al., 1985  Diet-induced thermogenesis (medium vs long chain triglycerides)  Reference  * indicates significant difference (p<0.05) Treatment fat represents quantity of medium versus long chain triglyceride in the meal. Postprandial thermogenesis was measured for 6 hours with the exception of Flatt et al. (1985) where measurement was 9 hours in duration.  21  Scalfi et al., 1991; n=40) indicated that postprandial energy expenditure (calculated relative to meal energy), was approximately 6% higher with medium chain versus long chain fatty acid test meal consumption. As diet-induced thermogenesis accounted for approximately 15% and 9% of energy present in medium chain and long chain fatty acid meals, respectively, diet-induced thermogenesis was approximately 63% higher with medium chain fatty acid feeding. The consumption of a diet high in unsaturated fat had no significant effect on postprandial energy expenditure in humans (Jones et al., 1988; 1992). In rats, oxygen consumption following a meal high in safflower oil exceeded that of a meal high in saturated fat (Shimomura et al., 1990).  2.2.2.2  ENERGY BALANCE DUE T OLONG T E R M CONSUMPTION O F DIFFERENT DIETARY FATS Long term energy balance studies have examined the effect of dietary fat  composition on energy balance, expressed as fluctuations in weight, fat deposition or energy retention. T A B L E 2-2 details long term energy balance studies involving medium chain fatty acid administration in rats. Similar long term studies in humans are less common, although Yost et al. (1989) found weight loss did not differ in obese women consuming 800 kcal/day liquid diets containing 24% of energy as medium chain or long chain triglycerides for up to 12 weeks. A number of long term studies have compared energy balance in rats fed commonly consumed fats. Increased fat accumulation occurred with consumption of  22  TABLE 2-2 LONG TERM ENERGY BALANCE IN RATS CONSUMING MEDIUM VERSUS LONG CHAIN TRIGLYCERIDES Treatment fat  Duration (days)  Energy balance (medium versus long chain triglyceride treatment)  Reference  63% (kcal)  44  M C T group consumed 13% fewer kcal and gained 28% less weight.  Crozier et al., 1987  45% (kcal)  42  M C T group gained 23% less weight, contained fat deposits which were reduced by 23%. Activity/intake were controlled.  Geliebter et al., 1983  32% (kcal)  45  M C T and low fat group gained 26% less weight than L C T group.  Chanz et al., 1991  50% (kcal)  42  Interscapular brown adipose tissue removed. M C T group gained less weight.  Baba et al., 1982  55% (kcal)  56  M C T group gained 10% less weight and had 40% less fat accumulation in fat pads.  Lavau et al., 1978  12% (wet weight)  21  M C T group consumed fewer kcal and gained less weight.  Whiley et al., 1973  Treatment fat represents quantity of medium versus long chain triglyceride in the diet.  23  beef tallow compared to corn oil (Mercer et al., 1987) or safflower oil (Shimomura et al., 1990). Conversely Awad et al. (1990) failed to note any effect of fat composition on long term energy balance in rats consuming menhaden oil, safflower oil or beef tallow. In summary, dietary fatty acid structure influences its intermediary metabolism and partitioning toward oxidation versus storage. This process, however, remains poorly understood in healthy meal fed humans consuming different dietary fatty acid blends.  24  3.  METHODOLOGY  3.1  STUDY SUBJECTS Eight, non-obese, male volunteers provided informed written consent to  participate in the study  (APPENDDX 3-1). All procedures utilized in the study were  approved by the Clinical Experimentation Ethical Review Conirnittee of the University of British Columbia. Subjects reported no history of chronic disease, abnormal triglyceride metabolism or abnormal cholesterol metabolism. Additionally, none of the subjects had atypical sleep patterns or atypical activity patterns. None of the subjects regularly consumed low fat or vegetarian diets.  3.2  STUDY DIETS Subjects were fed diets containing 22% of energy as either butter or tallow for  11 days. The diets, which provided 40%, 45%, and 15% of energy as fat, carbohydrate and protein, respectively, were fed at a level calculated to satisfy individual energy requirements. Energy requirements were calculated using the Harris Benedict equation (Harris et al., 1919) with an adjustment factor of 1.7 to compensate for additional energy needs of young men (Bell et al., 1985) and another adjustment for strenuous athletics. Formulated nutrient content of diets and component meals were calculated using Nutricomp (Robert Kok and Serge Trembley, Version 1, 1984), a computerized Canadian nutrient composition program. Diets were provided as a 2 day rotating menu of 3 meals per day. Meals were isocaloric in energy and fat.  25  Subjects were instructed to consume all food provided. The rotating menu was modified to ensure that, for all trials, day 1 of the rotating menu was used on pre-test days, and day 2 of the rotating menu was used on test days. Specific foods contained in the 2 day rotating menu, and a detailed nutrient analysis of each meal, are included in A P P E N D I X 3-2. To minimize natural product variation, a single homogenized batch of both butter and tallow were utilized; a single brand of packaged food was consistently purchased; whenever possible, meat and produce were purchased in a single batch; and finally, all foods were obtained from the identical source. Other than the prescribed diet, ingestion of any drugs or energy-containing substances were prohibited. A l l consumed foods were weighed to the nearest 0.5 g with the exception of treatment fats which were weighed to the nearest 0.1 g. Meals were prepared and consumed in the metabolic kitchen, Family and Nutritional Sciences Building, University of British Columbia. Subjects unable to consume a meal at the Family and Nutritional Sciences Building were given a packaged meal to be consumed at the regular meal time.  3.3  STUDY P R O T O C O L A cross-over design was used which included two, 11 day, randomized diet  cycles, separated by 7 days or more of habitual eating. Subjects were instructed to maintain typical and consistent sleep and exercise patterns during the study. Subjects removed footwear and outer clothing before daily weighing, which occurred prior to breakfast. On day 8 and 11 of each diet cycle, at 07:00 hour, following a 12 hour  26  fast, subjects reported to the metabolic unit at St Paul's Hospital (FIGURE 3-1). Following a brief relaxation period, continuous respiratory gas exchange and periodic breath sample collection were initiated. At 08:00 hour the breakfast meal was consumed by subjects. This was accompanied by a randomly assigned oral bolus of either [1- C]MA or [1- C]PA (Isotec Inc., Miamisburg, Ohio), fed at a level of 20 13  13  mg/kg body weight, and administered in gelatin capsule form. At 10:00 hour, of either day 8 or 11, and 24 hours later, a 10 ml blood sample was drawn in connection with an independent research project. At 12:20 hour, subjects consumed the lunch test meal. Continuous respiratory gas exchange was measured from 07:20 to 15:00 hour, 15:45 to 16:15 hour, and 16:45 to 17:15 hour. Respiratory gas exchange measurement was interrupted during test meal consumption, blood collection, and occasionally for washroom use. Baseline breath samples were collected at 07:30 and 09:00 hour. Similar hourly samples, from 10:00 to 17:00 hour were also collected. Breath sample collection was approximately 6 minutes in duration. During all measurements subjects reclined in a hospital bed. Reading, listening to music, or watching television were permitted; sleeping and physical movement beyond minor positional changes were discouraged.  27  FIGURE 3-1  STUDY PROTOCOL  DIET CYCLE MENUl D A Y  1  1  2  1  T  2  3  4  5  6  2  1  2  2  U  I  I  1  7  • — •  7am 8  Oh Oh  F I G U R E 3-2  9  lh  10  11  TEST DAY  MEAL  •  I  8 9  TEST D A Y BEGIN TEST  1  #  r  TEST DA7 END  MEAL  •  10 gh  i  11 3h  i  12 4h  i  1  5h  r  TEST  i  2  6h  3 7h  4  8h  5pm  9h  C A R B O N DIOXIDE PURIFICATION A P P A R A T U S  Pressure gauge V2  Sample bottle  V1  Dry ice methanol bath  28  3.4  ANALYTICAL PROCEDURES AND DATA ANALYSIS  3.4.1  RESPIRATORY GAS EXCHANGE MEASUREMENT Respiratory gas exchange measurements were performed using a Deltatrac  Metabolic Monitor (Sensormedics, Anaheim, CA). A transparent ventilated hood was placed over the subjects head, with a hose connecting the hood and analyzer system. Following a warm-up period, reference standards were used to calibrate the unit. A l l measurements were automatically corrected for environmental temperature, pressure, and humidity. Initial validation of this specific monitor with a lung model identified inaccuracies of 1.9% and 1.5% for oxygen consumption and carbon dioxide production determination, respectively (Phang et al., 1989).  3.4.2  RESPIRATORY GAS EXCHANGE ANALYSIS Respiratory gas exchange measurements were recorded each minute.  Measurements prior to the breakfast meal, and postprandially (commencing at 08:00 hour), were analyzed in 30 minute segments. Respiratory gas exchange data for the butter and tallow treatments were obtained on day 8 and 11 of each diet cycle. Resting metabolic rate was monitored for 30 minutes prior to consumption of the breakfast meal. Diet-induced thermogenesis was calculated as total energy expenditure minus resting metabolic rate. A constant protein oxidation rate of 0.7 g protein/kg fat free mass/day was assumed (Jones et al., 1988). Weir's formulas were used to calculate fat and carbohydrate oxidation (Weir, 1949).  29  3.4.3  COLLECTION OF C 0 BREATH SAMPLES 2  During respiratory gas exchange measurement, a portion of exhaled gas flowing through the metabolic analyzer system was directed toward a 100 cm spiral trap. The trap contained 10 ml of NaOH which collected all C 0 . Adequate C 0 2  2  collection  required approximately 6 minutes. This was monitored using a standard wall clock. Collected C 0 samples were stored in sealed vacutainers and frozen at -10 °C for later 2  analysis.  3.4.4  PURIFICATION O F C 0 B R E A T H SAMPLES 2  Carbon dioxide samples, effectively trapped in the NaOH solution as N a H C 0 , 3  were isolated and purified. In the initial step of this procedure, 1 ml of N a H C 0  3  sample solution was injected into a vacutainer containing 1 ml H P 0 . The resulting 3  4  solution was manually mixed prior to being submerged in a liquid N bath for 3 2  minutes. Following this, the rubber stopper of the vacutainer was connected to, but not penetrated by, a needle portion of the C 0 purification line (indicated by the 2  arrow, F I G U R E 3-2). Valves 1, 2, 4, and 5 of the C 0 purification line were opened 2  and the line was evacuated to a pressure of less than 100 millitorr. The rubber stopper of the vacutainer was then punctured by the needle, and the vacutainer was evacuated to less than 100 millitorr. Following evacuation, valves 1 and 2 of the C 0  2  purification line remained open, while valves 3, 4, and 5 were closed, thus creating a contained vacuumized portion of the line between valves 1 and 4. The liquid N bath, 2  which up to this point housed the vacutainer, was quickly replaced by a liquid  30  Nj/methanol slush bath. Valve 1, connecting the vacutainer to the contained portion of the C 0 purification line was opened, and C 0 gas proceeded out of the vacutainer, 2  2  down the line, through another Na/methanol slush bath, and finally to a liquid N bath. 2  This transfer required 5 minutes. Following the first transfer, valve 2 was closed and valve 4 opened, again creating a contained vacuumized portion of the C 0 line 2  between valve 2 and 5, which housed a liquid N bath containing the purified C 0 2  2  sample and a collecting tube/sample bottle. The liquid N bath was removed, the line 2  was heated and the purified C 0 gas proceeded down the line to the collecting 2  tube/sample bottle which was also bathed in liquid N . The second transfer also 2  required 5 minutes. The collecting tube was then flame-sealed. Overlap of sample purification was possible, resulting in a single sample purification time of approximately 11 minutes. Sample analysis was performed in duplicate and transfer time was controlled using a stop clock with alarm.  3.4.5  MASS SPECTROMETRY A dual inlet gas isotope ratio mass spectrometer (VG 903, V G Isogas Limited,  1988) was used to determine the C 0 enrichment of the purified C 0 breath samples. 1 3  2  2  The mass spectrometer was corrected for 0 and calibrated daily using reference 1 7  standards. Carbon dioxide was admitted to the sample side of the dual inlet system. The other side of the dual inlet system housed a C 0 reference gas of predetermined 2  composition (Boutton, 1991). Both gases flowed to the changeover valve assembly  31  and alternately into the ion source. During this procedure sample and reference gas pressures were matched. An electronic beam ionized the C 0 molecules and positive 2  ions were propelled down the analyzer tube. Ions passed through a curved sector within a magnetic field. As magnetic field strength and accelerating potential were maintained at a constant level, ion deflection was due to mass and energy. Carbon dioxide ions with less mass, thus less momentum, were deflected to a greater extent. Variability in C 0 mass produced 3 different ion beams, which struck separate 2  Faraday collector cups. As the ions were neutralized, electrical currents were produced. These were amplified and used to compute the stable isotope ratios. This procedure involved 6 measurements of standard and sample gases requiring approximately 10-15 minutes per sample. The actual measurement of interest was the difference between the ratio of C 0  2  of mass 44 (ie. CO^) and mass 45 (ie. "COj) in both the sample and reference gases. 12  Enrichment was measured in del  (7°°)» which means part per thousand.  %o was equivalent to the part per 1000 difference between the  1 3  Thus, C 1 3  C content of sample  and reference gases, calculated as follows: *  1 3  C %o = (R sample - R reference) x 1000 / R reference  where R = mass 45/44 C 0 ratio of a gas 2  Enrichment of C was expressed relative to a calcium carbonate standard 1 3  (PDB) with a clearly defined C / C ratio of 0.011 %>o, effectively normalizing the 1 3  1 2  data between individual laboratories. Conversion of del to atom percent excess (APE),  32  a more practical means of expressing enrichment data was accomplished through the following formula: *  A P E = (°/oo * R reference)/(°/oo * R reference + 1000)* 100% Background  1 3  C A P E , based on 07:30 and 09:00 hour baseline breath sample  enrichment was subtracted from  1 3  C APE breath sample enrichment of later samples to  determine enrichment due to [1- C]FA oxidation. 13  Percent of ingested [1- C]FA appearing in the breath as 13  1 3  C 0 was determined 2  using the following equation: *  mM C 0 * f *  1 3  2  C APE * 1.25/mM C administered 1 3  where f = fractional contribution of carbon mass to C 0 (Jones et al., 1985) 2  and 1.25 = correction factor for C 0 dilution in bicarbonate pools (Irving et al., 1983). 2  3.4.6  G A S LIQUID C H R O M A T O G R A P H Y Test meals were homogenized using a commercial blender. The fatty acid  content of treatment fats and breakfast test meal homogenates were detennined using gas liquid chromatography after lipid extraction (Folch et al., 1957) and boron trifluoride methylation (Bannon et al., 1982). The gas chromatograph was a Hewlett Packard (HP) 5890 Series II equipped with flame ionization detection. Separation was achieved on an HP-5, 30 m capillary column, 0.2 mm internal diameter, 0.33 um thickness. Spilt ratio was 100:1. Running conditions were: initial temperature 180 °C, ramp rc/minute, final temperature 210°C, hold for 30 minutes. Total run time was 60 minutes per sample.  33  3.4.7  DETERMINATION O F BREAKFAST M E A L F A T T Y ACID C O N T E N T AND OXIDATION The content of M A and PA in the test breakfast meals were calculated for  individual subjects using the following formula: *  fatty acid content (g) = breakfast meal fat (g) * concentration of corresponding fatty acid in the breakfast meal (g fatty acid/100 g fat) Net oxidation of dietary M A and PA was calculated as:  *  net oxidation (mg/9 hours) = breakfast meal fatty acid (mg) * fractional oxidation (%dose/9 hours)  3.5  STATISTICS Data were expressed as mean±SEM. Asymetrical distribution of data about the  mean was confirmed by testing for skewness. Fatty acid, fat and carbohydrate oxidation as well as energy expenditure time series data were analyzed using an A N O V A with repeated measures. Cumulative, peak and resting respiratory gas exchange and fatty acid oxidation data were analyzed using a paired t-test. Level of significance was defined as p<0.05.  34  4.0  RESULTS  4.1  STUDY SUBJECTS Subject age, height, weight and daily energy intake are reported in T A B L E  4-1. During diet cycles, no alterations in sleep, activity patterns or general health were reported. Weight change over 11 day diet cycles is reported in T A B L E 4-2 and ranged from 0.05-0.90 kg. Mean weight change was small (x±SEM; 0.08±0.17 kg and 0.13±0.25 kg, for the butter and tallow treatment, respectively), suggesting that calculated energy requirements were consistent with actual energy expenditure. Weight fluctuation over 11 day cycles did not differ significandy on the basis of treatment fat.  4.2  F A T T Y ACID ANALYSIS O F T R E A T M E N T F A T S A N D T E S T BREAKFAST MEALS Fatty acid analysis of butter, tallow and corresponding breakfast meal  homogenates are presented in T A B L E 4-3. Overall, 83% of fatty acids in the butter breakfast meal were compositionally the same as those in the tallow breakfast meal. Thus, of the approximately 47 g of lipid provided in either the butter or tallow breakfasts, 38.6 g were identical. Both butter and tallow test breakfast meals contained large quantities of PA and oleic acid but lesser amounts of stearic and linoleic acid. The butter breakfast meal contained more short and medium chain fatty acids up to and including M A (15.4 g and 6.6 g/100 g fat in butter and tallow breakfast meals,  35  T A B L E 4-1 Subject  SUBJECT PARAMETERS Age  Height  Initial Weight  Energy Intake  (years)  (cm)  (kg)  (kcal/day)  1  24  175  58.4  3400  2  25  187  71.6  3238  3  19  160  64.5  3039  4  28  174  61.3  2750  5  23  183  74.8  3278  6  21  173  71.8  3163  7  23  182  71.5  3600  8  31  174  69.2  3211  mean  24.3  176.0  67.9  3209.8  ±SEM  ±1.4  ±2.9  ±2.1  ±188.3  Energy intake refers to adjusted daily caloric consumption.  36  T A B L E 4-2 W E I G H T C H A N G E O V E R 11 D A Y D I E T C Y C L E S Butterfat treatment Subject  Initial  Final  Beef tallow treatment Change  Initial  Final  Change  (kg)  1  71.47  71.17  +0.31  72.18  72.30  -0.13  2  65.95  65.17  +0.78  61.10  61.30  -0.20  3  58.70  58.90  -0.20  72.30  71.40  +0.90  4  75.00  74.97  +0.33  64.50  64.20  +0.30  5  61.05  61.10  -0.05  74.30  74.80  -0.50  6  71.80  72.20  -0.40  58.25  57.88  +0.38  mean ±SEM  67.33 ±2.66  67.25 ±2.65  0.08 ±0.17  67.10 ±2.74  66.98 ±2.78  0.13 ±0.25  Initial weight represents mean weight of day 1-4. Final weight represents mean weight of day 7-11. Weight change for subjects 7 and 8 was within reported ranges. Specific values are no longer available.  37  T A B L E 4-3  Fatty acid  F A T T Y ACID ANALYSIS OF TREATMENT FATS AND TEST BREAKFAST MEALS  Butterfat  Beef tallow  Butterfat breakfast  Beef tallow breakfast  (g/100 g)  8:0  1.2  0  1.2  0.6  10:0  2.9  0  2.1  0.5  12:0  3.5  0  2.4  tr  14:0  11.4  3.9  9.7  5.1  14:1  1.7  1.0  1.2  0.6  16:0  32.4  27.1  28.6  23.5  16:1  1.6  3.4  1.5  3.4  18:0  11.1  17.2  10.8  13.2  18:1  21.8  40.0  25.9  33.8  18:lt  1.1  1.9  3.9  2.3  18:2  2.4  2.3  12.7  11.5  18:3w6  tr  tr  tr  tr  18:3w3  tr  tr  tr  tr  20:0  2.8  2.4  tr  tr  20:1  tr  0  tr  tr  20:4  tr  0  tr  tr  Breakfast measures include [l- C]fatty acids, tr indicates the presence of < 0.5 g/100 g of fat. 13  38  respectively). The concentration of PA was similarly high in the butter (28.6 g/100 g fat) and tallow (23.5 g/100 g fat) breakfast meal. The concentration of M A was lower in the butter (9.7 g/100 g fat) versus tallow (5.1 g/100 g fat) breakfast meal.  4.3  FRACTIONAL RECOVERY OF BUTTER AND T A L L O W B R E A K F A S T M E A L [1- C]FATTY ACIDS 13  Indirect measurement of fatty acid oxidation was based on C 0 enrichment in 1 3  2  collected breath samples. Mean internal and external precision (SEM) levels of the mass spectrometer were 0.014±0.002 and 0.44±0.04 °/oo, respectively, for mean sample enrichments of approximately -18.0210.43 °/oo.  4.3.1  FRACTIONAL RECOVERY OF BREAKFAST M E A L [1- C]MYRISTIC A C I D 13  The hourly recovery of C 0 from [1- C]MA was similar after consumption 1 3  13  2  of butter or tallow breakfast meals (FIGURE 4-1, for hourly values see APPENDDX 4-1).  After consumption of each test meal, [1- C]MA recovery increased rapidly from 13  3 to 6 hours post-dose. Recovery of [1- C]MA peaked at a level of 3.15±2.31% of 13  administered dose/hour, 6.00±0.37 hours after consumption of the butter breakfast meal and 2.4410.15% of administered dose/hour, 5.8310.31 hours after consumption of the tallow breakfast meal ( T A B L E 4-4).  Cumulative rates of recovery of  1 3  C0  2  from  [1- C]MA for 9 hours post-dose are shown in F I G U R E 4-2 and values are reported in 13  T A B L E 4-4. Cumulative recoveries of C 0 from [1- C]MA at 4 hours post-dose 1 3  13  2  39  Fig. 4-1 Hourly [1-13C] Fatty Acid Oxidation Over Nine Hours Postprandial 2.50 |  :  1  2  3  4  5  6  7  8  9  10  Hours After Dose (hr) -•—  Butter-M — • — Taliow-M - o-  Butter-P - ® -  Tallow-P  x ± S E M , n=8, n=7 [l- C]myristic acid (M) and [l- C]palmitic acid (P) recovered as breath C 0 per hour (%dose) following oral administration of [l- C]fatty acid and butter or tallow breakfast meal. Nine hour recovery of [1- C]M as breath C 0 differed from that of [1- C]P (p<0.001) and was not influenced by butter versus tallow feeding. Hourly recovery of [1- C]M and [1- C]P as breath C 0 differed from hour 4 to hour 7 post dose (p<0.01). 1  13  13  2  13  13  2  13  13  13  2  40  T A B L E 4-4  CUMULATIVE OXIDATION, PEAK OXIDATION AND HOUR OF P E A K OXIDATION FOR [1- C]FATTY ACID 13  ri- ClMvristic acid  ri- ClPalmitic acid  Butter trial  Tallow trial  Butter trial  4 hour recovery (% dose)  1.2610.36  1.9510.38  0.9710.47  0.6010.18  6 hour recovery (% dose)  4.6010.87  5.7410.66  2.0810.48  1.5310.45  9 hour recovery (% dose)  7.1311.04  8.6110.86  3.2610.73  3.0110.86  Peak recovery (% dose)  3.1512.31  2.4410.15  0.8310.16  0.7010.28  Time of peak recovery (hour)  6.0010.37  5.8310.31  5.5010.67  6.6710.62  13  13  1  Tallow trial  meaniSEM n=8 n=7 4/6/9 hour recovery refers to the cumulative oxidation of [l- C]fatty acid for 4/6/9 hours post isotope/breakfast administration in subjects fed corresponding diets for 11 days. Peak recovery is expressed as % dose recovered/hour. J  13  41  Fig. 4-2 Cumulative [1-13C] Fatty Acid Oxidation Over Nine Hours Postprandial  4  5  6  7  Hours After Dose (hr) But-P  Tal-M  But-M  Tal-P  x ± S E M , n=8, n=7 Cumulative [l- C]myristic acid (M) and [l- C]palmitic acid (P) recovered as breath C 0 over 9 hours (%dose) following oral administration of [l- C]fatty acid and butter or tallow breakfast meal. Cumulative 9 hour recovery of [1- C]M as breath C 0 exceeded that of [1- C]P (p<0.01) and was not influenced by butter versus tallow feeding. 1  13  13  13  2  13  2  13  42  were 1.26±0.36% for butter and 1.95±0.38% for tallow meals. A steady increase in cumulative recovery from [1- C]MA at 6 hours post-dose (4.60±0.87% for butter and 13  5.7410.66% for tallow meals, respectively), and at 9 hours post-dose (7.13±1.04% for butter and 8.61±0.86% for tallow meals, respectively) occurred and did not differ due to the source of dietary fat.  4.3.2  FRACTIONAL RECOVERY OF BREAKFAST M E A L [1- C]PALMITIC A C I D 13  The hourly recovery of C 0 from [1- C]PA was also similar following the 1 3  13  2  consumption of the butter and tallow breakfast meals ( F I G U R E 4-1, for hourly values see APPENDTX 4-1). For each treatment, recovery of C 0 from [1- C]PA 1 3  13  2  proceeded slowly, peaking at a level of 0.83±0.16% of administered dose/hour, 5.50±0.67 hours after the butter meal, and 0.70±0.28% of administered dose/hour, 6.67±0.62 hours after the tallow meal were consumed. Cumulative recovery of label from [1- C]PA for 9 hours post-dose are shown in F I G U R E 4-2 and values are 13  reported in T A B L E 4-4. Cumulative recovery of [1- C]PA at 4 hours post-dose 13  (0.97±0.47% for butter and 0.60±0.18% for tallow), 6 hours post-dose (2.08±0.48% for butter and 1.53±0.45% for tallow), and 9 hours post-dose (3.26±0.73% for butter and 3.01±0.86% for tallow) did not differ significantly as a function of dietary fat treatment.  43  4.3.3  FRACTIONAL RECOVERY OF BREAKFAST M E A L [1- C]MYRISTIC V E R S U S [1- C]PALMITIC 13  Fractional C 0  13  I 3  ACID  recovery rates of dietary [1- C]MA versus [1- C]PA differed 13  2  13  (p<0.001) from 4 to 7 hours post-dose. Peak recovery of dietary [1- C]MA exceeded 13  that of [1- C]PA (p<0.01), although peak recovery occurred at a similar time for both 13  dietary [l- C]fatty acids. Over the 9 hour period, the fractional recovery of dietary 13  [1- C]MA exceeded that of dietary [1- C]PA by more than 100% for both fat 13  13  treatments (p<0.01).  4.4  E N E R G Y EXPENDITURE AND SUBSTRATE OXIDATION PRIOR T O CONSUMPTION O F T H E B R E A K F A S T M E A L A N D F O R NINE HOURS  POSTPRANDIAL  Energy expenditure, carbohydrate oxidation, and fat oxidation prior to the breakfast meal and for 9 hours postprandial are reported in T A B L E 4-5.  All  respiratory gas exchange measurements were performed in duplicate. The inter-trial precision reflected the analytical error in respiratory gas measurement in combination with intra-subject variability. Replicate measures of 9 hour cumulative energy expenditure, carbohydrate oxidation and fat oxidation differed by (x±SEM; n=15) 3 0 ± 6 kcal, 2 2 ± 5 g and 11±2 g, respectively, for mean values of approximately 7 8 2 ± 2 0 kcal, 87±6 g and 4 5 ± 3 g, respectively.  44  T A B L E 4-5  ENERGY EXPENDITURE AND SUBSTRATE UTILIZATION PRIOR TO CONSUMPTION OF T H E TEST M E A L AND FOR NINE HOURS POSTPRANDIAL Butterfat treatment  Beef tallow treatment  P value  Resting (kcal/min)  1.14±0.05  1.16±0.05  0.67  Cumulative postprandial  783±26  781±31  0.94  169±7  157±13  0.43  Resting (g/min)  0.1010.02  0.1410.02  0.07  Cumulative postprandial  85±8  8917  0.72  32±7  1519  0.09  Resting (g/min)  0.0810.01  0.0610.01  0.12  Cumulative postprandial (g/9 hours)  4613  4414  0.83  Diet induced (g/9 hours)  314  1114  0.08  Energy expenditure:  (kcal/9 hour) Diet induced  (kcal/9 hours)  Carbohydrate oxidation:  (g/9 hours) Diet induced  (g/9 hours)  Fat oxidation:  meanlSEM, n=8 Resting metabolic measurements were recorded prior to consumption of the test breakfast meal. Cumulative postprandial measurements were recorded for 9 hours post breakfast administration. Diet induced measurements were calculated as total energy expenditure/substrate utilization minus resting energy expenditure/substrate utilization.  45  4.4.1  ENERGY EXPENDITURE Resting metabolic rate prior to consumption of the butter or tallow breakfast  meal was similar (1.14±0.05 kcal/minute and 1.1610.05 kcal/minute for butter and tallow treatment, respectively). Profiles of bi-hourly energy expenditure prior to the breakfast meal and for 9 hours postprandial are detailed in F I G U R E 4-3 (for bi-hourly values see A P P E N D I X 4-2) and did not differ as a result of treatment fat Following consumption of the breakfast meal the rate of energy expenditure increased rapidly to a level of 1.5 kcal/minute. This level was sustained for approximately an hour before gradually declining to approximately 1.4 kcal/minute, prior to lunch. In a similar manner to that occurring postbreakfast, posdunch the rate of energy expenditure rapidly increased to a level above 1.6 kcal/minute and gradually declined to 1.3 kcal/minute at hour 9. Cumulative energy expenditure for 9 hours postprandial was 783126 kcal and 781131 kcal for the butter and tallow treatment, respectively. Dietinduced thermogenesis for 9 hours postprandial was 16917 kcal and 157113 kcal for butter and tallow treatment, respectively. Resting metabolic rate, cumulative postprandial energy expenditure and diet-induced thermogenesis did not differ significantly due to fat treatment.  4.4.2  C A R B O H Y D R A T E OXIDATION The profile of bi-hourly carbohydrate oxidation prior to the breakfast meal and  for 9 hours postprandial is detailed in F I G U R E 4-4 (for bi-hourly values see A P P E N D I X 4-3) and did not differ as a result of fat treatment. Carbohydrate  46  Fig. 4-3 Bi-hourly Energy Expenditure Over Nine Hours Postprandial 1.75  1.64  c  H  1.53 h  o Q_  B o CO  1.42 h 1.31  1.20 4  —  • -  5 Time (hr)  Butter  i— Tallow  x ± S E M , n=8 Energy expended per minute following consumption of the butter or tallow breakfast meal. Energy expenditure over 9 hours was not influenced by butter versus tallow feeding.  47  Fig. 4-4 Bi-hourly Carbohydrate Oxidation Over Nine Hours Postprandial 8.00  6.80 h  0  1  2  3  4  5  6  7  8  9  Time (hr) -•—  Butter  - • -  Tallow  x ± S E M , n=8 Grams of carbohydrate oxidized per 30 minutes following consumption of the butter or tallow breakfast meal. Bi-hourly carbohydrate oxidation over 9 hours was not influenced by butter versus tallow feeding.  48  oxidation prior to the breakfast test meal was 0.10±0.02 g/minute and 0.1410.02 g/minute for the butter and tallow treatment, respectively.  Following consumption of  the test breakfast meal, for both fat treatments, the rate of carbohydrate oxidation increased rapidly to approximately 6 g/30 minutes. This level gradually declined to approximately 3 g/30 minutes prior to the lunch meal. Following lunch, the rate of carbohydrate oxidation increased rapidly to approximately 5.5-6 g/30 minutes. This was sustained for approximately 30 minutes before gradually declining to a level of approximately 3.5 g/30 minutes by hour 6 postprandial. Carbohydrate oxidation during hour 7 and 8 postprandial was not consistent with prior trends. This may have been influenced by the study protocol. Due to the lengthy nature of respiratory gas exchange measurements, subjects were permitted to leave the hood for 30 minute intervals during hours 7 and 8 postprandial. The resultant respiratory gas exchange values following break periods may have been influenced by (1) increased activity and/or (2) non-steady state because of leaving the hood. Cumulative carbohydrate oxidation for 9 hours postprandial totalled 8518 g and 8917 g for the butter and tallow treatment, respectively.  Diet-induced carbohydrate oxidation equalled 3217 g and  1519 g for the butter and tallow treatment, respectively. Although cumulative postprandial carbohydrate oxidation did not differ significantly between fat treatments, the difference between both resting carbohydrate oxidation and diet-induced carbohydrate oxidation approached statistical significance.  49  4.4.3  F A T OXIDATION The profile of bi-hourly fat oxidation prior to the breakfast meal and for 9  hours postprandial is detailed in F I G U R E 4-5 (for bi-hourly values see A P P E N D K 4-4) and did not differ as a result of fat treatment. Fat oxidation prior to the breakfast test meal was similar during both fat treatments (0.08±0.01 g/min and 0.06±0.01 g/min for the butter and tallow treatment, respectively).  Following consumption of the  breakfast meal, for both treatments, the rate of fat oxidation gradually rose from 2.02.2 g/30 minutes to a level of 3 g/30 minutes prior to lunch. Following lunch, the rate of fat oxidation dropped slightly and then continued to increase. Corresponding to the increase in carbohydrate oxidation reported during hours 7 and 8 postprandial, fat oxidation decreased. Cumulative fat oxidation for 9 hours postprandial was 4 6 ± 3 g and 4 4 ± 4 g for the butter and tallow treatment, respectively, while diet-induced fat oxidation was 3 ± 4 g and 11 ± 4 g for the butter and tallow treatment, respectively. Resting fat oxidation, cumulative postprandial fat oxidation, and diet-induced fat oxidation did not differ significantly due to fat treatment.  50  Fig. 4-5 Bi-hourly Fat Oxidation Over Nine Hours Postprandial 4.00  3.40  2.80  2.20 h T"-' 1.60  1.00  - • -  — •—  Butter  Tallow  x ± S E M , n=8 Grams of fat oxidized per 30 minutes following consumption of the butter or tallow breakfast meal. Bi-hourly fat oxidation over 9 hours was not influenced by butter versus tallow feeding.  51  4.5  D I E T A R Y F A T T Y ACID M E T A B O L I S M R E L A T I V E T O W H O L E BODY FAT METABOLISM  4.5.1  C O N T E N T AND NET OXIDATION O F B R E A K F A S T M E A L M Y R I S T I C A N D P A L M I T I C ACIDS Unlike the fractional oxidation of breakfast meal [l- C]fatty acid (expressed as 13  % of administered dose), net oxidation of breakfast meal fatty acid (expressed in mg) takes into account the breakfast meal fatty acid pool size. T A B L E 4-6 reports the content and net oxidation of breakfast meal M A and PA. M A contents of the butter and tallow meals were 4.6 g and 2.4 g, respectively, while PA contents were 13.6 g and 11.2 g, respectively. Net oxidation of dietary M A from the butter meal (329±45) exceeded (p<0.05) that from the tallow meal (212±25) over the postprandial period. In contrast, net oxidation of dietary PA for 9 hours did not differ between butter (441199) and tallow (348195 mg) meals.  4.5.1.1  B R E A K F A S T M E A L F A T T Y ACID OXIDATION R E L A T I V E T O FAT INTAKE In T A B L E 4-7, dietary fatty acid oxidation in 9 hours is compared with the  total amount of fat ingested in the breakfast meal. During the butter trial, oxidized breakfast meal M A and PA accounted for 0.710.1% and 1.010.2% of fat present in the breakfast meal, respectively. Similarly, oxidized breakfast meal M A and P A accounted for 0.510.1% and 0.710.25% of fat present in the tallow meal, respectively.  52  T A B L E 4-6  C O N T E N T AND NET OXIDATION O F B R E A K F A S T M E A L M Y R I S T I C A N D P A L M I T I C ACIDS Mvristic acid Butterfat breakfast  Beef tallow breakfast  Butterfat breakfast  Total fat  47±1 g  47±1 g  47±1 g  47±1 g  Fatty acid  5±0.1 g  2±0.1 g  14±0.4 g  11±0.3 g  Oxidized fatty acid  329±45 mg"  212±25 mg  441199 mg  348±95 mg  mean±SEM n=8 *n=7 Significandy different from  a  Palmitic acid  b  b  53  1  Beef tallow breakfast  T A B L E 4-7  B R E A K F A S T M E A L F A T T Y ACID OXIDATION R E L A T I V E T O FAT INTAKE  Myristic acid Butterfat trial  Palmitic acid Beef tallow trial  Butterfat trial 1  Beef tallow trial  (% of total breakfast fat)  Dietary F A , oxidized in 9 hour  0.7±0.1  mean±SEM n=8 n=7 significantly different from  a  0.5±0.1  x  a  b  54  b  1.0±0.2  0.7±0.2  Further, labelled fatty acids collectively represented 38.2% and 28.6% of butter and tallow breakfast meal fat, respectively, and 1.7% and 1.2% of oxidized butter and tallow breakfast meal fat, respectively. As a percentage of fat intake, postprandial oxidation of M A from the butter meal differed significantly (p<0.05) from that of the tallow meal, while postprandial oxidation of breakfast meal P A did not differ due to fat treatment.  4.5.1.2  B R E A K F A S T M E A L F A T T Y ACID OXIDATION R E L A T I V E T O POSTPRANDIAL F A T OXIDATION In T A B L E 4-8, 9 hour postprandial dietary fatty acid oxidation is compared  with 9 hour postprandial fat oxidation (dietary and endogenous). During the butter trial, dietary M A and PA accounted for 0.7±0.1% and 0.9±0.2% of 9 hour postprandial fat oxidation, respectively. Similarly, during the tallow trial, dietary M A and PA accounted for 0.5±0.1% and 0.8±0.2% of postprandial fat oxidation. Further, labelled fatty acids, which collectively represented 38.2% and 28.6% of butter and tallow breakfast meal fat, respectively, accounted for 1.7% and 1.3% of 9 hour postprandial fat oxidation after the butter and tallow breakfast meals, respectively. As a percent of postprandial fat oxidation, dietary M A or PA oxidation did not differ due to fat treatment.  55  T A B L E 4-8  B R E A K F A S T M E A L F A T T Y ACID OXIDATION R E L A T I V E T O POSTPRANDIAL F A T OXIDIZATION Myristic acid  Palmitic acid  Butterfat trial  Beef tallow trial  Butterfat trial  Beef tallow trial  Total fat, oxidized in 9 hours (g)  46±3  44±4  46±4  44±4  Dietary F A , oxidized in 9 hours (% of total fat oxidized in 9 hours)  0.7±0.1  0.5±0.1  0.9±0.2  0.8±0.2  mean±SEM n=8  56  1  5.  DISCUSSION  5.1  SUITABILITY O F T H E PRESENT P R O T O C O L T O D E T E R M I N E F A T T Y A C I D O X I D A T I O N IN H E A L T H Y H U M A N S Measurement of dietary C-fatty acid oxidation based on the evolution of 13  1 3  C0  2  depends on several assumptions involving the digestion and absorption of meal  fat and clearance of C 0 from tissues of oxidation into breath. In the present study, 1 3  2  it was assumed that digestion and absorption of fat was similar between diet fat treatments and that tracer and tracee absorption would be equivalent, despite the provision of tracee in the free fatty acid form. In support of the former assumption, Denke et al. (1991) reported similar fat absorption efficiencies in 10 healthy males fed butter or tallow (40% of energy) liquid formula diets. The absorption of P A (97% and 96% for butter and tallow treatment, respectively), stearic acid (90% and 94% for butter and tallow treatment, respectively), and oleic acid (99% and 99% for butter and tallow treatment, respectively) contained in the butter and tallow diets did not differ due to fat treatment, as determined using 3 day faecal collection. The fact that C I 3  triglycerides produced a similar cumulative oxidative response when administered either parenterally or orally (Metges et al., 1991), suggests high efficacy of digestion and absorption. In the case of M A and PA studied herein, differences in absorption were likely minor and thus would not account for the large oxidative differences observed in the present study (Jones et al., 1985b). Of greater concern is the potential for discrepancy between digestion and  57  absorption of tracer versus tracee fatty acids in the test meals. Labelled [l- C]fatty 13  acids were present in the free form, while corresponding unlabelled fatty acids (e.g., dietary lipids) were primarily esterified as triacylglycerols. It has been suggested that absorption of esterified saturated fatty acids may be greater than those in the free form due to a lack of monoacylglycerols which promote fatty acid emulsification and micellular solubulization during digestion (Jones et al., 1985a; DeShriver et al., 1991). As monoacylglycerols are in ample supply from the almost exclusive consumption of triglyceride-fatty acid in the test breakfast meal, saturated fatty acids in the free and esterified form should be equally absorbed in the present study.  5.2  S U I T A B I L I T Y O F T H E P R E S E N T ISOTOPIC M O D E L In the present study, non-compartmental analysis of respiratory  1 3  C0  2  enrichment was performed following the administration of a non-primed oral bolus of [l- C]fatty acid. The effectiveness of this and other commonly used models remains 13  controversial and will be discussed below. As described previously, oral versus intravenous administration of labelled fatty acid influenced early C 0 enrichment levels, but did not significantly affect overall 2  labelled C 0 recovery rate in the rat (Leyton et al., 1987) or the human (Schabe et al., 2  1964; Metges et al., 1991). Labelled fatty acid may be administered as a single bolus or constant infusion. Constant administration of labelled fatty acid produces a gradual rise in pre-oxidative fatty acid pool enrichment to a level where the concentration of infusate is equivalent  58  to that of the pre-oxidative pool, at which point, equilibrium is established. The constant infusion technique is advantageous in that, once equilibrium has been established, the pattern and complexity of whole body isotopic dilution becomes irrelevant. Additionally, a variable rate of substrate uptake or excretion from various compartments will not effect isotopic equilibrium, provided the enrichment of the infusate remains constant. Thus, isotopic steady state is not influenced by physiologic steady state. Similarly, the size and complexity of substrate pools, given an exchange of substrate, is not of concern. The question of whether labelled fatty acids should be administered using constant infusion or pulse chase approaches has been disputed. In recent years, inherent assumptions of the constant infusion technique regarding the attainment of isotopic equilibrium within pre-oxidative fatty acid pools has been criticized (Heiling et al., 1991). Thus, measurement of intracellular pre-mitochondrial fatty acid kinetics within organs such as heart, muscle, or liver in the human may be in error as isotopic equilibrium must be indirectly determined through the measurement of plasma substrate enrichment. Since a significant portion of oxidized fat appears to be derived from intracellular lipids, possibly not in equilibrium with corresponding plasma pools (Groop et al., 1991, Bonndanna et al., 1990, Heiling et al., 1991), tracer measurement of fat oxidation may have limited use under certain circumstances. The noncompartmental analysis of respiratory C 0 evolution following a non-primed oral 1 3  2  bolus of [l- C]fatty acid utilized in the present study is a stochastic approach where 13  1 3  C input and output are monitored without the complex analyses of whole body fatty  59  acid metabolism. While the present methodology is free of potentially inaccurate assumptions concerning isotopic equilibrium and endogenous fatty acid kinetics, other factors may limit the effectiveness of the present approach. Primarily, the present method provides no insight into endogenous fatty acid pool size, distribution and turnover, or to the relative amount of dietary/endogenous fatty acid oxidized, although these factors could potentially influence dietary fatty acid oxidation. In fact, differential dilution of label in endogenous fatty acid pools has been a major concern of researchers using a similar approach (Jones et al., 1985b; Toorop et al., 1979).  5.3  SUITABILITY O F T H E PRESENT P R O T O C O L T O D E T E R M I N E E N E R G Y E X P E N D I T U R E A N D S U B S T R A T E U T I L I Z A T I O N IN H E A L T H Y HUMANS The assessment of energy expenditure using indirect calorimetry is considered  accurate and shows close agreement with that of direct calorimetry (Livesey and Elia, 1988). In contrast, there is considerable debate concerning the validity of indirect calorimetry to estimate substrate utilization. Livesey and Elia (1988) estimated that both carbohydrate and fat oxidation are approximately 12-13 times more sensitive to error in respiratory gas exchange measurement compared to the heat equivalent of oxygen. This finding is consistent with results of the thesis where replicate measures of 9 hour postprandial energy expenditure differed by 3.8% compared to replicate measures of 9 hour postprandial carbohydrate and fat oxidation that differed by 25.1%  60  and 25.2%, respectively. Factors which may influence the accuracy of respiratory gas exchange measurement include the actual versus assumed composition of oxidized fuel, fluctuation in C 0 pool size, fluctuation in cutaneous gas exchange and erroneous 2  estimation of protein oxidation. Respiratory quotient is predictive of oxidized fuel composition, for which constant fat, carbohydrate and protein respiratory gas exchange values are assumed (Weir et al., 1949). In the present study, an unusual amount of medium chain fatty acid esters were consumed. The assumed respiratory quotient for fat oxidation is 0.710, based predormnanfly on the use of long chain triglyceride as fuel. The actual respiratory quotient for medium chain triglyceride is 0.737. However, for medium chain triglyceride oxidation, calculations using assumed values, differ from those using actual values, by a maximum factor of 0.3% (Elia and Livesey, 1988), suggesting that this issue is not of major concern in the present study. Fluctuation in C 0 pool size may result from hyper- and hypo-ventilation. This issue 2  is also of lesser concern if respiratory exchange is measured over a prolonged period of time (Jequier et al., 1987), as is the case in the present study. Cutaneous gas exchange, an additional problem, can be corrected through experimental protocol. Finally, non-protein respiratory gas exchange, based on a constant erroneous estimation of protein oxidation, will minimally influence estimation of non-protein heat production (Livesey and Elia, 1988).  61  5.4  C O M P A R A T I V E D I E T A R Y F A T T Y A C I D O X I D A T I O N IN H E A L T H Y HUMANS CONSUMING DIFFERENT DIETARY FATS Despite repeated observations using tracer studies that labelled medium chain  fatty acids are more rapidly converted to carbon dioxide than longer chain fatty acids, the response of  1 3  C 0 excretion from ingested tracer labelled fatty acid to changing 2  diet fat has not been examined. The present study is the first to follow the oxidation to C0  2  of labelled saturated fatty acids in response to medium chain versus long chain  fatty acid feeding. Results show that over a 9 hour postprandial period, independent of the amount of fatty acid in the test meal and precedent diet, almost identical proportions of dietary C - M A (about 8%) and C - P A (about 3%) were recovered as 1 3  13  respiratory C 0 . While the fractional oxidation of dietary M A and PA were 1 3  2  independent of butter or tallow fat treatment, rates were markedly influenced by even a minor decrease in acyl chain length. The greater net oxidation of M A with butter feeding was due to its higher dietary level in contrast to PA where pool sizes were equivalent across dietary treatments. The demonstration of structure-dependent differences in the fractional oxidation of [l- C]fatty acid is not inconsistent with results from previous experiments in 13  humans using a single diet approach (Jones et al., 1985b; Watkins et al., 1982; Metges and Wolfram, 1991). However, although tracer studies have compared oxidation of various labelled fatty acids using olive (Cenedella and Allen, 1969; Leyton et al., 1987), corn (Mead et al., 1956) or soybean (Coots, 1964) oils, systematic inter-oil  62  comparisons have not been carried out. The finding that labelled carbon dioxide profiles for a given fatty acid appear to be independent of meal pool size is novel, affording some insight into mechanisms governing the relative partitioning of fats through processes of absorption, transport and oxidation. A consistent proportion of label appearing in breath suggests that discrimination of fatty acids for oxidation versus partitioning into storage pools is fatty acid-specific and is not influenced by the amount of any given fatty acid in the diet. However, because the extent of discrimination varies across dietary fats, changing relative levels in the diet produces the overall effect of shifting the incremental partitioning towards or away from oxidation. Therefore the concentration of specific fatty acids becomes the primary determinant of net oxidation potential of any dietary fat source. Chain length dependent-differences in the fractional oxidation of dietary fatty acids observed in our study have been reported over a range of conditions, however the extent of fractional oxidation appears to be highly dependent on the physiological state of the organism (e.g., exercising, fasted, fed) and the fat/carbohydrate composition of the test meal. For example the oxidation of a dietary C-palmitate 14  bolus over 8 hours in the rat has been shown to decrease from 35% to 7% when accompanied by a high carbohydrate meal (Toorop et al., 1979). It is interesting to note, however, that regardless of variability in experimental protocol, the tendency toward rapid versus slow fractional oxidation of dietary fatty acid is somewhat predictable. This is illustrated in  TABLE 5-1, where the fractional oxidation of fatty  acids varied a great deal among studies, while the comparative ratio of fractional  63  T A B L E 5-1  C O M P A R A T I V E F A T T Y ACID OXIDATION B E T W E E N STUDIES  Fatty Acid Comparison  Oxidation Rate (%)  Ratio  8:0 vs 16:0  45 vs 9  5.0  1  Satabin et al., 1987  28 vs 6.6  4.2  1  Watkins et al., 1982  34 vs 25  1.4  1  Metges et al., 1991  90 vs 45  2.0  1  Johnson et al., 1990  28 vs 11  2.5  1  Watkins et al., 1982  32 vs 57  0.5  1  Leyton et al., 1987  6.6 vs 11  0.6  1  Watkins et al., 1982  10 vs 15 vs 3  3.3  5 :1  Jones et al., 1985  48 vs 57 vs 25  1.9  2.3 : 1  Leyton et al., 1987  8:0 vs 18:1  16:0 vs 18:0  18:2 vs 18:1 vs 18:0  64  Study  oxidation of fatty acids within studies remained more consistent, under a wide variety of circumstances. With experimental conditions which resembled those of our study, Jones et al. (1985) found that approximately 15%, 10%, and 3% of  1 3  C oleic, linoleic and stearic  acid, respectively, in a mixed meal containing largely saturated fat were recovered in the breath of resting healthy humans after 9 hours. Watkins et al. (1982) investigated fatty acid oxidation in healthy children and reported that 27%, 11% and 6.6% of labelled trioctanoic, triolein and palmitic acid, respectively, as components of Lipomul, were recovered in resting subject breath after 6 hours. Thus, the level of dietary fatty acid oxidation observed in our study is similar to that of other studies involving healthy resting humans in the fed state. Further, it is apparent that when the level of 9 hour postprandial fatty acid oxidation of healthy, resting humans is considered in relation to the amount of fat ingested, the majority of dietary fatty acids are directed toward storage. Thus, at any time the majority of fat that is actively being oxidized following a meal is derived from endogenous stores. The qualitative effect of dietary fat in influencing tissue levels has been well established in animals (Jones et al., 1995) and humans (Field et al., 1985). Mechanisms through which chain length-dependent differences in dietary fatty acid oxidation express themselves is beyond the scope of the present study. The potential influence of physiologic processes (chylo-portal partitioning and/or penetration of the mitochondrial membrane) or methodological considerations (differential dilution and/or oxidation of individual fatty acid in endogenous pools)  65  remains to be determined.  5.5  C O M P A R A T I V E S U B S T R A T E U T I L I Z A T I O N IN H E A L T H Y HUMANS CONSUMING DIFFERENT DIETARY FATS In the present study no significant difference in 9 hour postprandial energy  expenditure, fat oxidation and carbohydrate oxidation was observed following consumption of butter or tallow breakfast meals. This is in contrast to several studies comparing the postprandial utilization of medium chain and long chain fatty acids, where significant difference in energy expenditure (Hill et al., 1989; Seaton et al., 1986: Scalfi et al., 1991) and fat oxidation (Mascioli et al., 1991) have been observed. These studies differ a great deal, however, in meal contents of dietary fat, dietary medium chain versus long chain fatty acids, and energy. For example, with a 400 kcal meal consisting almost entirely of medium chain versus long chain triglyceride, a 300% difference in diet-induced thermogenesis was observed (Seaton et al., 1986). Studies which were more consistent with normal feeding situations, where mixed meals containing between 858 and 1300 kcal of which 30-40% was fat were used (Hill et al, 1989; Scalfi et a l , 1991), reported differences of 10-30% in diet-induced thermogenesis from medium versus long chain triglyceride consumption. In these studies, however, medium chain triglycerides (25-40% of meal energy) were derived from commercial formulas, as it is difficult to obtain this level of medium chain triglycerides in foodstuffs. In the present study, which was designed to resemble a normal feeding  66  situation, all fat was obtained from foodstuffs.  The butter and tallow breakfast meals  differed in fat composition and macronutrient composition by 17.0% and 6.8% of total energy, respectively. This level is much lower than that of previously mentioned studies. Given minor compositional differences between butter and tallow breakfast meals, the absence of significant difference in postprandial energy expenditure and substrate oxidation was not surprising. Differences between butter and tallow meals included the content of medium chain fatty acid and PA. The medium chain fatty acid content, up to and including M A , accounted for 3.2% more energy in the butter breakfast meal. The PA content accounted for 1.9% more energy in the butter meal. When one considers that relative to unsaturated long chain fatty acid, medium chain fatty acid is rapidly oxidized (Leyton et al., 1987; Scalfi et al., 1991) and P A is slowly oxidized (Leyton et al., 1987), it is entirely possible that elevated postprandial thermogenic/oxidative effects of dietary medium chain fatty acids were masked by depressed thermogenic/oxidative effects of PA after the butter breakfast meal. When one considers that foodstuffs are typically heterogeneous in fatty acid content, low in medium chain fatty acid concentration, and abundant in other dietary fatty acid which are also rapidly oxidized (ie. unsaturated fatty acid), the implications of chain lengthdependent differences in dietary fat composition for short term energy expenditure/substrate utilization in healthy humans consuming a mixture of foodstuffs appears to be negligible. In the present study, resting carbohydrate oxidation tended to be marginally lower, and diet-induced carbohydrate oxidation marginally higher, with the butter  67  compared to the tallow treatment. These findings are difficult to interpret, primarily because only a few studies of this nature have included pre-feeding. Elevated carbohydrate oxidation following medium chain versus long chain fatty acid consumption has been observed in intravenously fed dogs (Sato et al., 1992) and in women on low calorie diets (Yost et al., 1989). In a 7 day pre-feeding study examining postprandial substrate utilization of test meals differing in fat saturation, a saturated fat treatment was associated with a nonsignificant decrease in resting carbohydrate oxidation and a nonsignificant increase in diet-induced carbohydrate oxidation (Jones et al., 1988). The authors proposed that increased diet-induced carbohydrate oxidation depleted glycogen stores. As a result, substrate oxidation following an overnight fast tended to favour fat oxidation. This proposal is consistent with findings in the present study where elevated diet-induced carbohydrate oxidation with the butter treatment was also accompanied by elevated resting fat oxidation.  5.6  THE CONTRIBUTION OF DIETARY FAT TO POSTPRANDIAL FAT OXIDATION. In the present study, dietary M A or PA accounted for less than 1% of  postprandial fat oxidation (dietary and endogenous) over 9 hours. Obviously the majority of meal fat is directed toward storage. A considerable proportion of labelled dietary fatty acid may be rapidly diluted in endogenous pools, both intra- and extracellular, in the triglyceride-fatty acid cycle. In the triglyceride-fatty acid cycle, fatty acids released during lipolysis are directed toward re-esterification rather than  68  oxidation. Wolfe et al. (1990), using sophisticated isotopic and respiratory gas exchange methodologies, measured the intra- and extracellular re-esterification of palmitate. At rest, Wolfe et al. (1990) reported that approximately 70% of all released fatty acids were re-esterified. Dilution of dietary fat within pre-oxidative pools was characterized by Leyton et al. (1987) who reported cumulative recoveries of 37%, 57%, and 70% for [114  C]linoleic acid at 6, 24 and 120 hours after isotope administration. This result  indicated that over half of the dietary fat released as C 0 in 5 days was recovered 1 4  2  during the first 6 hours. Jones et al. (1985) reported that isotopic enrichment of oleic and stearic acids were detectable 24 hour after isotopic ingestion. Watkins et al. (1982) noted that 3 out of 9 subjects tested with PA, 3 out of 10 subjects tested with triolein and 1 out of 9 receiving tri-octanoin, all exhibited elevated baseline enrichment on consecutive days. Clearly, endogenous fat is the major contributor to fat oxidation following a meal. Improved understanding of the interaction between the consumption of fat and subsequent oxidation of endogenous fat stores would certainly contribute to our understanding of the physiologic implications of fat selection.  69  6.  CONCLUSIONS Based on the findings of the present study, it can be concluded that in healthy,  normally fed humans, postprandial oxidation of dietary fatty acids is enhanced when fats possessing even a minor decrement in fatty acid chain length are fed. Furthermore, fractional oxidation of dietary fatty acids is constant and independent of the mixture of fatty acids consumed in the diet. Increased net oxidation of dietary MA occurring after consumption of the butter meal appears to have resulted from the presence of a larger dietary pool of that fatty acid in the butter compared to tallow meal. 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"Tracers in Metabolic Research: Radioisotope and Stable Isotope / Mass Spectrometry Methods." New York: Liss, 1984. Wolfe, RR., Klein, S., Carraro, F., Weber, JM. Role of triglyceride-fatty acid cycle in controlling fat metabolism in humans during and after exercise. American Journal of Physiology 258:E382-E389, 1990. Yamazaki, Maiz, A . , Sobrado, J. Hypocaloric lipid emulsions and amino acid metabolism in injured rats. Journal of Enteral and Parenteral Nutrition 8:361-366, 1984. Yost, TJ., Eckel, RH. Hypocaloric feeding in obese women: metabolic effects of medium-chain triglyceride substitution. American Journal of Clinical Nutrition 49:326-30, 1989.  78  A P P E N D I X 3-1 CONSENT BY SUBJECT OF R E S E A R C H P R O T O C O L Protocol #:  Patient Name:  Research Protocol:  Effect of Milk Fat on Fatty Acid Utilization in Vivo.  Researcher:  Kitts D.D., Jones P.J., MacDougall, D. Ph:  I, , the undersigned, hereby consent to participate as a subject in the above-named research project conducted by the University of British Columbia. The nature of the procedure or treatment, its risks and/or benefits, and possible alternatives follow: •'  I.  i  N A T U R E AND DURATION O F PROCEDURE:  The objective of the study is to examine the way milk fat is taken up by the body. You will be requested to consume for two 11 day cycles a test diet containing normal foods at a level which is intended to keep your weight constant. You should refrain from consuming any extraneous food, caffeine or alcohol over this period. On days 8 and 11 of each cycle you will remain within the Metabolic Research Area from 7 A M to 5:30 P M . On each of these days a capsule containing a fatty acid tag will be consumed with your breakfast meal. Breath samples will be collected on each of these days every hour for 9 hours after consuming the capsule.  II.  P O T E N T I A L RISKS A N D / O R B E N E F I T S : i  There is no known hazard associated with the use of the fatty acid tags in the present procedure. The substance of the project and procedures associated with it have been fully explained to me, and all experimental procedures have been identified. I have had the opportunity to ask questions concerning any and all aspects of the project and any procedures involved. I am aware that I may withdraw my consent at any time. I acknowledge that no guarantee or assurance has been given by anyone as to results to be obtained. Confidentiality of records concerning my involvement in this project will be maintained in an appropriate manner. I understand that I will receive $100 upon 79  completion of the study. If I decide to withdraw before completion, I will receive an appropriated pro-rated fraction of this amount.  I acknowledge receiving a copy of this consent form and all appropriate attachments. Doctor: Witness: Date:  Signature of Subject If relative or legal representative signs, please indicate relationship or other authority  80  --//  APPENDIX 3-2  FOOD/NUTRIENT ANALYSIS OF MEALS  BUTTERFAT BREAKFAST DAY 1  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 100 .00 G 100 0 ORANGE J U I C E , FROZEN CONCENTRATE, UNDILUTED 244 .00 G 244 0 MILK, PARTLY SKIMMED, ( 2 % F A T ) , FLUID 34 .00 G 34 0 CEREALS, READY-TO-EAT, CHEERIOS 50 .00 G 50 0 EGG, CHICKEN, WHOLE, FRESH AND FROZEN, RAW 66 .00 G 66 0 EGG, CHICKEN, WHITE, FRESH AND FROZEN, RAW 30 .00 G 30 0 TOMATOES, RED, RIPE, RAW 15. 00 G 15 0 PEPPERS, SWEET, RED AND GREEN, FROZEN, CHOPPED 10 .00 G 10 0 ONIONS, RAW 13 .00 G 13 0 CRANBERRIES, RAW 14. 00 G 74 0 MUFFINS, HOME RECIPE, PLAIN 7 .00 G 7 0 MARGARINE, TUB, SUNFLOWER + UNSPECIFIED OILS 23 .50 G 23 0 BUTTER, REGULAR NUTRIENT FAT (TOTAL LIPID) . CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES) . . . . . . MOISTURE  PHOSPHORUS POTASSIUM SODIUM ZINC VITAMIN A (INTERNATIONAL UNITS) . VITAMIN C . . . . THIAMIN TOTAL NIACIN (406+407 OR 406+408) PANTOTHENIC ACID . . . . . . . .  TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  81  INPUT TOTAL 34. 40 45.10 111.00 980.00 469.00 495.00 8.05 127.00? 537.00 1530.00 1200.00 2.91? 2480.00 157.00 0.65 1.09 12.30 3.39 0.62 244.00 1.95 0.39? 1.17? 1.54? 2.35? 1.72? 0.76? 0.53? 1.46? 1.11? 1.82? 1. 61? 0.65? 383.00 19 .60 7 . 47  COOKED TOTAL 34 . 40 45.10 111.00 980.00 469.00 495.00 8.05 127.00? 537.00 1530.00 1200.00 2.91? 2480.00 157.00 0.65 1.09 12.30 3.39 0.62 244.00 1.95 0.39? 1.17? 1.54? 2.35? 1.72? 0.76? 0.53? 1.46? 1.11? 1.82? 1.61? 0.65? 383.00 19 .60 7 . 47  B U T T E R F A T BREAKFAST DAY 2  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 100 100 . 00 G 0 ORANGE J U I C E , FROZEN CONCENTRATE, UNDILUTED 125 . 00 G 125 0 YOGURT, FRUIT VARIETIES, FRUIT BOTTOM, 50 . 00 G 50 0 EGG, CHICKEN, WHOLE, FRESH AND FROZEN, RAW 66 . 00 G 66 0 EGG, CHICKEN, WHITE, FRESH AND FROZEN, RAW 25 . 00 G 25 0 SIRUPS: TABLE BLENDS:CANE AND MAPLE 31 . 00 G 31 0 BUTTER, REGULAR 100 . 00 G 100 0 BREADS: WHOLE-WHEAT, ( 2 % SKIM MILK POWDER) 13 .00 G 13 0 PORK, CURED, BACON, BROILED, PAN-FRIED  NUTRIENT PROTEIN FAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES) . . . . . . IRON MACNESIUM PHOSPHORUS SODIUM ZINC VITAMIN A (INTERNATIONAL UNITS) . VITAMIN C THIAMIN TOTAL NIACIN (406+407 OR PANTOTHENIC ACID . VITAMIN B-6 FOLACIN VITAMIN B-12  406+408)  THREONINE ISOLEUCINE METHIONINE PHENYLALANINE TYROSINE VALINE ARGININE HISTIDINE CHOLESTEROL TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  82  INPUT TOTAL 34 .10 44.90 126.00 1020.00 205.00? 324.00 4.70? 109.00? 547.00 1380.00 1230.00 3.06? 1740.00 146.00 0 . 70 0.84 11.60? 2 . 51? 0.43? 254.00? 1. 08? 0.25? 0.79? 0.99? 1. 46? 1.17? 0.56? 0.37? 0.96? 0.66? 1.18? 1.19? 0.43? 356.00? 20.20? 3 . 40?  COOKED TOTAL 34 .10 44.90 126.00 1020.00 205.00? 324.00 4 . 70? 109.00? 547.00 1380.00 1230.00 3.06? 1740.00 146.00 0.70 0.84 11.60? 2.51? 0. 43? 254.00? 1. 08? 0.25? 0.79? 0.99? 1.46? 1.17? 0.56? 0.37? 0.96? 0.66? 1.18? 1.19? 0. 43? 356.00? 20.20? 3.40?  BEEF TALLOW BREAKFAST DAY 1  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS l O o . 00 G 100 0 ORANGE J U I C E , FROZEN CONCENTRATE, UNDILUTED 244 .00 G 244 0 MILK, PARTLY SKIMMED, ( 2 % F A T ) , FLUID 34 .00 G 34 0 CEREALS, READY-TO-EAT, CHEERIOS 50 .00 G 50 0 EGG, CHICKEN, WHOLE, FRESH AND FROZEN, RAW 66 .00 G 66 0 EGG, CHICKEN, WHITE, FRESH AND FROZEN, RAW 30 .00 G 30 0 TOMATOES, RED, RIPE, RAW 15. 00 G 15 0 PEPPERS, SWEET, RED AND GREEN, FROZEN, CHOPPED, 10 .00 G 10 0 ONIONS, RAW 13 .00 G 13 0 CRANBERRIES, RAW 74 .00 G 74 0 MUFFINS, HOME RECIPE, PLAIN 7. 00 G 7 0 MARGARINE, TUB, SUNFLOWER + UNSPECIFIED OILS, 17 .00 G 17 0 ANIMAL FAT, BEEF TALLOW NUTRIENT UNIT PROTEIN G FAT (TOTAL L I P I D ) . . . G CARBOHYDRATE, TOTAL (BY DIFF) . . G ENERGY (KILOCALORIES) . . . . . . CAL G MG IRON MG MG PHOSPHORUS MG POTASSIUM MG MG ZINC . . MG VITAMIN A (INTERNATIONAL UNITS) . IU VITAMIN C MG THIAMIN MG RIBOFLAVIN MG TOTAL NIACIN (406+407 OR 406+408) NE PANTOTHENIC ACID MG VITAMIN B-6 MG MCG VITAMIN B-12 MCG TRYPTOPHAN G THREONINE G ISOLEUCINE G G G METHIONINE G G PHENYLALANINE G TYROSINE G VALl NE G ARQlNINE G HISTIDINE G CHOLESTEROL MG TOTAL SATURATED FATTY ACIDS . . . G TOTAL POLYUNSATURATED FATTY ACIDS G  83  INPUT TOTAL 34 . 20 43.10 111.00 965.00 465.00 489 .00 8.02 126.00? 531.00 1520.00 1000.00 2.89? 1760.00 157.00 0.65 1.08 12.20? 3.36 0.62 244.00 1.92 0.39? 1.16? 1.53? 2.33? 1.71? 0.75? 0.53? 1.45? 1.10? 1.81? 1.61? 0.64? 350.00 16.20 7.45  COOKED TOTAL 34 . 20 43.10 111.00 965.00 465.00 489.00 8.02 126.00? 531.00 1520.00 1000.00 2.89? 1760.00 157.00 0.65 1. 08 12.20? 3.36 0. 62 244.00 1.92 0.39? 1.16? 1.53? 2.33? 1.71? 0.75? 0.53? 1.45? 1.10? 1.81? 1.61? 0.64? 350.00 16 . 20 7 .45  BEEF TALLOW BREAKFAST DAY 2 AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS l O o . 00 G 100 0 - ORANGE J U I C E , FROZEN CONCENTRATE, UNDILUTED 125. 00 G 125 0 YOGURT, FRUIT VARIETIES, FRUIT BOTTOM, 50. 00 G 50 0 EGG, CHICKEN, WHOLE, FRESH AND FROZEN, RAW 66 .00 G 66 0 EGG, CHICKEN, WHITE, FRESH AND FROZEN, RAW 25. 00 G 25 0 SIRUPS: TABLE BLENDS:CANE AND MAPLE 24 .50 G 24 0 ANIMAL FAT, BEEF TALLOW 100 .00 G 100 0 BREADS: WHOLE-WHEAT, ( 2 % SKIM MILK POWDER) 13. 00 G 13 0 PORK, CURED, BACON, BROILED, PAN-FRIED  NUTRIENT FAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) ENERGY (KILOCALORIES)  . .  PHOSPHORUS ZINC VITAMIN A (INTERNATIONAL UNITS) . THIAMIN TOTAL NIACIN (406+407 OR PANTOTHENIC ACID . VITAMIN B-6 . . . FOLACIN VITAMIN B-12  406+408)  THREONINE ISOLEUCINE  PHENYLALANINE  CHOLESTEROL TOTAL SATURATED FATTY ACIDS . . . TOTAL. POLYUNSATURATED FATTY ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  84  INPUT TOTAL 33.80 44.20 126.00 1020.00 200.00? 317.00 4.65? 108.00? 540.00 1370.00 970.00 3.05? 789.00 146.00 0. 70 0.82 11.50? 2.48? 0.43? 253.00? 1.04? 0.24? 0.77? 0.97? 1.44? 1.15? 0.56? 0.37? 0.95? 0.65? 1.17? 1.18? 0.42? 315.00? 16.80? 3. 45?  COOKED TOTAL 33.80 44.20 126.00 1020.00 200.00? 317.00 4.65? 108.00? 540.00 1370.00 970.00 3.05? 789.00 146.00 0.70 0.82 11.50? 2.48? 0.43? 253.00? 1. 04? 0.24? 0. 77? 0.97? 1.44? 1.15? 0.56? 0.37? 0.95? 0.65? 1.17? 1.18? 0.42? 315.00? 16.80? 3.45?  BUTTERFAT LUNCH DAY 1  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS Z45.00 245 0 ~~ G - R I C E : WHITE: ENRICHED .'COMMON COMMERCIAL VARIETIES 75.00 G 75 0 BEEF: ROUND AND HEEL OF ROUND, TOTAL EDIBLE, 75. 00 G 75 0 BROCCOLI, RAW 20 . 00 G 20 0 ONIONS, RAW 25.00 G 25 0 MUSHROOMS, RAW 75.00 G 75 0 CARROTS, RAW 20 . 00 G 20 0 WATERCHESTNUTS, CHINESE, CANNED 30 . 00 G 30 0 SAUCES, READY-TO-SERVE, SOY 45.00 G 45 0 STRAWBERRIES, FROZEN, SWEETENED, SLICED 100.00 G 100 0 I C E CREAM, VANILLA, REGULAR, ( 1 0 % F A T ) , 31.00 G 31 0 BUTTER, REGULAR  NUTRIENT FAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY D I F F ) ENERGY (KILOCALORIES)  VITAMIN  . .  A (INTERNATIONAL  UNITS) .  TOTAL NIACIN (406+407 OR PANTOTHENIC ACID VITAMIN B-6  406+408)  VITAMIN  B-12  THREONINE ISOLEUCINE  . . . .  METHIONINE  .  PHENYLALANINE  HISTIDINE CHOLESTEROL T O T A L SATURATED FATTY ACIDS TOTAL POLYUNSATURATED FATTY  . . . ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  85  INPUT TOTAL 30. 70 46.00 114.00 978.00 539.00 247.00 7.26 66.10? 516.00 1300.00 3080.00 1.91? 23600.00 98.90 0.56 0.73 15.60 2.60 0.80 113.00 1.56 0.10? 0.31? 0.38? 0.56? 0.52? 0.14? 0.06? 0.31? 0.27? 0.42? 0.35? 0.18? 164.00 26.90? 1.80?  COOKED TOTAL 30.70 46.00 114.00 978.00 539.00 247.00 7.26 66.10? 516.00 1300.00 3080.00 1.91? 23600.00 98.90 0.56 0.73 15. 60 2.60 0.80 113.00 1. 56 0.10? 0.31? 0.38? 0.56? 0.52? 0 .14? 0.06? 0.31? 0.27? 0.42? 0.35? 0.18? 164.00 26.90? 1.80?  BUTTERFAT LUNCH DAY 2  MT UNIT WT(G) RETENTION RECIPE INGREDIENTS 3 5 . 00 G 35 0 CHEESE, CHEDDAR 75. 00 G 75 0 BREADS: WHOLE-WHEAT, ( 2 % SKIM MILK POWDER) 110 .00 G 110 0 BANANAS, RAW 244 .00 G 244 0 MILK, PARTLY SKIMMED, ( 2 % F A T ) , FLUID 65. 00 G 65 0 COOKIES: OATMEAL WITH RAISINS 44 .00 G 44 0 HAM, SLICED, EXTRA LEAN (APPROXIMATELY 5% FAT) 17 .00 G 17 0 BUTTER, REGULAR  NUTRIENT ^AT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) ENERGY (KILOCALORIES)  . .  71TAMIN A (INTERNATIONAL UNITS) .  TOTAL NIACIN (406+407 OR  406+408)  TRYPTOPHAN  TC a SATURATED FATTY ACIDS . . . TO" -L POLYUNSATURATED FATTY ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  86  INPUT TOTAL 38.50 45.00 122.00 1020.00 375.00 651.00 5.39 127.00? 770.00 1450.00 1610.00 4.35? 1510.00 23.90 0.94 0.97 14.20 2.01? 1.10? 95.50 1.53? 0.34? 1.10? 1.45? 2. 40? 2.16? 0.67? 0.27? 1.27? 1.13? 1.56? 1.23? 0.92? 141.00 22.80 4.34  COOKED TOTAL 38. 50 45.00 122.00 1020.00 375.00 651.00 5.39 127.00? 770.00 1450.00 1610.00 4.35? 1510.00 23.90 0.94 0.97 14. 20 2.01? 1.10? 95.50 1. 53? 0.34? 1.10? 1.45? 2.40? 2.16? 0.67? 0.27? 1.27? 1.13? 1. 56? 1.23? 0.92? 141.00 22.80 4.34  BEEF TALLOW LUNCH DAY1  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 245. 00 G 245 0 RICE: WHITE:ENRICHED:COMMON COMMERCIAL VARIETI 75. 00 G 75 0 BEEF: ROUND AND HEEL, OF ROUND, TOTAL EDIBLE 75. 00 G 75 0 BROCCOLI, RAW 20. 00 G 20 0 ONIONS, RAW 25. 00 G 25 0 MUSHROOMS, RAW 75. 00 G 75 0 CARROTS, RAW 20 .00 G 20 0 WATERCHESTNUTS, CHINESE, CANNED, 30 .00 G 30 0 SAUCES, READY-TO-SERVE, SOY 45. 00 G 45 0 STRAWBERRIES, FROZEN, SWEETENED, SLICED 100 .00 G 100 0 ICE CREAM, VANILLA, REGULAR, ( 1 0 % F A T ) , 24 .50 G 24 0 ANIMAL FAT, BEEF TALLOW  NUTRIENT PAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES) CALCIUM PHOSPHORUS POTASSIUM VITAMIN A (INTERNATIONAL UNITS) . VITAMIN C RIBOFLAVIN . . . . . TOTAL NIACIN (406+407 OR 406+408) PANTOTHENIC ACID VITAMIN B-12  . . . .  THREONINE  CYSTINE PHENYLALANINE . . . .  TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  87  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  INPUT TOTAL 30. 40 45. 40 114.00 977.00 534.00 240.00 7.21 65.50? 509.00 1290.00 2820.00 1.89? 22700.00 98.90 0.56 0.72 15.50? 2.56 0.80 112.00 1.52 0.10? 0.30? 0.37? 0.54? 0.50? 0.13? 0.06? 0.29? 0.26? 0. 41? 0.34? 0.17? 122.00 23.40? 1. 84?  COOKED TOTAL 30.40 45.40 114.00 977.00 534.00 240.00 7.21 65.50? 509.00 1290.00 2820.00 1.89? 22700.00 98.90 0.56 0.72 15.50? 2.56 0.80 112.00 1.52 0.10? 0.30? 0.37? 0.54? 0. 50? 0.13? 0.06? 0.29? 0.26? 0.41? 0.34? 0.17? 122.00 23.40? 1.84?  BEEF TALLOW LUNCH DAY2  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 35. 00 G 35 0 CHEESE, CHEDDAR 75. 00 G 75 0 BREADS: WHOLE-WHEAT, ( 2 % SKIM MILK POWDER) 110. 00 G 110 0 BANANAS, RAW 244 .00 G 244 0 MILK, PARTLY SKIMMED-, ( 2 % F A T ) , FLUID 65. 00 G 65 0 COOKIES: OATMEAL WITH RAISINS 44 .00 G 44 0 HAM, SLICED, EXTRA LEAN (APPROXIMATELY 5% FAT) 10. 50 G 10 0 ANIMAL FAT, BEEF TALLOW NUTRIENT PAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES)  PHOSPHORUS  VITAMIN A (INTERNATIONAL UNITS) .  TOTAL NIACIN (406+407 OR 406+408) PANTOTHENIC ACID FOLACIN VITAMIN B-12  . . . .  THREONINE  TYROSINE MISTIDINE TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  88  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  INPUT TOTAL 38.40 41.80 122.00 991.00 372.00 647.00 5.37 127.00? 766.00 1450.00 1470.00 4.34? 992.00 23.90 0.94 0.96 14.20? 1.99? 1.10? 95.10 1.51? 0.34? 1.09? 1.44? 2.38? 2.14? 0.67? 0.27? 1.26? 1.12? 1.55? 1.23? 0.92? 115.00 19.50 4.25  COOKED TOTAL 38.40 41.80 122.00 991.00 372.00 647.00 5. 37 127.00? 766.00 1450.00 1470.00 4.34? 992.00 23.90 0.94 0.96 14.20? 1.99? 1.10? 95.10 1. 51? 0.34? 1.09? 1.44? 2. 38? 2.14? 0.67? 0.27? 1.26? 1.12? 1.55? 1.23? 0.92? 115.00 19.50 4.25  B U T T E R F A T SUPPER D A Y 1  A^toUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 75.00 G 75 0 TURKEY, ALL CLASSES, LIGHT MEAT ONLY 50.00 G 50 0 CRANBERRY SAUCE, CANNED, SWEETENED 75.00 G 75 0 CARROTS, RAW 210.00 G 210 0 POTATOES, BOILED, COOKED WITHOUT SKIN 244.00 G 244 0 MILK, PARTLY SKIMMED, ( 2 % F A T ) , FLUID 100.00 G 100 0 ICE CREAM, VANILLA, REGULAR, (10% F A T ) , 250.00 G 250 0 PEACHES, CANNED HALVES/SLICES, WATER PACK 31.00 G 31 0 BUTTER, REGULAR  NUTRIENT CARBOHYDRATE, TOTAL  (BY DIFF)  . .  VITAMIN A (INTERNATIONAL UNITS) .  TOTAL NIACIN (406+407 OR  VITAMIN  406+408)  B-12  TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  89  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  INPUT TOTAL 40 .00 43. 60 120 .00 1010 .00 825. 00 494 .00 3 .21 136. 00 649 .00 2000. 00 572. 00 4 .52 24300 .00 33. 60 0. 49 0. 90 18 .50 3. 16? 1. 29 57. 60? 1. 67 0. 49? 1. 74? 2. 10? 3. 27? 3. 35? 1. 04? 0. 41? 1. 69? 1. 64? 2 .29? 2 .23? 1. 14? 183. 00 26. 10? 2. 37?  COOKED TOTAL 40.00 43.60 120.00 1010.00 825.00 494.00 3.21 136.00 649.00 2000.00 572.00 4.52 24300.00 33.60 0.49 0.90 18.50 3.16? 1.29 57.60? 1.67 0. 49? 1.74? 2.10? 3.27? 3 . 35? 1.04? 0 . 41? 1.69? 1.64? 2.29? 2.23? 1.14? 183.00 26.10? 2.37?  B U T T E R F A T SUPPER D A Y 2  AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 7 5 . 00 G 75 0 BEEF: HAMBURGER, LEAN, COOKED 65. 00 G 65 0 TOMATO PASTE, CANNED 85. 00 G 85 0 TOMATO SAUCE, CANNED 25. 00 G 25 0 MUSHROOMS, RAW 160. 00 G 160 0 SPAGHETTI: ENRICHED:COOKED,TENDER 5. 00 G 5 0 CHEESE, PARMESAN, GRATED 58. 00 G 58 0 BREADS: FRENCH OR VIENNA 100 .00 G 100 0 SHERBET, ORANGE 31. 00 G 31 0 BUTTER, REGULAR 15. 00 G 15 0 CHEESE, CREAM 20. 00 G 20 0 CELERY, RAW NUTRIENT PROTEIN FAT (TOTAL L I P I D ) CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES) MOISTURE CALCIUM [RON MAGNESIUM PHOSPHORUS POTASSIUM 30DIUM ZINC VITAMIN A (INTERNATIONAL UNITS) . VITAMIN C THIAMIN RIBOFLAVIN TOTAL NIACIN (406+407 OR 406+408) PANTOTHENIC ACID VITAMIN B-6 FOLACIN VITAMIN B-12 TRYPTOPHAN THREONINE ISOLEUCINE LEUCINE CYSINE METHIONINE CYSTINE PHENYLALANINE TYROSINE VAUI NE AR^TNINE HIST IDINE CHOLESTEROL TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  90  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  INPUT TOTAL 40.10 45.60 120.00 1040.00 425.00 232.00 9.13 98.90? 513.00 1580.00 1400.00 2.41? 3770.00 42.80 0. 74 0 .84 19.80 2.20 0.50 73.00 1. 00 0. 10? 0. 30? 0. 35? 0. 58? 0. 56? 0. 15? 0. 06? 0. 34? 0. 30? 0. 40? 0. 27? 0.23? 168.00 25.70 2.23  STAGE  COOKED TOTAL 40.10 45.60 120.00 1040.00 425.00 232.00 9.13 98.90? 513.00 1580.00 1400.00 2.41? 3770.00 42.80 0.74 0.84 19.80 2.20 0.50 73.00 1. 00 0. 10? 0. 30? 0. 35? 0. 58? 0. 56? 0. 15? 0. 06? 0. 34? 0. 30? 0. 40? 0. 27? 0. 23? 168.00 25.70 2.23  B E E F T A L L O W SUPPER D A Y 1 AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 75 . 00 G 75 0 50 .00 G 50 0 75 .00 G 75 0 CARROTS, RAW 244 .00 G 244 0 MILK, PARTLY SKIMMED, ( 2 % F A T ) , FLUID 210 .00 G 210 0 POTATOES, BOILED, COOKED WITHOUT SKIN, 100 .00 G 100 0 ICE CREAM, VANILLA, REGULAR, ( 1 0 % F A T ) , 250. 00 G 250 0 PEACHES, CANNED HALVES/SLICES, WATER PACK 24 .50 G 24 0 ANIMAL FAT, BEEF TALLOW NUTRIENT CARBOHYDRATE, TOTAL  (BY DIFF)  . .  VITAMIN A (INTERNATIONAL UNITS) .  TOTAL NIACIN (406+407 OR  406+408)  VITAMIN B-6  VALINE HISTIDINE  . . . . .  TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  91  INPUT TOTAL 39.70 42.90 120.00 1010.00 820.00 487.00 3.16 136.00 642.00 1990.00 316.00 4.51 23300.00 33.60 0. 49 0.89 18.40? 3.12? 1.29 56.70? 1.64 0.49? 1.73? 2.08? 3.25? 3.33? 1.03? 0.40? 1.68? 1.63? 2.27? 2.22? 1.13? 141.00 22.70? 2.42?  COOKED TOTAL 39.70 42.90 120.00 1010.00 820.00 487.00 3.16 136.00 642.00 1990.00 316.00 4.51 23300.00 33.60 0. 49 0.89 18.40? 3.12? 1.29 56.70? 1.64 0.49? 1.73? 2.08? 3.25? 3.33? 1.03? 0.40? 1.68? 1.63? 2.27? 2.22? 1.13? 141.00 22.70? 2.42?  BEEF TALLOW SUPPER DAY 2 AMOUNT UNIT WT(G) RETENTION RECIPE INGREDIENTS 75.00 G 75 0 BEEF: HAMBURGER, LEAN, COOKED 65.00 G 65 0 TOMATO PASTE, CANNED 85.00G 85 0 TOMATO SAUCE, CANNED 25.00 G 25 0 MUSHROOMS, RAW 160.00 G 160 0 SPAGHETTI: ENRICHED:COOKED,TENDER 5.00 G 5 0 CHEESE, PARMESAN, GRATED 58.00 G 58 0 BREADS: FRENCH OR VIENNA 100.00 G 100 0 SHERBET, ORANGE 15.00 G 15 0 CHEESE, CREAM 20.00 G 20 0 CELERY, RAW 24.50 G 24 0 ANIMAL FAT, BEEF TALLOW NUTRIENT PROTEIN FAT (TOTAL L I P I D ) . . . CARBOHYDRATE, TOTAL (BY DIFF) . . ENERGY (KILOCALORIES) MOISTURE CALCIUM IRON MAGNESIUM . PHOSPHORUS POTASSIUM SODIUM ZINC VITAMIN A (INTERNATIONAL UNITS) . VITAMIN C THIAMIN . RIBOFLAVIN TOTAL NIACIN (406+407 OR 406+408) PANTOTHENIC ACID VITAMIN B-6 FOLACIN VITAMIN B-12 TRYPTOPHAN . . THREONINE ISOLEUCINE LEUCINE LYSINE METHIONINE CYSTINE PHENYLALANINE TYROSINE VALINE AP^TNINE H1STIDINE CHOLESTEROL TOTAL SATURATED FATTY ACIDS . . . TOTAL POLYUNSATURATED FATTY ACIDS  92  UNIT G G G CAL G MG MG MG MG MG MG MG IU MG MG MG NE MG MG MCG MCG G G G G G G G G G G G G MG G G  INPUT TOTAL 39.80 44.90 120.00 1040.00 420.00 224.00 9.08 98.20? 506.00 1580.00 1150.00 2.39? 2820.00 42.80 0.74 0.83 19.70? 2.16 0.50 72.10 0.97 0.09? 0.28? 0.33? 0.56? 0.54? 0.14? 0.06? 0.33? 0.29? 0.39? 0.26? 0.22? 127.00 22.30 2.27  STAGE  COOKED TOTAL 39.80 44.90 120.00 1040.00 420.00 224.00 9.08 98.20? 506.00 1580.00 1150.00 2.39? 2820.00 42.80 0.74 0.83 19.70? 2.16 0.50 72.10 0.97 0.09? 0.28? 0.33? 0.56? 0.54? 0.14? 0.06? 0.33? 0.29? 0.39? 0.26? 0.22? 127.00 22.30 2.27  APPENDIX 4-1  HOURLY [1- C]FATTY ACID R E C O V E R Y FOR BUTTERFAT AND BEEF T A L L O W TREATMENTS 13  ri- Clmvristic acid  ri- Clpalmitic acid  Butterfat treatment  Butterfat treatment  13  Postbreakfast  hours  13  Beef tallow treatment  Beef tallow treatment  (% dose recovered)  1:00  0.00±0.00  0.0010.00  0.0010.00  0.0010.00  2:00  0.10±0.09  0.3210.07  0.1310.11  0.1210.04  3:00  0.39±0.14  0.5410.14  0.4210.20  0.1710.06  4:00  0.78±0.15  1.1010.19  0.4210.18  0.3110.10  5:00  1.64±0.39  2.0210.33  0.5310.13  0.5010.16  6:00  1.70±0.25  1.7610.26  0.5810.10  0.4310.12  7:00  1.4810.14  1.3910.34  0.3910.09  0.5610.12  8:00  0.6010.24  0.8110.18  0.4910.17  0.5210.21  9:00  0.4610.15  0.6710.17  0.3010.09  0.4810.26  meanlSEM n=8  93  A P P E N D I X 4-2  Time after breakfast meal  POSTPRANDIAL E N E R G Y EXPENDITURE FOR BUTTERFAT AND BEEF T A L L O W TREATMENTS  Butterfat treatment  hours:minutes  Beef tallow treatment  (kcal/minute)  0:00-0:29  1.5110.07  1.5110.07  0:30-0:59  1.5210.07  1.5110.07  1:00-1:29  1.4810.06  1.4710.07  1:30-1:59  1.4510.05  1.4310.07  2:00-2:29  1.4010.06  1.4110.07  2:30-2:59  1.4510.07  1.4110.07  3:00-3:29  1.4310.06  1.3810.07  3:30-3:59  1.4110.04  1.3710.06  4:00-4:29  1.3710.04  1.3510.06  4:30-4:59  1.6410.05  1.6110.06  5:00-5:29  1.6410.05  1.6210+06  5:30-5:59  1.5410.04  1.5610.06  6:00-6:29  1.4610.04  1.4810.06  6:30-6:59  1.4210.06  1.4510.05  7:45-8:15  1.3710.05  1.4010.05  8:45-9:15  1.3110.05  1.3310.07  meanlSEM n=8  94  A P P E N D I X 4-3  Time after breakfast meal  hoursrminutes  POSTPRANDIAL C A R B O H Y D R A T E OXIDATION F O R BUTTERFAT AND BEEF T A L L O W T R E A T M E N T S  Butterfat treatment  Beef tallow treatment  (g/30 minutes)  0:00-0:29  6.29±0.88  5.9110.75  0:30-0:59  6.20±0.82  5.9910.62  1:00-1:29  5.0810.74  5.5010.45  1:30-1:59  4.6910.57  4.5510.32  2:00-2:29  4.2410.27  4.1410.40  2:30-2:59  4.6210.35  4.6410.54  3:00-3:29  4.0810.44  4.0710.39  3:30-3:59  3.6210.54  3.7310.54  4:00-4:29  3.3210.53  3.0610.44  4:30-4:59  5.5510.54  5.8610.73  5:00-5:29  5.4710.53  5.8910.69  5:30-5:59  3.8610.67  3.8410.63  6:00-6:29  3.6310,50  3.4210.48  6:30-6:59  3.9210.55  4.1110.50  7:45-7:15  5.7610.82  6.3610.37  8:45-9:15  4.5910.65  5.7310.42  meanlSEM n=8  95  A P P E N D I X 4-4  Time after breakfast meal  hours:minutes  POSTPRANDIAL F A T OXIDATION F O L L O W I N G BUTTERFAT AND BEEF T A L L O W TREATMENTS  Butterfat treatment  Beef tallow treatment  (g/30 minutes)  0:00-0:29  2.0110.31  2.2310.25  0:30-0:59  2.0710.31  2.2010.22  1:00-1:29  2.5210.33  2.2810.28  1:30-1:59  2.5710.30  2.5710.25  2:00-2:29  2.6410.20  2.7010.30  2:30-2:59  2.6010.24  2.4610.29  3:00-3:29  2.8410.32  2.6310.29  3:30-3:59  2.8810.30  2.7410.36  4:00-4:29  2.9610.28  2.9710.32  4:30-4:59  2.8110.25  2.6010.36  5:00-5:29  2.8410.24  2.6110.36  5:30-5:59  3.2710.28  3.3010.36  6:00-6:29  3.1210.23  3.2410.26  6:30-6:59  2.7710.23  2.8410.29  7:45-8:15  1.8710.26  1.6910.23  8:45-9:15  2.0310.24  1.7610.30  meanlSEM n=8  96  A P P E N D I X 4-5  Subject  C U M U L A T I V E F R A C T I O N A L OXDDATION O F F A T T Y ACIDS F O R I N D I V I D U A L S U B J E C T S Butter-MA  Tallow-MA  Butter-PA  1 3  C-  Tallow-PA  (%dose/9h)  1  9.73  7.34  2.16  1.43  2  10.32  5.65  2.60  0.56  3  9.41  9.72  6.41  0.41  4  5.04  10.71  0.59  2.97  5  3.05  6.05  2.72  7.45  6  8.40  11.65  5.11  2.37  7  3.25  6.76  n/a  4.83  8  7.87  10.98  3.20  4.69  mean+SEM  7.13±1.04  8.6110.86  3.2610.73  97  3.0110.86  1  A P P E N D I X 4-6  C U M U L A T I V E SUBSTRATE UTDLIZATION A N D E N E R G Y E X P E N D I T U R E F O R INDIVDDUAL S U B J E C T S AFTER T H E BUTTER M E A L Carbohydrate Oxidation  Fat oxidation  (kcal/9 hr)  (gr/9 hr)  (gr/9 hr)  1  866  78  58  2  650  72  38  3  834  87  48  4  794  120  32  5  739  85  42  6  780  50  61  7  728  79  43  8  868  111  44  Subject  Energy Expenditure  98  A P P E N D I X 4-7  C U M U L A T I V E SUBSTRATE UTILIZATION AND E N E R G Y EXPENDITURE F O R INDIVIDUAL SUBJECTS AFTER THE TALLOW MEAL Carbohydrate Oxidation  Fat oxidation  (kcal/9 hr)  (gr/9 hr)  (gr/9 hr)  1  808  103  41  2  664  105  25  3  822  75  55  4  837  83  53  5  697  67  45  6  673  88  33  7  878  68  64  8  864  122  39  Subject  Energy Expenditure  99  

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