@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Dendy, Shauneen Marguerite"@en ; dcterms:issued "2010-10-07T19:40:15Z"@en, "1990"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "The purpose of this study was to investigate whether increased endogenous cholesterol synthesis contributes to the elevated plasma cholesterol levels observed in type III hyperlipoproteinemia (type III HLP). Eight apolipoprotein (apo) E2 subjects with type III HLP and 8 apo E2 non-hyperlipidemic control subjects (controls) were given a priming bolus dose of deuterium oxide (D₂O) (0.7 g D₂O/kg body H2O). Daily M1 (central) pool free cholesterol fractional synthetic rate (FSR) was calculated as the incorporation rate of deuterium from body water into plasma free cholesterol. Blood samples were collected one half hour prior to, and at 12 hour intervals over 48 hours following, the bolus D₂O dose. Drinking water labelled at 1.4 and 0.7 g D₂O/liter H₂O was given on the fed and fasted days, respectively. Over 0-24 hours, subjects consumed a diet of three isocaloric meals which, in composition, approximated average North American intakes. Subjects fasted over 24-48 hours. The deuterium enrichment of plasma free cholesterol and plasma water was determined by isotope ratio mass spectrometry. When all subjects were included, mean (±SEM) free cholesterol overall FSR in type III HLPs (0.031 ± 0.006 per day) was not significantly different from controls (0.037 ± 0.004 per day). Estimated Ml total cholesterol pool size in type III HLPs (26.1 ± 1.9 g) and controls (24.9 ± 0.6 g) was not significantly different. When free cholesterol net synthesis was calculated as the absolute amount of cholesterol synthesized per day, based on Ml total cholesterol pool size, overall free cholesterol net synthesis in type III HLPs (0.304 ± 0.034 g/day) was not significantly different from controls (0.364 ± 0.035 g/day). When all subjects were included, overall free cholesterol FSR and overall free cholesterol net synthesis were significantly greater (p<0.001) in the fed (0.066 ± 0.006 day⁻¹ and 0.655 + 0.048 g/day, respectively) as compared to the fasted state (0.001 ± 0.004 day⁻¹ and 0.010 ± 0.037 g/day, respectively). In the fed state, type III HLPs tended to synthesize cholesterol at a lower rate and in a lower absolute amount as compared to controls, while the reverse was observed in the fasted state. These results suggest that: (1) the elevated plasma cholesterol levels observed in type III HLPs are not due to excess de novo cholesterol synthesis; (2) fasting significantly reduces cholesterol synthesis from the fed state."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/28979?expand=metadata"@en ; skos:note "CHOLESTEROL SYNTHESIS IN TYPE III HYPERLIPOPROTEINEMIC AND NON-HYPERLIPIDEMIC INDIVIDUALS by SHAUNEEN MARGUERITE DENDY B.Sc, University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DIVISION OF HUMAN NUTRITION SCHOOL OF FAMILY AND NUTRITIONAL SCIENCES We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1990 ^)Shauneen Marguerite Dendy, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of HUMAN NUTRITION The University of British Columbia Vancouver, Canada Date APRIL 30, 1990 DE-6 (2/88) ABSTRACT The purpose of this study was to investigate whether increased endogenous cholesterol synthesis contributes to the elevated plasma cholesterol levels observed in type III hyperlipoproteinemia (type III HLP). Eight apolipoprotein (apo) E2 subjects with type III HLP and 8 apo E2 non-hyperlipidemic control subjects (controls) were given a priming bolus dose of deuterium oxide (D2O) (0.7 g D207kg body H2O). Daily M l (central) pool free cholesterol fractional synthetic rate (FSR) was calculated as the incorporation rate of deuterium from body water into plasma free cholesterol. Blood samples were collected one half hour prior to, and at 12 hour intervals over 48 hours following, the bolus D2O dose. Drinking water labelled at 1.4 and 0.7 g D2U/liter H2O was given on the fed and fasted days, respectively. Over 0-24 hours, subjects consumed a diet of three isocaloric meals which, in composition, approximated average North American intakes. Subjects fasted over 24-48 hours. The deuterium enrichment of plasma free cholesterol and plasma water was determined by isotope ratio mass spectrometry. When all subjects were included, mean (±SEM) free cholesterol overall FSR in type III HLPs (0.031 ± 0.006 per day) was not significantly different from controls (0.037 ± 0.004 per day). Estimated M l total cholesterol pool size in type III HLPs (26.1 ± 1.9 g) and controls (24.9 ± 0.6 g) was not significantly different. When free cholesterol net synthesis was calculated as the absolute amount of cholesterol synthesized per day, based on M l total cholesterol pool size, overall free cholesterol net synthesis in type III HLPs (0.304 ± 0.034 g/day) was not significantly different from controls (0.364 ± 0.035 g/day). When all subjects were included, overall free cholesterol FSR and overall free cholesterol net synthesis were significantly greater (p<0.001) in the fed (0.066 ± 0.006 day\" 1 and 0.655 + 0.048 g/day, respectively) as compared to the fasted state (0.001 ± 0.004 day\"1 and 0.010 ± 0.037 g/day, respectively). In the fed state, type III HLPs tended to synthesize cholesterol at a lower rate and in a lower absolute amount as compared to controls, while the reverse was observed in the fasted state. These results suggest that: (1) the elevated plasma cholesterol levels observed in type III HLPs are not due to excess de novo cholesterol synthesis; (2) fasting significantly reduces cholesterol synthesis from the fed state. ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Figures vi List of Tables vii Acknowledgement viii 1. Introduction 1 2. Literature Review 3 2.1 Function of Apolipoprotein E 3 2.1.1 Transport of Dietary Lipids to the Liver 3 2.1.2 Transport of Endogenous Lipids to Peripheral Cells 4 2.1.3 Transport of Lipids from Peripheral Tissues to Liver 4 2.2 Apo E Polymorphism 5 2.3 Structural Basis For Apo E Polymorphism .....6 2.4 Impact of Allelic Variation on Functional Characteristics of Apo E 7 2.5 Metabolic Consequences of Apo E2/2 Phenotype - Possible Mechanism 11 2.6 Association of the Apo E2/2 Phenotype With Type III Hyperlipoproteinemia 12 2.7 Historical Background of Type III Hyperlipoproteinemia 12 2.8 Clinical and Pathologic Features of Type III Hyperlipoproteinemia 13 2.9 Genetic Mode of Inheritance of Type III Hyperlipoproteinemia 15 2.10 Treatment of Type III Hyperlipoproteinemia 15 2.11 Factors Modulating the Effects of the Apo E2 Defect in the Phenotypic Expression of Type III Hyperlipoproteinemia 16 2.12 Measurement of Cholesterol Synthesis 18 2.13 Deuterium Incorporation Methodology 21 2.14 Assumptions of Deuterium Incorporation Methodology 22 2.15 Summary 23 3. Methods 25 3.1 Experimental Design 25 3.1.1 Phase I: Initial Subject Screening 25 3.1.2 Phase II: Experimental Trial 26 3.2 Analytical Procedures 29 3.2.1 Phase I: Initial Subject Screening 29 3.2.2 Phase II: Experimental Trial 33 3.3 Free Cholesterol Fractional Synthetic Rate and Net Synthesis Calculations 36 3.4 Statistics 38 iii 4. Results 39 4.1 Phase I: Initial Subject Screening 39 4.2 Phase II: Experimental Trial 44 5. Discussion 62 5.1 Phase I: Initial Subject Screening 62 5.2 Phase II: Experimental Trial 66 5.3 Effect of Group on Cholesterol FSR and Cholesterol Net Synthesis 71 5.4 Effect of Feeding State on Cholesterol FSR and Cholesterol Net Synthesis 76 5.5 Evaluation of Deuterium Incorporation Methodology 77 5.5.1 Metabolic Considerations 77 5.5.2 Methodologic Considerations 79 5.6 Conclusions 80 Appendix One Informational Letter Sent to Type III Hyperlipoproteinemics 82 Appendix Two Subject Information Sheet for Individuals Screened for Type III HLP Test Group 83 Appendix Three Subject Information Sheet for Individuals Screened for Control Group..84 Appendix Four Consent by Subject of Research Protocol 85 Appendix Five Description of Research Project Protocol 86 Appendix Six Food Record Instructions 87 Appendix Seven Diet Fed to Control and Type III HLP Subjects on Feeding Day of Experimental Trial 88 Appendix Eight Estimated Daily Caloric Requirements of Control and Type III HLP Subjects Administered on Feeding Day of Experimental Trial 89 Appendix Nine Estimated Total Body Water Content of Control and Type III HLP Subjects and Corresponding Deuterium Oxide Bolus Dose and Deuterium Labelled Drinking Water Administered During Experimental Trial 91 Appendix Ten Results of Apo E Phenotype and Plasma Lipid Concentration Analyses in Subjects Screened for Control Group 92 Appendix Eleven Body Weight Fluctuations of Control and Type III HLP Subjects Throughout Experimental Trial 93 Appendix Twelve Plasma Water Deuterium Enrichment in Blood Sample Drawn from Control and Type III HLP Subjects During Experimental Trial 94 Appendix Thirteen Plasma Free Cholesterol Deuterium Enrichment in Blood Samples Drawn from Control and Type III HLP Subjects During Experimental Trial 95 iv Appendix Fourteen Plasma Free Cholesterol Deuterium Enrichment Blood Samples Drawn from Control and Type III HLP Subjects At 12 Hour Intervals During Experimental Trial 96 Appendix Fifteen Cholesterol Fractional Synthetic Rate in Control and Type III HLP Subjects Over 12 Hour Time Intervals During Experimental Trial 97 Appendix Sixteen Individual Free Cholesterol Net Synthesis Per Day Based on Individual Ml (Central) Total Cholesterol Pool Size in Control and Type III HLP Subjects Over 12 Hour Time Intervals During Experimental Trial 98 Appendix Seventeen Cholesterol Fractional Synthetic Rate in Control and Type III HLP Subjects Over 12 Hour Time Intervals During Experimental Trial 99 Appendix Eighteen Individual Free Cholesterol Net Synthesis Per Day Based on Individual Ml (Central) Total Cholesterol Pool Size in Control and Type III HLP Subjects Over 12 Hour Time Intervals During Experimental Trial 100 Bibliography 101 v LIST OF FIGURES Figure 1 One-dimensional isoelectric focusing technique showing the three homozygous apo E phenotypes. The amino acid differences among the three major polymorphic forms of apo E are given for comparison 8 Figure 2 Pathogenesis of type III HLP: interaction between genes, environment, and a specific apo E genotype 19 Figure 3 Effect of group (controls versus type III HLPs) on free cholesterol fractional synthetic rate (0-48 hr) for all subjects 50 Figure 4 Effect of group (controls versus type III HLPs) on free cholesterol fractional synthetic rate when subject EK is excluded 52 Figure 5 M l (central) total cholesterol pool size in controls and type III HLPs 54 Figure 6 Effect of group (controls versus type III HLPs) on free cholesterol net synthesis (0-48 hr) for all subjects 57 Figure 7 Effect of feeding state on free cholesterol net synthesis for all subjects 59 Figure 8 Mean free cholesterol fractional synthetic rate for all control and type III HLP subjects during the fed and fasted state 60 Figure 9 Mean free cholesterol net synthesis for all control and type III HLP subjects during the fed and fasted state 61 vi LIST OF TABLES Table 1 Summary of apolipoprotein E phenotype and plasma lipid concentration results in subjects screened for control group 40 Table 2 Apolipoprotein E and plasma lipid profile of age-sex matched control and type III HLP subjects selected for experimental trial 41 Table 3 Anthropometric, medicinal and hormonal profile of control and type III HLP subjects selected for experimental trial 43 Table 4 Analysis of control and type III HLP subjects' usual dietary intake as reported by 3 day food records 45 Table 5 Plasma total cholesterol determinations in control and type III HLP subjects during the experimental trial 47 Table 6 Cholesterol fractional synthetic rate in control and type III HLP subjects during experimental trial 49 Table 7 Estimation of M1 (central) total cholesterol pool size in control and type III HLP subjects 53 Table 8 Individual free cholesterol net synthesis per day based on individual M l (central) total cholesterol pool size in control and type III HLP subjects 56 Table 9 Summary of repeated measures analysis of variance for free cholesterol fractional synthetic rate and free cholesterol net synthesis based on M l (central) total cholesterol pool size in control and type III HLP subjects 58 vii ACKNOWLEDGEMENT I would like to thank the members of my thesis committee, Dr. Peter Jones (Supervisor) of the Division of Human Nutrition, University of British Columbia, Dr. J i r i Frohlich, Director of the University Hospital, Shaughnessy Site Lipid Clinic, Dr. Sheila Innis of the Department of Paediatrics, University of British Columbia, Dr. David Kitts of the Department of Food Science, University of British Columbia, and Dr. Linda McCargar of the Division of Human Nutrition, University of British Columbia, for their competent instruction and consultation, and thoughtful guidance during my thesis project. I am especially indebted to the 16 subjects who enthusiastically volunteered their time to participate in, and provide the data for, this research project. I would like to acknowledge the following individuals for their generous and efficient assistance with various aspects of the research project: Alan Klima and Robin King for phlebotomizing with finesse to obtain the blood samples during the experimental trial; Wendy Van Wermeskerken for fastidious weighing and preparation of the food for the experimental diet for each trial; Dr. Dale Schoeller of the Stable Isotope Laboratory, Clinical Nutrition Research Center, University of Chicago and Dr. Katie Leitch and Jennifer Wang for their time in the reduction procedures and mass spectrometric analyses of my cholesterol and plasma water samples; and Ruth Grierson, Supervisor of the University Hospital, Shaughnessy Site Lipid Clinic for her helpful instruction and overseeing of my plasma cholesterol and triglyceride level analyses. Final gratitude is expressed to my family and my husband, Richard, for their patience, encouragement and support throughout the completion of this degree. Personal and project support for this research was received from the British Columbia Health Care Research Foundation. viii 1. INTRODUCTION Individuals with the apo E 2/2 phenotype, who comprise approximately 1-2% of the population (Brown et al. 1983b, Innerarity et al. 1986), display primary dysbetalipoproteinemia, a lipoprotein abnormality characterized by low levels of LDL and traces of fasting p-VLDL (Utermann et al. 1977). It is postulated that impaired lipoprotein clearance (Utermann 1986), due to the abnormal binding of the E2 apoprotein to hepatic lipoprotein receptors (Weisgraber et al. 1982, Schneider et al. 1981), is the primary basis for these plasma lipoprotein alterations. The resultant effect of such abnormal binding is normal or subnormal plasma cholesterol levels. The same processes occur in the approximately 10% of the population with the apo E3/2 phenotype (Davignon et al. 1988), yet to a more moderate degree, due to possession of only a single E2 apoprotein (Weintraub et al. 1987, Havel et al. 1986). Although accurate prevalence data are scarce, it is estimated that 1 in 1,000 to 10,000 individuals have a confirmed diagnosis of type III hyperlipoproteinemia (type III HLP) (Breslow et al. 1986), a relatively rare fasting hyperlipidemia characterized by highly elevated concentrations of fasting ^-VLDL, xanthomatosis and premature coronary artery disease (Mahley et al. 1984b). Untreated, plasma cholesterol levels may range from 7.8-26 mmol/1 (Innerarity et al. 1986). Paradoxically, type III HLP, and the elevated cholesterol levels observed, most commonly develop in individuals with the apo E2/2 phenotype (Mahley et al. 1984b), yet individuals with other apo E phenotypes (Gregg et al. 1983, Havel et al. 1983, Smit et al. 1987, Rail et al. 1989) and apo E deficiency (Schaefer et al. 1986, Mabuchi et al. 1989) have been identified with this disorder. The apo E2:receptor binding abnormality is considered the principle defect in type III HLP (Rail et al. 1983a). However, as this disease does not develop in all apo E2 individuals, a multifactorial etiology has been proposed (Utermann et al. 1986), suggesting an interaction of other genetic and/or environmental factors with the underlying apo E2 defect to precipitate the expression of type III HLP (Davignon et al. 1988). 1 It is hypothesized that excess primary de novo cholesterol synthesis may be a factor which interacts with the abnormal E2 apoprotein to cause the elevated plasma cholesterol levels observed in type III HLP. In order to test this hypothesis, two primary objectives were addressed: 1) to determine whether apo E2 type III hyperlipoproteinemic subjects (type III HLPs) synthesize cholesterol at an increased rate as compared to apo E2 non-hyperlipidemic control subjects (controls). 2) to determine whether cholesterol synthetic rate in apo E2 type III hyperlipoproteinemic subjects (type III HLPs) and apo E2 non-hyperlipidemic control subjects (controls) is influenced by feeding state. 2 2. LITERATURE REVIEW 2.1 FUNCTION OF APOLIPOPROTEIN E Apolipoprotein E (apo E) is a 299 amino acid, 34,000 molecular weight glycoprotein (Mahley et al. 1984a), synthesized in humans primarily in the liver, while secondary sources include the brain, adrenals, kidney and spleen (Utermann et al. 1986, Davignon et al. 1988). Apo E was first identified in 1973 by Shore and co-workers (Shore et al. 1973). As a surface component of several plasma lipoproteins, apo E functions as a ligand which mediates the interaction between specific cellular lipoprotein receptors and the plasma lipoproteins (Brewer et al. 1983). This apoprotein plays a critical role in three major pathways of lipid transport (Mahley et al. 1988) including transport of dietary lipids to the liver, transport of endogenous lipids to peripheral cells, and transport of lipids from peripheral tissues to the liver. 2.1.1 Transport of Dietary Lipids to the Liver Apo E is present in chylomicrons, which transport dietary cholesterol and triglyceride from intestinal mucosal cells through the thoracic duct lymph to the liver. Apo E is acquired by chylomicrons upon secretion into lymph. Following entry into the bloodstream, chylomicrons are metabolized to chylomicron remnants within extra hepatic tissues, through lipolytic action on chylomicron core triglycerides by the enzyme lipoprotein lipase (LPL). Chylomicron remnants are rapidly extracted from the bloodstream by hepatocytes, via receptor-mediated endocytosis. Hepatic intracellular cholesterol metabolism is in turn regulated by the degradation of the incoming cholesterol and triglyceride containing lipoproteins, through effects on 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase activity, the rate limiting enzyme of the cholesterol biosynthetic pathway, and on the low density lipoprotein (LDL) receptor, or apo B,E receptor, synthesis and expression (Brown and Goldstein 1983a, Andersen et al. 1979, Brown et al. 1981). The binding of chylomicron remnants to the hepatic chylomicron remnant receptor, or apo E receptor, present only on hepatic cells, is mediated by apo E (Gregg et 3 al. 1986), yet the specific uptake mechanism is unknown (Mahley 1988). In contrast to the LDL (apo B,E) receptor, found on liver and extrahepatic cells, the activity of the remnant (apo E) receptor is unregulated (Brown and Goldstein 1983a, Mahley 1984b). Although apo E of chylomicron remnants may be recognized by the hepatic LDL (apo B,E) receptor, normally most chylomicron remnants are cleared by the liver through the remnant (apo E) receptor (Brown and Goldstein 1983a, Sutherland et al. 1988). 2.1.2 Transport of Endogenous Lipids to Peripheral Cells Secondly, apo E is a constituent of very low density lipoproteins (VLDL). Endogenous triglycerides and cholesterol, synthesized in the liver, are transported by VLDL secreted from hepatocytes. Similar to the catabolic course of chylomicrons, VLDL remnants, also referred to as lipoproteins of intermediate density (IDL), are produced by LPL hydrolysis of VLDL core triglycerides. The subsequent processing of VLDL remnants diverges at this point from that of chylomicrons. Firstly, VLDL remnants (IDL) may be removed directly into the liver via either the remnant (apo E) receptor or the LDL (apo B,E) receptor. Secondly, VLDL remnants may be further catabolized via LPL to LDL. In normal human metabolism, most VLDL remnants undergo conversion to LDL (Utermann 1986, Gregg et al. 1986). Apo E appears to play a role in both of these catabolic routes of VLDL metabolism, yet the precise mechanism whereby apo E functions is undefined (Utermann 1986). LDL do not contain apo E, and 60-80% of these lipoproteins are therefore catabolized directly by the LDL (apo B,E) membrane receptors of hepatocytes or peripheral tissues (Meddings et al. 1987). The remaining 20-40% of LDL are removed from the plasma by nonspecific receptor-independent endocytosis in hepatic and extrahepatic tissues. 2.1.3 Transport of Lipids from Peripheral Tissues to Liver In normal human plasma, the majority of reverse cholesterol transport is thought to occur via cholesterol ester transfer protein (CETP) transport of cholesterol esters from HDL to either 4 chylomicrons and VLDL (and/or their remnants), or primarily to LDL, followed by LDL (apo B,E) and remnant (apo E) receptor hepatic uptake (Havel 1988). Some of the typical high density lipoproteins (HDL) without apo E become enriched with unesterified cholesterol from peripheral tissue cell membranes and/or the surfaces of lipoproteins (Havel 1988), and acquire apo E, forming apo E-HDLc (Mahley et al. 1988). It has been speculated, that in humans, apo E-HDLc functions as an element in reverse cholesterol transport, carrying a fraction of the cholesterol obtained from peripheral tissues directly to the liver through an apo E:LDL (apo B,E) receptor interaction (Mahley et al. 1988). Clearly, apo E is critically important as a component of chylomicron and VLDL remnants, as well as apo E-HDLc, in the recognition process of these plasma lipoproteins by lipoprotein receptors. Apo E is thus indirectly crucial to the control of intracellular lipid metabolism, as these lipoproteins are subsequently removed from the plasma. Any variation in the functional role of apo E, as a result of genetic alteration of this polypeptide, would have significant effects on cholesterol homeostatic control in humans. 2.2 APO E POLYMORPHISM Utermann et al. (1977) were the first to demonstrate that human apo E exists as three major isoforms using the phenotyping technique of isoelectric focusing of VLDL apo E. In subsequent studies, Zannis and Breslow (1981) hypothesized that the polymorphic nature of apo E is due to genetic variation at the apo E gene locus. These investigators predicted that synthetic control of apo E is determined by three independent alleles of the gene for apo E, designated £4, £3, and £2, each allele coding for a major gene product, referred to as the E4, E3 and E2 isoforms, respectively (Zannis et al. 1981). The three alleles result in six genotypes: 3 homozygous (£4/4, £3/3, and9.2/2) and 3 heterozygous (£4/3, £3/2, and £4/2). Six corresponding phenotypes arise from the expression of any one of the genotypes: E4/4, E3/3, E2/2 and E4/3, E3/2, and E4/2 (Davignon 1988). 5 The relative frequency with which the three common alleles £4, £3, and £2, occur displays within and between population variability. As reviewed by Boerwinkle (1987), frequencies ranging from 12-18%, 72-89% and 0-13% for the£4,£3, and£2 alleles respectively, were estimated from 11 populations of differing ethnic origin or geographical location. Relative frequencies of these common alleles among Caucasians (weighted averages, from Germany, Scotland, Canada, Netherlands, France, Finland and New Zealand) and for Canadians (Ottawa) have been estimated by Davignon (1988) as 15.0%, 76.9% and 8.0% and 15.2%, 77.0%, and 7.8% for the£4, £3, and£2 alleles, respectively. Recent studies have determined the approximate frequencies of 3.9%, 61.7%, 2.0%, 20.6%, 9.8%, and 2.0% for the E4/4, E3/3, E2/2, E4/3, E3/2, and E4/2 phenotypes, respectively, for a sample of Canadians living in Ottawa (Davignon et al. 1988). 2.3 STRUCTURAL BASIS FOR APO E POLYMORPHISM As the homozygous E3/3 phenotype predominates within the population, the £3/3 genotype is considered the wild type or parent apo E isoform, from which the aberrant E4 and E2 isoforms are derived (Mahley et al. 1984b). The structural basis for apo E polymorphism has been explained through amino acid sequencing of the major apo E isoforms (Rail et al. 1982b). Weisgraber et al. (1982) have demonstrated point mutations at residues 112 and 158, resulting in alterations of the primary structure of the apo E gene. Apo E4 differs from apo E3 by a single substitution of the basic amino acid arginine in place of the neutral amino acid cysteine at residue 112 in the apo E3 isoform, noted as \"E4 Cys 112 --> Arg\". This amino acid interchange to form the E4 mutant, results in a +1 charge difference relative to the E3 isoform. The most common form of apo E2 differs from apo E3 by a single substitution of the amino acid cysteine in place of arginine at residue 158 in the apo E3 isoform, noted as \"E2 Arg 158 --> Cys\". This amino acid interchange to form the E2 mutant, results in a -1 charge difference relative to the E3 isoform (Weisgraber et al. 1982). These cysteine-arginine interchanges in the parent apo E3 isoform, which is noted as \"112 Cys, 158 Arg\", provide the basis for separation of the three major isoforms 6 by polyacrylamide gel isoelectric focusing according to differing isoelectric points of 6.2 to 5.7, ranging from basic (E4) to acidic (E2) (Rail et al. 1986) (Figure 1). In addition to the three major, common apo E isoforms, several rare apo E mutant isoforms have been described, among which include E2* (E2 145 Arg --> Cys) and E2** (E2 146 Lys --> Gin) (Breslow et al. 1986, Ehnholm et al. 1986). Homozygosity for the apo E2 phenotype may therefore result from a combination of any two of the £2, £2* or £2** alleles (Breslow et al. 1986). 2.4 IMPACT OF ALLELIC VARIATION ON FUNCTIONAL CHARACTERISTICS OF APO E Recent studies by Weisgraber et al. (1982) and Schneider et al. (1981) have demonstrated that the apo E2 isoform exhibits abnormal binding activity to LDL (apo B,E) lipoprotein receptors, due to the cysteine-arginine substitution at residue 158. Innerarity et al. (1986) have described, the molecular basis for this decreased binding affinity. Alterations in the molecular conformation of the apo E receptor binding domain, comprised of amino acid residues 140-150, may result from amino acid substitutions in regions outside the actual binding site, such as residue 158. These conformational alterations may be deleterious to normal binding activity. Heterogeneity in the defective lipoprotein.-receptor binding activity of individuals with the apo E2/2 phenotype (Rail et al. 1982b) may be explained by the genetic heterogeneity within this phenotype. Impaired binding activity is exhibited by all of the apo E2 mutant forms, varying from <2%, 40%, and 45% of apo E3 normal binding for the E2, E2**, E2* mutants, respectively (Mahley et al. 1984a, Mahley et al. 1984b). The defective receptor binding of the apo E2 variants (E2** and E2*) may be explained by disruption of an apoprotein:receptor direct ionic interaction with the substitution of neutral for basic amino acids at residues 145 and 146 in the receptor binding domain (Lalazar et al. 1988). 7 E 3 : E 2 + E 2 / 2 E 3 / 3 E 4 / 4 Relative o +1 + 2 Charge Residue 112 Cys Cys Arg Residue 158 Cys Arg Arg Figure 1 One-dimensional isoelectric focusing technique showing the three homozygous apo E phenotypes. The amino acid differences among the three major polymorphic forms of apo E are given for comparison (Rail et al. 1986). 8 The impact of this allelic variation on the functional role of apo E is manifest in defective receptor mediated transport of triglyceride and cholesterol containing lipoproteins in human plasma. A defect in the high affinity binding of apo E containing chylomicron remnants, VLDL remnants (IDL), and apo E-HDLc to both the hepatic LDL (apo B,E) receptor and the remnant (apo E) receptor would be expected to result in hyperlipidemia, due to an accumulation of these particles in the plasma (Utermann 1986, Utermann et al. 1984). Individuals homozygous for the E2/2 phenotype do display impaired lipoprotein clearance and accumulation of plasma cholesterol and triglyceride enriched chylomicron and VLDL remnants and apo E-HDLc (Davignon et al. 1988, Havel et al. 1980a), yet only to a moderate degree. Paradoxically, gross elevations in cholesterol and triglyceride levels in most of these subjects do not occur, while subnormal lipid levels are often observed (Utermann et al. 1977, Utermann et al. 1979a). Utermann et al. (1979a), comparing serum lipid levels in the three phenotypic groups, apo E-N (apo E3/3), apo E-ND (apo E3/2), and apo E-D (apo E2/2), observed a hypocholesterolemic effect in individuals homozygous for the E2/2 phenotype, whose serum cholesterol levels were approximately 1.0 mmol/1 (40 mg/dl) lower than E3/3 subjects. The effects of apo E polymorphism on normal plasma lipid and lipoprotein concentration were confirmed and extended through further investigation by Sing and Davignon (1985). Based on the previous finding that interindividual genetic variability accounts for 50% of the fluctuation in normal serum cholesterol levels (Sing et al. 1978), the fractional contribution of the common apo E alleles to plasma cholesterol levels was estimated to be 14% of the genetic variance, and 7% of the total variance for a population sample in Ottawa, Canada (Sing and Davignon 1985). These results, in combination with data from other studies (Utermann 1986, Bouthillier et al. 1983), have established the \"cholesterol-lowering\" effect of the£2 allele, measured against the wild type £3 allele, and the \"cholesterol-raising\" effect of the £4 allele (Davignon et al. 1988). A difference of approximately 1.6 mmol/1 (60 mg/dl) has been observed between mean total cholesterol levels of individuals with the E2/2 and E4/4 phenotypes, averaging approximately 3.6 and 5.2 mmol/1 (140 and 200 mg/dl), respectively (Utermann 1986). Individuals heterozygous for the apo £2 allele exhibit plasma 9 cholesterol levels between values obtained for £2 homozygotes and individuals who do not possess the £2 allele (Utermann et al. 1984). Therefore, the £\"4, £3 , and £2 alleles in apo E homo- and heterozygotes effect an overlapping array of phenotypically related plasma cholesterol concentrations in the population (Utermann 1986). Mean plasma cholesterol levels for apo E phenotypes observed in a study in Ottawa, Canada (Davignon 1988) were 3.53 (E2/2), 4.17 (E3/2), 4.51 (E3/3), 4.62 (E4/2), 4.77 (E4/3), and 4.67 (E4/4) mmol/1. The homozygous E2 phenotype occurs in approximately 1 (Brown et al. 1983b) to 2% (Innerarity et al. 1986) of Europeans and North Americans. As shown by Utermann, despite inheritance of this mutant form of apo E, most individuals displaying the E2/2 phenotype actually have subnormal cholesterol levels (Utermann et al. 1979a, Utermann et al. 1979b). The metabolic consequences of the apo £2 allele may be summarized as: hypocholesterolemic, hypertriglyceridemic and dyslipoproteinemic (Utermann et al. 1979a). Most of the individuals who are homozygous for this allele display primary dysbetalipoproteinemia (Utermann et al. 1977), a lipoprotein abnormality characterized by the E2/2 phenotype, LDL deficiency, and evidence of ^-VLDL, also known as \"floating -^lipoproteins\". Three basic characteristics differentiate ^,-VLDL particles from normal VLDL, also referred to as o(cVLDL. Firstly, j^-VLDL exhibit -^mobility on electrophoresis, as opposed to the normal pre-^ mobility ofcA-VLDL, hence the term dysbetalipoproteinemia. On most types of electrophoretic systems,c<-VLDL usually migrate more rapidly than LDL, also referred to as£-lipoproteins. Carlson and Carlson (1975) have classified VLDL with slow electrophoretic mobility as \"£-VLDL\" or \"slow pre-^ j\" VLDL. Secondly, VLDL exhibit an increased ratio of cholesterol, mainly cholesterol ester, to triglycerides (Sata et al. 1972), compared to normal VLDL, which are triglyceride-rich. Thirdly, the total amount of apo E in £-VLDL is absolutely increased (Havel et al. 1973, Havel 1980b) and the ratio of apolipoprotein C:apo B is decreased (Brown et al. 1983b, Havel et al. 1973), relative to normal VLDL. It has been shown that £-VLDL are composed of two distinct fractions (Fainaru et al. 1982). The first subfraction has been suggested to correspond to chylomicron remnants, as 10 evidenced by their supposed intestinal origin, large 700-800 angstrom diameter, and apo E, apo B-48 content. The second subfraction resembles VLDL cholesterol enriched remnants, identified by their supposed origin in the liver, smaller 400 angstrom diameter, and apo E, apo B-100 content. These defective metabolic consequences exhibited in individuals who carry the mutant£2 allele, have recently been confirmed by Weintraub et al. (1987) in a study of the effect of apo E polymorphism on dietary fat clearance. Apo E3/2 phenotypic individuals were documented to display, yet to a more moderate degree, the delayed chylomicron remnant clearance observed in E2/2 homozygotes. Havel et al. (1986) have also reported such moderate accumulations of$-VLDL in E3/2 heterozygotes. 2.5 METABOLIC CONSEQUENCES OF APO E2I2 PHENOTYPE - POSSIBLE MECHANISM Utermann (1986) has proposed a model for the mechanism of apo E2 homozygosity effects on plasma cholesterol. Direct uptake of chylomicron remnants and apo E-HDLc into hepatocytes, is mediated by high affinity binding of their surface apo E ligand primarily to remnant (apo E) receptors, and secondarily to LDL (apo B,E) receptors. Possession of the mutant £2 allele results in defective lipoprotein:lipoprotein receptor interaction and an increase in plasma chylomicron remnant, VLDL remnant and apo E-HDLc concentrations. As a result, the decreased uptake of cholesterol derived from the diet and peripheral tissues into the liver stimulates an up-regulation of hepatic LDL receptors. A corresponding decrease in the level of plasma cholesterol rich LDL particles, and therefore a decrease in plasma total cholesterol levels, results due to an increased LDL uptake proportional to the increased LDL receptor availability. A second component of this model, which may explain the reduced LDL levels and presence of VLDL observed in apo E2 individuals, is the inhibitory role of apo E2 in the normal conversion of VLDL remnants to LDL (Ehnholm et al. 1984, Chait et al. 1977). It has been suggested that normal apo E3 is required for this interconversion, while the variant E2 impedes this process. The resultant effect on plasma lipid levels of these altered lipoprotein metabolic pathways is to increase the amount of remnant lipoproteins rich in cholesterol and triglyceride and 11 decrease the concentration of cholesterol rich LDL in the plasma. This corresponds to net lowered plasma cholesterol concentrations and prolonged raised plasma triglycerides postprandially in apo E2/2 individuals (Davignon et al. 1988). 2.6 ASSOCIATION OF THE APO E2I2 PHENOTYPE WITH TYPE III HYPERLIPOPROTEINEMIA Paradoxically, of the 1-2% of the population homozygous for the £2 allele, approximately 2-10% (Rail et al. 1989) develop a relatively rare fasting hyperlipidemia termed type III hyperlipoproteinemia (HLP). Type III HLP, also known as broad-beta disease, differs from the primary dysbetalipoproteinemia exhibited in most E2/2 homozygotes, in that the characteristic E2/2 phenotype and evidence of fasting plasma B-VLDL are accompanied by a quantitative increase in plasma lipoprotein remnants with resultant hypercholesterolemia and hypertriglyceridemia, and the appearance of distinguishable clinical signs. 2.7 HISTORICAL BACKGROUND OF TYPE III HYPERLIPOPROTEINEMIA In 1952, Gofman et al. (1954) were the first to describe some of the clinical features of type III HLP, while in 1967, Fredrickson et al. instituted the nomenclature \"type III hyperlipoproteinemia\" to designate patients presenting with this disorder from the five other hyperlipoproteinemia classifications (Fredrickson et al. 1978). In these early clinical and biochemical studies, the diagnosis of type III HLP was based on two abnormal characteristics of VLDL from fasting blood: ^ -electrophoretic mobility, increased VLDL cholesterol:total triglyceride ratio greater than 0.30, and a measurement of 3.9-25.9 mmol/1 (150-1000 mg/dl) for plasma triglycerides (Zannis 1986). The significance of the regulatory involvement of apolipoprotein E was first evidenced in 1975, by Havel and Kane's observation that the VLDL of type III HLP patients contained a greater total amount of this arginine-rich protein than that occurring in normal oL-VLDL (Havel and Kane 1973). Utermann et al. in 1975, implementing the technique of 12 isoelectric focusing, demonstrated that type III HLP patients displayed the E2/2 phenotype (Utermann et al 1975). In most of the studies recorded to date, a strong association of the E2/2 phenotype with type III HLP has been suggested, as 90% of individuals with this disorder have been determined homozygous for the£2 allele (Breslow et al. 1982, Utermann 1987, Sutherland et al. 1988). The remaining 10% of individuals diagnosed with type III HLP, are not apo E2 homozygotes, but exhibit defective apo E:receptor binding, due to heterozygosity for the apo £2 allele, or possession of an unusual and functionally abnormal variant of apo E (Utermann 1986, Rail et al. 1982b). Individuals with the E3/2 (Smit et al. 1987), and E3/3 (Havel et al. 1983, Rail et al. 1989, Havekes et al. 1984, Havekes et al. 1986) phenotypes have been identified with this disorder. Type III HLP has also been associated with apo E deficiency (Schaefer et al. 1986, Mabuchi et al. 1989). 2.8 CLINICAL AND PATHOLOGIC FEATURES OF TYPE III HYPERLIPOPROTEINEMIA Characteristic features of type III HLP may be summarized as: hypercholesterolemia; hypertriglyceridemia, most commonly an E2/2 phenotype, presence of ^-VLDL, increased plasma apo E levels, and clinical findings including xanthomatosis and atherosclerosis (Mahley et al. 1984b). With respect to clinical biochemistry, typical plasma cholesterol levels observed in untreated type III HLP patients are 7.8 mmol/1 (300 mg/dl) and may even extend as high as 26 mmol/1 (1000 mg/dl), while triglyceride values typically range from 2.3 mmol/1 (200 mg/dl), up to 9.0 mmol/1 (800 mg/dl) (Mahley et al. 1984b, Innerarity et al. 1986). In addition to plasma chylomicrons, 0C.-VLDL, LDL and HDL normally present in type III HLP subjects, the most prevalent abnormal plasma lipoproteins in these individuals are £-VLDL particles (Brown et al. 1983b). These lipoproteins comprise the broad beta band observed on paper electrophoresis of whole plasma. Higher resolution techniques of starch block electrophoresis delineate both pre £-VLDL, or normal 0(:VLDL, and ^ -VLDL, in this fraction 13 (Innerarity et al. 1986). The j£-VLDL are composed of two heterogeneous subgroups as described above. LDL levels are usually decreased, while the concentration of HDL in the plasma may be at or below normal levels (Mahley et al. 1984b). The most prominent clinical manifestation of type III HLP is the development of xanthomas, characterized as slightly elevated, soft, rounded nodules formed by deposition of lipid in tissues. In a study of 115 patients, 50% exhibited these clinical features (Brewer et al. 1983). Xanthomata striata palmaris, lipid deposits in the creases of the palm, identified by an orange or yellow pigmentation, are the most common xanthomas, and are considered pathognomonic for type III HLP (Mahley et al. 1984b). Additional types of xanthomas observed include tuberoeruptive xanthomas, raised tuberous tissue on the elbows and knees, and xanthelasmas, slightly raised yellowish tumors occurring on the upper and lower eye lids, found in less than 25% of type III HLP patients. These pathologic tissue characteristics have been suggested to occur as a result of macrophage accumulation of lipids. This accumulation is thought to result from discharge of massive loads of cholesterol to various tissue macrophages by intestinal and hepatic ^-VLDL, resulting in induction of cholesterol ester synthesis and storage in these phagocytic cells (Fainaru et al. 1982). The foam cells of tuberous xanthomas are postulated to originate from such macrophages (Carlson et al. 1988, Assmann 1984). Combined data from four major studies (Brewer et al. 1983, Zannis 1986) indicated that premature coronary artery disease occurred in 33% of type III HLP patients, while peripheral vascular atherosclerosis was detectable in one third of patients. Coronary artery disease appeared earlier in men, on average in the late thirties, and in women, in the late forties. Age of onset of type III HLP has been documented to range from 16-95 years (Brown et al. 1983b), although cases of this disorder in adolescents have been reported (Mabuchi et al. 1989). Men tend to present at a mean age of 39, with women exhibiting onset of the disorder at a mean age of 49. Four major studies have demonstrated the occurrence of type III HLP in men as compared to women is 2:1 (Brewer et al. 1983). 14 2.9 GENETIC MODE OF INHERITANCE OF TYPE III HYPERLIPOPROTEINEMIA The mode of inheritance for the type III HLP disorder is a subject of debate. Confusion has existed as to the autosomal recessive or dominant genetic nature of the disease (Utermann et al. 1979b, Morganroth et al. 1975). The proposal that type III HLP in most cases is inherited as an autosomal recessive trait has been supported by observations that individuals with the E2/2 phenotype and type III HLP are born from parents who possess the £2 allele, but do not exhibit type III HLP. Also, children born from one parent with the E2/2 phenotype plus type III HLP, and one parent without the £2 allele, have been shown to lack the apo E2/2 phenotype and be free from the type III HLP disease (Breslow et al. 1986). Therefore, as type III HLP is most commonly associated with inheritance of two apo £2 alleles, it is typically thought of as a recessive trait. Observance of type III HLP in patients heterozygous for the normal apo E3 protein and an apo E variant protein defective in lipoprotein receptor binding, suggests a truly autosomal dominant mechanism of transmission in some individuals (Breslow et al. 1986, Havekes et al. 1986, Rail et al. 1989, Smit et al. 1987). 2.70 TREATMENT OF TYPE III HYPERLIPOPROTEINEMIA Of all the familial hyperlipoproteinemias, treatment of type III is the most successful. Successful treatment is determined by the reduction of plasma cholesterol and triglyceride levels to more normal concentrations (Brown et al. 1983b). Exacerbating secondary factors, such as hypothyroidism and obesity, must first be eliminated before effective therapy is begun. The hyperlipidemia is exceptionally responsive to diet control. Effective measures in the dietary management of this disorder include restriction of total caloric intake until ideal body weight is reached, as well as reduction of cholesterol, saturated fat and alcohol intake (Brown et al. 1983b, Mahley et al. 1984b, Brewer et al. 1983). In addition, drug therapy is often implemented in the treatment of type III HLP patients, to achieve and maintain normal plasma lipid levels. Although ji-VLDL will always be present in 15 individuals homozygous for the £2 allele, the exceptional effects of two common medications administered to these patients, clofibrate (2 g/day) and nicotinic acid (2-3 g/day), are demonstrated by regression of xanthomatous lesions after a few weeks (Mahley et al. 1984b, Brown et al. 1983b, Brewer et al. 1983) and the subjective inhibition of atherosclerotic progression (Fredrickson etal. 1978). 2.11 FACTORS MODULATING THE EFFECTS OF THE APO E2 DEFECT IN THE PHENOTYPIC EXPRESSION OF TYPE III HYPERLIPOPROTEINEMIA The apo E2:receptor binding abnormality is considered the principal defect in type III HLP (Rail et al. 1983a). However as all apo E2 individuals do not develop the gross hyperlipidemia and disease phenotypic ally expressed as type III HLP (Utermann 1987), additional factors must act to precipitate the pathogenesis of the disease. Recent studies by Rail et al. (1983a) have supported the concept for a multifactorial etiology of type III HLP. It was speculated that apo E2 from hypo- and normolipidemic individuals may not exhibit the defective receptor binding activity observed in E2/2 homozygotes with type III HLP. Rail demonstrated, however, a 158 cysteine residue and a functional abnormality in apolipoprotein E2 in all hypo-, normo- and hypercholesterolemic subjects. Apo E:LDL receptor binding in vitro was equally defective in all cases. Utermann et al. (1979b) have proposed a permissive role for the mutant apo £2 allele in individuals who develop type III HLP. The apo£2 allele, or apo E variant with similar defective receptor binding, while necessary, is not sufficient to cause this disease. Additional genetic or environmental factors must act to modulate this inborn metabolic error and precipitate the phenotypic expression of type III HLP (Utermann 1987). Several precipitating factors have been suggested to promote type III HLP, when superimposed most commonly on the £2/2 genotype (Davignon et al. 1988, Utermann et al. 1979b, Utermann 1987). Firstly, coincidental inheritance of a gene for hyperlipidemia, such as familial-combined hyperlipoproteinemia or familial hypertriglyceridemia, has been suggested to modulate the effect 16 of the apo E2 defect and result in the phenotypic expression of type III HLP. Although the molecular mechanism is poorly understood, inheritance of an additional defect in lipid metabolism may overload the already compromised degradative pathway of VLDL conversion to VLDL remnants and LDL (Utermann et al. 1979b), with resultant elevations in plasma lipid levels. Secondly, endogenous endocrine perturbations such as decreases in circulating estrogen levels with menopause or hypothyroidism, may augment the apo E2 metabolic abnormality and precipitate type III HLP disease (Davignon et al. 1988, Utermann et al. 1979b, Utermann 1987). Estrogens have been shown in animals to increase the affinity or number of hepatic LDL (apo B,E) receptors which bind apo E, therefore increasing VLDL remnant catabolism and lowering plasma lipid levels (Brewer et al. 1983, Stuyt et al. 1986). Hypothyroidism is often associated with type III HLP. The pathophysiological relationship of hypothyroidism to type III HLP may occur through the diminished receptor-mediated lipoprotein removal observed in the hypothyroid state (Mahley et al. 1984b, Thompson et al. 1981), as a result of decreased hepatic LDL (apo B,E) receptor activity (Mahley et al. 1985). Thirdly, elements such as obesity, another clinical characteristic often associated with type III HLP may unmask or exacerbate the effects of the apo E genetic disorder (Brewer et al. 1983), although the physiological association of obesity with the pathogenesis of type III HLP has yet to be determined. Fourthly, metabolic alterations occurring with age (Sutherland et al. 1988, Rail et al. 1983a, Mahley et al. 1984b, Mahley et al. 1985, Meddings et al. 1987) may be responsible for the manifestation of type III HLP. Hepatic LDL (apo B,E) receptor expression in animals has been shown to be age-dependent. Adult animals, in contrast to their young, express hepatic LDL receptors at very low levels (Mahley et al. 1984b). Although the biochemical basis for the age-related expression of type III HLP has not been definitely resolved, age dependent hepatic apo B,E receptor expression may be responsible for influencing ^ -VLDL concentration in type III HLP (Sutherland et al. 1988, Mahley et al. 1984b). 17 This multifactorial model for the phenotypic expression of type III HLP, relating the interaction of the primary molecular defect of apo E2 with secondary genetic and/or environmental triggering factor(s), has been summarized graphically by Davignon et al. (1988) (Figure 2). A final possible mechanism for the paradoxical effects of homozygosity for the£2 allele and the development of the clinical expression of type III HLP from primary dysbetalipoproteinemia, is excessive primary de novo synthesis of cholesterol by the liver (Mahley et al. 1984b, Innerarity et al. 1986, Rail et al. 1983a, Gregg etal. 1983). The basis for excessive primary de novo synthesis of cholesterol in type III HLP individuals may relate to the rate determining step in hepatic cholesterol biosynthesis, the conversion of HMG CoA to mevalonic acid. This step is catalyzed by the enzyme HMG CoA reductase. The activity of this enzyme, and therefore the rate of hepatic cholesterogenesis, may be increased in apo E2 type III HLPs as compared to apo E2 non-hyperlipidemic individuals. Additional possible explanations for increased rates of hepatic cholesterogenesis, however, include secondary causes such as obesity (Mahley et al. 1984b, Rail et al. 1983a). Several studies have demonstrated elevated hepatic cholesterol biosynthesis in obese humans (Angelin 1985), although mechanisms are unknown. Using a chromatographic sterol balance technique, Meittinen has demonstrated that increasing body weight is significantly correlated with increased cholesterol synthetic rate (Meittinen et al. 1971). 2.12 MEASUREMENT OF CHOLESTEROL SYNTHESIS Precision measurement of the rate of human cholesterol synthesis necessitates an accurate, quantitative, non-hazardous methodology. Three types of procedures for determination of lipid synthetic rates have been studied extensively in experimental animals. The first of these methods, the determination of HMG-CoA reductase activity, a well established indicator of cholesterol synthesis, has not been widely applied to human study, as fresh liver biopsy tissue is required (Sodhi et al. 1979) and, as a qualitative method, only relative rates of cholesterol synthesis are obtained (Dietschy et al. 1984). 18 Figure 2 Pathogenesis of type III HLP: interaction between genes, environment, and a specific apo E genotype (Davignon et al. 1988). 19 Secondly, measurement of the incorporation rate of -^C-labelled cholesterol precursors such as acetate (Turley et al. 1976), glucose or pyruvate (Dietschy et al. 1984), has been carried out in animal models. However, the utility of this technique is limited in humans, due to the radioactivity associated with this isotope of carbon and the potential underestimation of cholesterol synthetic rate measurements due to dilution of the acetyl CoA intracellular pool specific activity, through mixing of labelled precursor or the acetyl CoA produced from the precursor, with unlabeled substrates. Rates of incorporation of various l^C-labelled substrates therefore provide only relative, rather than absolute, rates of sterol synthesis. Thirdly, measurement of the incorporation of tritium atoms from tritiated water in the endogenous synthesis of cholesterol (Jeske et al. 1980) and fatty acids (Jungas 1968, Wadke et al. 1973) has been successfully implemented in animals to determine lipid biosynthesis, but the radiation hazard associated with this technique eliminates its wide use in studies using humans. Available methods to determine de novo cholesterol biosynthesis in humans include measurement of plasma and 24-h urinary mevalonic acid (MVA) concentration (Parker et al. 1984). Measurement of MVA formed from HMG-CoA in an irreversible reaction catalyzed by HMG-CoA reductase and NADPH, provides an accepted estimate of cholesterol synthesis, as it is generally concluded that most MVA produced is converted to cholesterol (Sodhi et al. 1979). Sterol balance techniques (Nestel et al. 1973, Bennion et al. 1975) have been used, based on the premise that fecal cholesterol and its metabolites indicate whole body net cholesterol synthesis when exogenous cholesterol intake is eliminated in the steady state, assuming the feces are the major route of elimination of endogenous cholesterol and its metabolites from the body. Cholesterol turnover and synthesis have also been measured by decay of plasma cholesterol specific activity after injection of ^C-labelled cholesterol, followed by compartmental analysis using three pool theoretical modelling (Goodman et al. 1980). Of the above methods available for measuring human cholesterol synthesis, the former procedure provides only a qualitative, indirect measure of cholesterol synthesis, while the two 20 latter techniques are laborious and lengthy, with the experimental period often spanning several months (Sodhi et al. 1979). 2.13 DEUTERIUM INCORPORATION METHODOLOGY Direct, short-term measurement of human plasma cholesterol synthesis has been accomplished by deuterium incorporation methodology (Jones et al. 1988, Jones et 1990). The technique involves oral administration of deuterium oxide, an isotope of water occurring naturally in the body. Deuterium oxide contains non-radioactive, deuterium or \"heavy hydrogen\" atoms, so named by virtue of a single neutron in the atomic nucleus, in place of isotopic protium or \"light hydrogen\" atoms, containing no neutrons in the atomic nucleus. The incorporation of deuterium atoms into endogenously synthesized cholesterol molecules in place of protium atoms, permits quantitative determination of cholesterol fractional synthetic rate (FSR) over short time periods, without radiation risk. Initial studies employing deuterium labelling for measuring human de novo cholesterol synthesis were carried out by Taylor et al. in 1966. Deuterium enrichment of body water was maintained at 5.0-6.0 g deuterium oxide/kg body water, above normal deuterium body water baseline levels which are 0.145 g deuterium oxide/kg body water. This high level of deuterium enrichment was required to adequately label de novo synthesized cholesterol because of the lack of precision of isotope ratio mass spectrometers then available. Approximately 40 days were required to achieve maximum deuterium oxide enrichment. Also, toxic side affects, such as severe dizziness, were observed in subjects given such large amounts of deuterium oxide (140-250 g). Recent methodological advances have increased the precision of protium and deuterium relative abundance measurements in plasma cholesterol samples analyzed by differential isotope ratio mass spectrometry. Assessment of cholesterol deuterium enrichment within 12 hours post oral dosage of deuterium, and reduction in the required level of deuterium enrichment of body water, which eliminates toxic side effects, are now possible (Jones et al. 1988, Jones et al. 1990, Schoeller et al. 1983). Labelling of the body water pool at 0.5 g deuterium oxide/kg body water 21 has been used successfully to detect cholesterol deuterium enrichment over a 12 hour period (Jones et al. 1988). 2.14 ASSUMPTIONS OF DEUTERIUM INCORPORATION METHODOLOGY Whole body cholesterol turnover has been characterized by a three pool mathematical model which describes the rates at which the three major groups of exchangeable cholesterol in the body equilibrate with plasma cholesterol (Goodman et al. 1973). This quantitative methodology involves periodic evaluation of plasma cholesterol specific activity (SA) over a period of several months following a labeled cholesterol injection. The decay curve of the radioactive cholesterol, obtained by plotting SA versus time, is analyzed by a multicompartmental system which suggests that the long-term disappearance of the tracer can best be explained by a three pool model. The three cholesterol pools do not describe physiological compartments within the body, rather they separate the exchangeable cholesterol within body tissues into three categories, based on how quickly the SA of the tissue cholesterol equilibrates with plasma cholesterol. The rapidly exchangeable central pool, pool 1 (Ml), which is estimated to be 24 grams in size in non-hyperlipidemic individuals (Goodman et al. 1980), consists mainly of plasma, liver, intestine, red blood cell, pancreas, spleen, kidney and lung cholesterol. Side pools 2 (M2) and 3 (M3) consist of tissue cholesterol which equilibrates at intermediate and slow rates with plasma cholesterol, respectively. Pool 2 is comprised of some visceral tissue cholesterol and a portion of peripheral tissue cholesterol. Pool 3 includes mostly peripheral tissue cholesterol such as skeletal muscle, adipose tissue, connective tissue and arteries (Goodman et al. 1980). The three pool cholesterol model assumes that losses of cholesterol from the body, by catabolism to bile acids or direct excretion through the feces, occur only via pool 1 (Goodman et al. 1973, Sodhi et al. 1979). It is also assumed that the introduction of new cholesterol occurs solely through pool 1, via absorption of dietary cholesterol or endogenous cholesterol synthesis (Dietschy et al. 1970, Goodman et al. 1973) and that exchange of cholesterol between pools 2 and 3 is 22 conducted only through pool 1 (Goodman et al. 1973). In the three pool model, all de novo cholesterol synthesis is therefore assumed to take place in the tissues of the M l central pool. The deuterium incorporation methodology states that cholesterol FSR may be validly determined through measurement of deuterium enrichment of plasma free cholesterol and plasma water contained within the M l pool, based on Goodman's three pool model and the following rationale. Upon administration of the priming bolus deuterium oxide dose, deuterium oxide equilibrates with the extracellular plasma water pool. After rapid migration across all cellular membranes, deuterium atoms of deuterium oxide equilibrate with the protium atoms of intracellular water pools, present within cholesterol synthetic tissues (Dietschy et al. 1984), mainly the liver and intestine of the M l pool, which synthesize approximately 90% of total body cholesterol (Sodhi et al. 1979). In subsequent cellular de novo cholesterol synthesis, constituent hydrogen atoms are drawn from the stable isotope labelled intracellular water pool, and deuterium isotopes, rather than protium isotopes, are permanently incorporated into the molecular structure. Unesterified, deuterium labelled, de novo synthesized cellular cholesterol molecules, and those incorporated into lipoproteins, exchange rapidly with other plasma lipoproteins, and quickly equilibrate with the extracellular plasma free cholesterol component of the M1 pool (Norum et al. 1983). Therefore, applying the three assumptions of Goodman's three-pool model (Goodman et al. 1973), with the additional assumption that movement of free cholesterol to and from the M2 and M3 side pools will be slow (Jones et al. 1988), due to the slow rates of equilibration of these pools with plasma cholesterol (Ml pool) (Goodman et al. 1973), the fraction of the total M l pool synthesized de novo in a day (cholesterol FSR), may be calculated from the free cholesterol and water deuterium enrichment of a sample of plasma from the M l pool. 2.15 SUMMARY At present, the factors which interact with the genetically abnormal E2 apolipoprotein to precipitate the clinical manifestations of type III HLP are speculative. It is suggested that environmental factors, such as an increase in the rate of de novo cholesterol synthesis, 23 superimposed on this genetic background, may be responsible. Recent methodological advances have made available the non-hazardous technique of deuterium incorporation for short-term measurement of plasma cholesterol synthetic rate. It is anticipated that analysis of cholesterol synthetic rate in both apo E2 type III H L P and non-hyperlipidemic individuals, and comparison of these values determined in both the fed and fasted state, will provide information contributing to elucidation of mechanisms responsible for elevated cholesterol levels observed in type III H L P . 24 3. METHODS 3.1 EXPERIMENTAL DESIGN 3.1.1 Phase I: Initial Subject Screening Two subject groups, test (n = 8) and control (n = 8), were identified. The test group consisted of hyperlipidemic individuals with the apo E2/2 or E3/2 phenotype, diagnosed with type III HLP (type III HLPs). The control group consisted of non-hyperlipidemic individuals with the apo E2/2 or E3/2 phenotype, without type III HLP disease and one non-hyperlipidemic type III HLP individual (controls). The type III HLP test subjects were recruited from the University Hospital, Shaughnessy Site Lipid Disorders Clinic. Over 300 patient charts were reviewed to identify potential test subjects. Contact with 53 selected patients was initiated by letter (Appendix One). Forty replies were returned by mail, and 30 interested respondents were invited by phone to a follow-up informational meeting. Eighteen individuals attended the meeting, and 8 of the most appropriate were selected for the experimental trial. The following selection criteria were used, based on information obtained from patient charts and a completed subject information sheet (Appendix Two): (i) male or female, (ii) 35-75 years of age, (iii) non-obese, defined as weight not greater than 20% above ideal weight (Burtis et al. 1988), (iv) free of restrictive food allergies or diet limitations, (v) easy access to UBC campus, (vi) free of chronic ailments, (vii) diagnosed with type III HLP, (viii) willingness to discontinue use of lipid lowering drug treatment or any other medication 4 weeks before and during the experimental trial (Grundy 1978, Carlson 1988, Kuo 1988). Few type III HLP subjects met all of the above criteria. Thus selection was modified to include subjects with the following less desirable characteristics: (i) obesity (NN, ES, MKu, DP), (ii) taking medication for high blood pressure (WJ, EK) and hypothyroidism (MKu) (maintained during the experimental trial) and (iii) heterozygous apo E2 phenotype (JB, AT, DP, EK). The normolipidemic control subjects were recruited in 4 ways. Firstly, one apo E2 homozygote previously identified at the Lipid Disorders Clinic with type III HLP, yet maintaining 25 normal lipid levels through diet control, was asked to participate. Secondly, one subject previously identified as an apo E2 homozygote in studies at the University of British Columbia, was contacted, rescreened, and asked to participate. Thirdly, six first degree relatives of type III HLP patients were contacted and four were screened. Fourthly, to match the controls with the type III HLPs for age and sex, advertisements for study volunteers were placed at local senior citizens' centers. Over 70 respondents were prescreened by phone, and 32 were selected for screening. Six more of the most appropriate subjects screened were selected as control subjects for the experimental trial, based on information obtained from laboratory results and a completed subject information sheet (Appendix Three), against the following criteria: (i) male or female, (ii) 35-75 years of age, (iii) homozygous for the apo E2 phenotype, (iv) fasting plasma total cholesterol levels between 2.80-6.35 mmol/1 (110-245 mg/dl) and triglyceride levels between 0.79-2.26 mmol/1 (70-200 mg/dl) (Hoeg et al. 1987, Brown et al. 1987), (v) no previous history of raised plasma cholesterol or triglyceride levels, (vi) non-obese, (vii) willingness to discontinue use of any medication 4 weeks before and during the experimental trial, (viii) free of reported chronic ailments, (ix) free of reported restrictive food allergies or diet limitations, and (x) easy access to the UBC campus. Due to the relatively rare occurrence of apo E2/2 homozygotes in the population (1-2%) (Brown et al. 1983b), apo E3/2 heterozygous individuals, which comprise approximately 10% of the population (Lenzen 1986) were necessarily included in the control group (JG, DD, EM, RS, HD, MW). Additionally, 2 subjects taking hypothyroid medication (JG, MW) (maintained during the experimental trial), and one obese subject (MW), were included, due to the difficulty of age-sex matching the elderly type III HLP subjects with elderly individuals who are free of all medications and at normal body weight. 3.1.2 Phase II: Experimental Trial Subjects were divided into groups of 3 or 4. Each group underwent a 3 day experimental trial. Two weeks before commencement of the trial, subjects met at the UBC Human Nutrition 26 Metabolic Laboratory, for orientation. At this time, height, weight and daily activity information was obtained, consent forms (Appendix Four) were signed, and a description of the research project protocol (Appendix Five) and food record instructions (Appendix Six) were given. Subjects recorded intake of all foods and beverages consumed for seven days preceding the trial. Subjects were instructed to determine portion sizes from household measuring devices such as measuring cups, spoons, and scales, as well as recording product package weights. Recipes were included when appropriate and available. During the trial, food records were reviewed with subjects, and any clarification needed regarding brands, types, portions, preparation methods or omissions of foods, was obtained. Three food record days (Thurs., Fri., and Sat.) (Stuff et al. 1983) were selected for determination of carbohydrate (CHO), protein, total fat, alcohol, energy, and polyunsaturated:saturated fat (P:S) intake, as an estimate of habitual dietary consumption. Computerized nutritional analysis was carried out using the PC Nutricom V2.0 program (1986, Smart Engineering Ltd., Vancouver, B.C.). Foods were entered into the program using Canadian Nutrient File food descriptions and Nutrition Canada food codes (Canadian Nutrient File, Food Name Subfile, 1986, Department of National Health and Welfare). Data for cholesterol, polyunsaturated fatty acids (PUFA) and saturated fatty acids (SFA) not available in Nutricom for some foods entered, were obtained from food composition tables (Pennington et al. 1985, Nutrient Value of Some Common Foods, 1987). The diet fed to subjects during the experimental trial was intended to approximate the average North American consumption of 40%, 45% and 15% total energy intake from fat, CHO and protein respectively, 200 mg cholesterol/1000 kcal, and a P:S ratio of 0.4 (US Dept of Health and Human Services, 1986). A standard 3000 kcal diet consisting of isocaloric breakfast, lunch and dinner meals, previously designed by a research dietitian using data from the Canadian nutrient file (Verdier et al. 1984), was modified slightly in the present study (Appendix Seven) for practicality of packaging meals for take-out eating. The modified trial diet consisted of 40% fat, 45% CHO, 15% protein, 220 mg cholesterol/1000 kcal and a P:S ratio of 0.7, as computed on PC 27 Nutricom. The caloric value of the diet was adjusted to meet the estimated energy requirements determined for each subject, by multiplication of each food item by the appropriate conversion factor (estimated energy need (kcal)/30Q0 kcal). Subjects' daily caloric requirements were estimated (Appendix Eight) with the aid of the Mayo Clinic Nomogram (Mayo Clinic Diet Manual, 1961). Body surface area (m2) was first determined from the nomogram using ideal weight and height. The nomogram was then used to obtain basal metabolic rate (BMR) (kcal/24 hrs) from body surface area, age and sex. Individual daily energy requirements (kcal/24 hrs) were then calculated by multiplying BMR by an activity factor of 1.5 or 1.7, depending on subject reported activity (Bogert et al. 1973, Bell et al. 1985). Non-perishable menu items were bought in bulk and frozen or stored, while perishable items were purchased from a local grocer before each experimental trial. Careful weighing of food items, meal preparation and cooking of meals was carried out before each trial in the UBC Human Nutrition Metabolic Kitchen. Breakfast and dinner meals were served in the Metabolic Kitchen, while the lunch meal was packaged for take-out. On day 0, the day before the experimental trial began, subjects were asked to fast after consuming the evening meal. At 7:00 am on day 1 of the trial, subjects reported to the Division of Human Nutrition Metabolic Laboratory, UBC, when the first 28 ml blood sample was drawn for determination of baseline plasma water and cholesterol deuterium enrichment. All blood samples taken during the experimental trial were drawn by a registered laboratory technician. At 7:30 am, a \"priming\" bolus dose of deuterium oxide (99.9 atom % excess deuterium (A%E)), MSD Isotopes, Montreal, Canada and Cambridge Isotope Labs, Woburn, Mass.) was administered orally to each subject at a level of approximately 0.7 g D20/kg estimated total body water (Appendix Nine), followed by 50 ml distilled H2O as a rinse. Individual total body water determinations were calculated based on a body water content estimation of 60% of body weight (Gamble et al. 1954). At 8:00 am, (hr 0 post bolus D2O dose), timing of four 12 hr intervals began (12, 24, 36, and 48 hr post bolus D2O dose). A 28 ml blood sample was drawn at each interval. Drinking water labelled at approximately 1.4 and 0.7 g D2OA H2O was given on day 1 and day 2, 28 respectively, to maintain deuterium body water enrichment (Appendix Nine). The amount of deuterium label in the drinking water on the feeding day was double that on the fasting day to compensate for dilution of deuterium body water enrichment by the unlabelled water consumed in the experimental diet. At hr 48, additional 28 ml blood samples were drawn from the type III HLPs for apo E re-phenotyping. This was done to validate the phenotype determined previously for these individuals by the University Hospital, Shaughnessy Site Lipid Clinic, using the same method. Additional 28 ml blood samples were also drawn from 6 subjects for thyroxine (T4) and thyroid stimulating hormone (TSH) assays, in order to determine thyroid status at the time of the experimental trial. Thyroid status tests were not done on all subjects as 3 experimental trials had been completed before the decision to acquire this data was made. Throughout the 3 day trial, subjects consumed only the test meals provided, and were weighed daily. At 8:00 am on day 1 (feeding day) subjects were fed the experimental trial breakfast meal. Subjects were given the packaged lunch meal and permitted to leave the Metabolic Laboratory until 7:00 pm, at which time they returned and consumed the dinner meal. Subjects were instructed to eat all of the food provided at each meal. Subjects reported to the Metabolic Laboratory at 8:00 am on day 2 (fasting day), and remained there under supervision, involved in various quiet activities, until 8:00 pm. Subjects consumed no food on day 2, although calorie free, caffeine free soft drinks were permitted. On the final day of the study, day 3, subjects returned to the metabolic unit at 8:00 am after 36 hours of fasting. 3.2 ANALYTICAL PROCEDURES 3.2.1 Phase I: Initial Subject Screening A single 12 hr fasting 28 ml blood sample was drawn from each subject screened into test tubes containing EDTA (Becton Dickinson, Miss., Ont.) by a registered laboratory technician at the Department of Laboratory Medicine, University Hospital, UBC campus. Blood samples were 29 stored for less than 4 hrs at 4 °C before plasma was obtained by centrifugation for 15 min at 3000 rpm. Plasma samples were stored at -20 °C. Total cholesterol and triglyceride level analyses were carried out for each subject at the University Hospital, Shaughnessy Site Lipid Clinic. All analyses were performed on the automated Technicon RA-500 (Technicon Instruments, Corp., Tarrytown, NY). Plasma samples (300-400jd) were assayed for single determination of total cholesterol and triglyceride concentration. Samples were assayed in batches of approximately 30 with two controls (Technicon Diagnostics Testpoint Assayed Chemistry Controls 1 and 2, Technicon Instruments Corp., Tarrytown, NY) run at the beginning and end of each batch assay. Within-run variability reported for cholesterol assays performed on this machine approximates 1.6%. An enzymatic colorimetric method (Cholesterol C-system, Boehringer Mannheim, Montreal, Quebec) was used for the determination of total plasma cholesterol. Free cholesterol was liberated from cholesterol esters by cholesterol esterase. Delta-4-cholestenone and hydrogen peroxide was then produced from the oxidation of free cholesterol by cholesterol oxidase. Hydrogen peroxide, 4-aminophenazone and phenol, in the presence of peroxidase, combined to form a chromagen, 4-phenazone, which absorbed maximally at 500 nm. The concentration of total cholesterol in the sample was directly proportional to the intensity of the color produced (Katterman et al. 1984). Triglycerides were assayed using the Technicon RA-500 systems triglycerides enzymatic method. Plasma triglycerides were hydrolyzed to glycerol and free fatty acids by LPL. Glycerol-3-phosphate was formed by glycerol kinase and ATP. Hydrogen peroxide was then produced by reaction with glycerol phosphate-oxidase. The chromagen produced by the reaction of hydrogen peroxide with 4-chlorophenol and 4-aminoantipyrine in the presence of peroxidase, had maximal absorbance at 500 nm. The triglyceride concentration of the sample was directly proportional to the intensity of the color produced (Fossati et al. 1982). The apo E phenotype of each subject screened was determined by isoelectric focusing on a single polyacrylamide cylindrical gels, according to the methods of Bouthillier et al. (1983) and 30 Warnick et al. (1979). Density gradient preparative ultracentrifugation of 10-12 ml plasma was used to separate out the VLDL fraction in each sample. Duplicate samples for each subject of 5-6 ml plasma were injected below 3.0 ml of 0.15 mole/1 NaCl into a 10 ml ultracentrifuge tube, using a 21 gauge 1 1/2\" needle. The tube was filled to a final volume of 9.0 ml by carefully layering saline on top. VLDL was isolated by ultracentrifugation in a Beckman L3-50 ultracentrifuge with type 50 rotor (Beckman Instruments, Palo Alto, CA) at 16 °C for 18-20 hrs at 40,000 rpm. The duplicate VLDL samples for each subject were removed by pipetting off the top milky layer and pooled for a second wash in a third ultracentrifuge tube, with final volume adjusted to 9.0 ml using 0.15 M/l saline. The VLDL was then recentrifuged under the same conditions. The washed VLDL was recovered by pipetting off the top 3 ml into a 12 ml culture tube and freezing at -20 °C until delipidation. Delipidation of VLDL was carried out by drop wise transfer of VLDL into 10 ml of acetone:ethanol (1:1 v/v). The mixture was stored for 4 hrs or overnight at -20 °C, after which the protein was sedimented by centrifugation for 15 min at 3000 rpm at 4 °C. The solvent was discarded, the protein pellet resuspended in another 10 ml of ethanohacetone by thorough vortexing, and the mixture was stored for 2 hrs at -20 °C. After centrifugation at 3000 rpm for 15 min, the solvent was discarded and the protein pellet resuspended in 5 ml of cold diethyl ether by thorough vortexing and stored for 1 hr at -20 °C. The solvent was discarded after a final centrifugation at 3000 rpm for 15 min and the protein dried under nitrogen gas (N2) before storage at -20 °C. Single polyacrylamide gel isoelectric focusing was carried out at the University Hospital, Shaughnessy Site Lipid Clinic. Polyacrylamide gel solution for 10 samples, including one previously phenotyped control sample, was prepared for each focusing experiment by combining 1.50 g acrylamide (electrophoresis purity, BDH, Toronto, Ont.), 40 mg BIS, 9.6 g urea, and 1.0 ml carrier ampholytes, pH 4-6 (LKB Bromma, Fisher Scientific, Toronto, Ont.) made up to a final volume of 20 ml with distilled water in a graduated cylinder. The polymerization reaction was activated by the addition of 10/A TEMED and 40/A ammonium persulfate. The gel solution was quickly pipetted into gel tubes sealed with parafilm at one end, to a level of 8.0 cm. To form a 31 smooth interface, the gel solution was overlaid with 1 drop of distilled H2O and left to polymerize for 1/2 hr. 200jul of a solubilization buffer consisting of 2.4 g urea, 16 mg DTT and 5.0 ml of 10 mM Tris-HCl was added to each dried protein sample, vortexed thoroughly and stored at 4 °C for 1.5 hrs. Meanwhile the polymerized gel tubes were loaded into the electrophoresis chamber (Model 150 A, Bio-Rad Laboratories) containing a 10 mM orthophosphoric acid buffer in the lower anode reservoir and 20 mM NaOH buffer in the upper cathode reservoir. After 1 hr pre-focusing at 110 V, the upper reservoir buffer was discarded, the protein samples were loaded carefully into the tube gels and overlaid with 200 fA of sample overlay buffer containing 1.0 ml solubilization buffer, 1.0 ml distilled H2O and 20/ please give details. Do you have any food allergies? Do you have any food restrictions &/or strong food preferences? Do you take any medication on a regular basis? No, I am not interested in participating in the 3 day research project. ALL INFORMATION WILL BE KEPT STRICTLY CONFIDENTIAL Yes, I would like to participate in the 3 day research project. Month(s) available Days available 83 APPENDIX THREE SUBJECT INFORMATION SHEET FOR INDIVIDUALS SCREENED FOR CONTROL GROUP CHOLESTEROL SYNTHESIS RESEARCH PROJECT SUBJECT INFORMATION SHEET Name Age Address Phone (home) (work) Occupation/Place of Employment^ Social Insurance # Ethnic Background Height Weight Please answer the following questions. If your answer to any question is \"yes\", please give details. Do you have any food allergies? Do you have any food restrictions &/or strong food preferences? Do you take any medication on a regular basis? Have you ever been diagnosed as having a problem with you lipid (fat) metabolism? (e.g. high cholesterol or high fat levels? Availability: Month(s) Days 84 APPENDIX FOUR CONSENT BY SUBJECT OF RESEARCH PROTOCOL CHOLESTEROL SYNTHESIS RESEARCH PROJECT CONSENT FORM Protocol #: Subject Name: Research Protocol: Cholesterol Synthesis in Type III Hyperlipoproteinemic and Non-Hyperlipidemic Individuals. Research Directors: Shani Dendy Dr. Peter Jones Dr. Jiri Frohlich 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. Nature and Duration of Procedure: The objective of the study is to compare the synthesis of cholesterol in individuals with normal lipid levels and individuals with type III hyperlipoproteinemia. This study involves your eating a prepared diet for 1 out of 3 days. This diet will be fed at a level which should maintain your normal body weight. On the second day you will fast for the entire day. You will be given drinking water containing a stable tracer. You will be required to remain in the testing facility from 8 am to 8 pm on day 2. Blood samples of 28 ml will be collected on 5 occasions during the three day study. II. Potential Risks and/or Benefits: There is no known hazard associated with the use of the stable labelled tags in the procedure. There are no risks of the procedure other than that normally associated with blood-taking. 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 the results to be obtained. Confidentiality of the records concerning my involvement in this project will be maintained in an appropriate manner. I understand that I will receive $100.00 upon completion of the study. If I decide to withdraw before completion of the study, I will receive an appropriate prorated fraction of this amount. I acknowledge receiving a copy of this consent form and all appropriate attachments. Doctor: Witness: Date: Time am/pm Signature of Subject If relative or legal representative signs, please indicate relationship or other authority. 85 APPENDIX FIVE DESCRIPTION OF RESEARCH PROJECT PROTOCOL CHOLESTEROL SYNTHESIS RESEARCH PROJECT DESCRIPTION OF RESEARCH PROJECT PROTOCOL Research Directors: Shani Dendy, Dr. Peter Jones, Dr. Jiri Frohlich Division of Human Nutrition / University Hospital, Shaughnessy Site Lipid Clinic The University of British Columbia THE RESEARCH PROJECT WILL CONSIST OF TWO PHASES: PHASE I: INITIAL SUBJECT SCREENING (Fall, 1988) - To identify appropriate test and control subjects for the experimental trial - ALL volunteers will complete a Subject Information Sheet - SOME volunteers will donate a small sample (28 ml = 2 tbsp) of blood - NOT ALL volunteers screened will be asked to participate in the experimental trial PHASE II: EXPERIMENTAL TRIAL (Fall, 1988 and Spring, 1989) The experimental trial will take place over a period of 3 consecutive days, at the University of British Columbia (UBC) Metabolic Laboratory/Kitchen, most likely on a Friday, Saturday and Sunday. A. TWO WEEKS BEFORE THE EXPERIMENTAL TRIAL: 1. Subjects will meet at the UBC Metabolic Laboratory/Kitchen for an orientation meeting - height, weight, and daily activity information will be obtained - food record instructions will be given B. DURING THE EXPERIMENTAL TRIAL: 1. Subjects will consume only the test meals provided by the Research Directors - Day 0 (day before study begins): subjects will fast after consuming the evening meal - Day 1: 3 complete meals will be provided - Day 2: FAST from evening meal on Day 1 until the breakfast meal on Day 3 - Day 3: breakfast snack provided 2. Subjects will be given a priming bolus dose of deuterium oxide (D2O) on Day 1 and drinking water that contains deuterium on Days 1 and 2 3. Subjects will donate 5 small samples of blood (28 ml each = 2 tbsp) - Day 1: Blood Sample #1 = before consuming deuterium oxide (D2O) bolus dose Blood Sample #2 = HOUR 12 post D 2 0 bolus dose - Day 2: Blood Sample #3 = HOUR 24 post D 2 0 bolus dose Blood Sample #4 = HOUR 36 post D 2 0 bolus dose - Day 3: Blood Sample #5 = HOUR 48 post D 2 0 bolus dose 4. Subjects will report to the UBC Metabolic Laboratory/Kitchen at the following times: - Day 1: 7:00 am-8:30 am and 7:00 pm-8:30 pm - Day 2: 8:00 am-9:00 pm (all day) - Day 3: 8:00 am-9:00 am 86 APPENDIX SIX FOOD RECORD INSTRUCTIONS CHOLESTEROL SYNTHESIS RESEARCH PROJECT FOOD RECORD INSTRUCTIONS PLEASE READ CAREFULLY: 1. Write down EVERYTHING you eat and drink each day. Be sure to include all SNACKS and ALCOHOL. Record immediately after each meal and snack to ensure accuracy. 2. Write down HOW MUCH you eat and drink each day: A. Use VOLUME measures (cup, tbsp, tsp, or ml) for cereals, cooked rice and pasta, vegetables, canned fruit, peanut butter, mayonnaise, salad dressing, butter, margarine, sauces, gravies, soups, sugar, jam, beverages, etc. B. Use COOKED WEIGHTS (ounces or grams) for meat, fish, and poultry. Note: the weight of meat, poultry and fish decreases by about 25% during cooking. Examples: 4 oz. raw beef shrinks to 3 oz. cooked beef 6 oz. raw cod shrinks to 4.5 oz. cooked cod C. Use SIZE for raw fruits, muffins, crackers, cakes, pies, cookies, desserts, etc. Give dimensions. Example: 1 oatmeal cookie, 1\" in diameter D. Be specific about the TYPE OF FOOD, BRAND NAME IF APPLICABLE, HOW THE FOOD WAS PREPARED, AND CONTENT OF MIXED DISHES. E. For combination items, list each item separately. Example: a cheese cheeseburger would be described as: bun, cooked ground beef, processed cheese, butter, relish, etc. F. IF THE FOOD IS PREPARED BY SOMEONE OTHER THAN YOURSELF, please try to estimate the portion size and describe the contents of the dish that is served to you. G. Don't forget the EXTRAS! Examples: sugar on cereal or in coffee dressing on salad candy, soft drinks, alcohol EXAMPLE: TIME FOOD ITEM DESCRIPTION AMOUNT LOCATION Breakfast 2% milk 1/2 cup Smitty's Restaurant whole wheat bread 2 slices margarine 2 tsp strawberry jam 2 tsp omelette: eggs 2 large cheese 1/2 oz. orange juice 6 oz 87 APPENDIX SEVEN DIET FED TO CONTROL AND TYPE III HLP SUBJECTS ON FEEDING DAY OF EXPERIMENTAL TRIAL Meal Food Item Quantity Breakfast: whole wheat bread 60 g 2% milk 260 ml soft sunflower margarine 15 g Kellogg's bran flakes 55 g seedless raisins 25 g apple juice 145 ml omelette: brick cheese 35 g egg whites 1.5 whites egg yolk 0.5 yolk butter 6.5 g soft sunflower margarine 4.5 g Lunch: whole wheat bread 50 g soft sunflower margarine 10 g dip: 1.55% yogurt 25 g Miracle Whip 15 g macaroni and cheese: cooked macaroni 160 g evaporated 2% milk 100 ml cheddar cheese 35 g soft sunflower margarine 13 g egg 0.5 egg chopped onion 60 g carrot sticks 60 g celery sticks 60 g fresh pear 1 6.4 cm x 8.9 cm Dinner: whole wheat bread 30 g soft sunflower margarine 10 g cooked chicken thigh (no skin) 150 g hollandaise sauce: egg yolk 0.38 yolk lemon juice 3.75 ml butter 10 g cooked rice 150 g frozen peas 75 g frozen carrots 75 g vanilla ice milk 75 g Hershey's chocolate syrup 35 g Cool Whip 15 ml . *diet contains 3000 kcal and food item quantities were modified to meet the estimated daily caloric requirements for each subject. 88 APPENDIX EIGHT ESTIMATED DAILY CALORIC REQUIREMENTS OF CONTROL AND TYPE III HLP SUBJECTS ADMINISTERED ON FEEDING DAY OF EXPERIMENTAL TRIAL Subject by Body Surface Group Area 1 Basal Metabolic Rate2 Reported Activity Activity Factor^ (meters )^ 1.90 1.84 1.91 1.84 1.86 1.99 1.66 1.64 Daily Caloric Requirement4 Controls: CD MKa JG DD EM RS HD MW Mean ( + SEM) Type III HLPs: NN WJ ES JB AT MKu DP EK Mean (±SEM) 1.85 2.25 1.77 1.88 1.90 1.64 1.69 1.48 (kcal/24 hrs) 1730 1670 1660 1596 1620 1730 1370 1343 1675 1800 1540 1630 1645 1320 1360 1210 -run, 3 miles,5x/wk 1.7 -weights, light, 3x/wk -hockey, .5hr, 2x/wk -WORKING -bike, walk, 4x/wk 1.7 -WORKING -swim, 15 min, 2x/wk 1.5 -bike, 10 min, 2x/wk -RETIRED -YMCA aerobics, 1.7 45 min,5x/wk -WORKING -walk, periodically 1.5 -SEMI-RETIRED -swim, 1 hr, 4x/wk 1.5 -walk, 4 km/wk -RETIRED -walk, 1 mile, 2x/wk 1.5 -WORKING -walk, 10 blks/day 1.5 -RETIRED -bike, 45 min/day 1.7 -sit-ups, push-ups/day -WORKING -bike, lOOkm/wk -row, 15 min/day -weights, sit-ups, 10 min/day -WORKING -RETIRED -WORKING -walk, 1.5 hrs, 2x/wk -sit-ups, 500/day -RETIRED -bike, 10 min/day -walk, lhr/day -swim, .5 hr/day -RETIRED -walk, 3 miles/day -WORKING -walk, 2 miles/wk -RETIRED 1.7 1.5 1.5 1.5 1.5 1.5 1.5 (kcal/24 hrs) 2914 2839 2490 2713 2430 2595 2055 2015 2506.4 (117.9) 2847 3060 2310 2445 2468 1980 2040 1815 2370.6 (151.9)5 89 ^obtained from Mayo Clinic Nomogram using ideal weight and height; 2obtained from Mayo Clinic Nomogram using surface area, age and sex; ^determined from references Bogert et al. 1973 and Bell et al. 1985; 4basal metabolic rate x activity factor;^p = 0.49 90 APPENDIX NINE ESTIMATED TOTAL BODY WATER CONTENT OF CONTROL AND TYPE III HLP SUBJECTS AND CORRESPONDING DEUTERIUM OXIDE BOLUS DOSE AND DEUTERIUM LABELLED DRINKING WATER ADMINISTERED DURING EXPERIMENTAL TRIAL Total D 2 0 Deuterium Labelled Subject by Body Bolus Dose3 Drinking Water Group Weight1 Water2 Theor. Exp. Fed Fasted (kg) (kg) (g) ( g D 2 0 / l H 2 0 ) Controls: CD 83.2 49.9 34.9 34.96332 1.4 0.7 MKa 79.5 47.7 . 33.4 33.43536 1.4 0.7 JG 78.3 47.0 32.9 32.92700 1.4 0.7 DD 79.6 47.8 33.4 33.43000 1.4 0.7 EM 75.5 45.3 31.7 31.72262 1.4 0.7 RS 74.9 44.9 31.5 31.51710 1.4 0.7 HD 57.7 34.6 24.2 24.23900 1.4 0.7 MW 77.6 46.6 32.6 32.63749 1.4 0.7 Type III HLPs: NN 84.2 50.5 35.4 35.41171 1.4 0.7 WJ 91.3 54.8 38.3 38.32930 1.4 0.7 ES 86.0 51.6 31.0 31.01200 1.23 0.63 JB 84.2 50.5 35.4 35.40389 1.4 0.7 AT 84.7 50.8 35.6 35.60550 1.4 0.7 MKu 71.3 42.8 29.9 29.91295 1.4 0.7 DP 109.3 65.6 39.4 39.43600 1.2 0.6 EK 54.9 32.9 23.0 23.02661 1.4 0.7 -^ obtained at orientation meeting prior to experimental trial; 260% of body weight; 30.7 g D20/kg total body water, however 0.6 g D20/kg total body water given when deuterium supply limited; 3due to limited deuterium supply 91 APPENDIX TEN RESULTS OF APO E PHENOTYPE AND PLASMA LIPID CONCENTRATION ANALYSES IN SUBJECTS SCREENED FOR CONTROL GROUP Subject by Phenotype Total Cholesterol Triglyceride E3:2 Ratio Age Sex (mmol/1) (mmol/1) (years) E2/2 CD 4.12 1.19 35 M E3/2 RD SO KJ MB BT LE AD ML DT BS HD GB MW ED AM JG DD EM JO-R JJ JC RS M-LS 4.09 4.11 6.25 5.20 5.35 6.15 6.09 6.38 6.01 6.35 5.19 4.76 7.46 5.75 4.89 5.66 6.07 4.52 5.80 4.99 4.87 8.36 0.82 0.82 2.52 2.54 1.29 1.12 1.54 1.95 0.94 1.11 .53 .25 .68 .81 .04 .08 .38 1.23 2.56 1.60 2.11 3.34 1. 2. 1. 1. 1. 1. 1. 0.99 0.97 1.14 0.97 0.86 0.98 0.94 1.11 0.70 1.18 0.97 1.06 0.82 1.11 1.11 0.91 0.83 0.97 0.91 0.79 1.08 1.03 0.84 28 26 38 45 38 63 57 56 28 42 52 68 73 66 69 68 53 62 61 68 71 67 60 M F F M M M F F F M F M F F F M M M F M M M F E3/3 WW AT RR 4.27 5.28 5.14 0.55 1.03 1.67 1.49 1.26 1.22 37 32 62 F M M E4/3 MS EE WM DS MC TF MT DR BR 3.43 5.41 4.60 4.49 4.70 5.88 5.82 6.59 4.96 0.85 1.33 0.82 1.17 4.56 1.77 1.99 1.33 1.38 40 42 40 36 53 40 61 60 66 F F M M F M F F F 92 APPENDIX ELEVEN BODY WEIGHT FLUCTUATIONS OF CONTROL AND TYPE III HLP SUBJECTS THROUGHOUT EXPERIMENTAL TRIAL Subject by Group Day 1 (Feeding) Day 2 (Fasting) Day 3 (Fasting) Feeding Weight Change 1 CV Total 2 CV (kg) (%) (%) Controls: CD 81.5 82.5 81.0 1.0 0.86 -0.5 0.94 MKa 81.6 81.8 80.8 0.2 0.17 -0.8 0.65 JG 77.4 76.8 75.8 -0.6 0.55 -1.6 1.05 DD 80.1 80.0 78.9 -0.1 0.09 -1.2 0.84 E M 74.8 74.1 73.4 -0.7 0.67 -1.4 0.95 RS 73.8 74.5 72.3 0.7 0.67 -1.5 1.53 HD 59.0 58.5 58.0 -0.5 0.60 -1.0 0.86 MW 77.3 77.2 75.8 -0.1 0.09 -1.5 1.09 Mean 75.7 75.7 74.5 0.0 0.46 -1.2 0.99 ±SEM 2.6 2.7 2.6 0.2 0.11 0.1 0.09 Type III HLPs: NN 82.6 82.6 81.3 0.0 0.00 -1.3 0.91 WJ 92.5 90.1 89.6 -2.4 1.86 -2.9 1.71 ES 86.0 86.4 84.9 0.4 0.33 -1.1 0.91 JB 83.3 82.6 81.3 -0.7 0.60 -2.0 1.23 AT 85.1 - 83.5 - - -1.6 1.34 MKu 68.4 68.3 66.4 -0.1 0.10 -2.0 1.67 DP 108.3 108.0 106.0 -0.3 0.20 -2.3 1.16 EK 54.6 54.1 53.1 -0.5 0.65 -1.5 1.42 Mean 82.6 81.7 80.8 -0.5 0.53 -1.8 1.29 + SEM 5.6 6.4 5.5 0.3 0.24 0.2 0.11 p = 0.28 p = 0.38 p = 0.32 p = 0.23 p = 0.78 p = 0.02p = 0.05 *Day 2-Day 1; 2Day 3-Day 1 93 APPENDIX TWELVE PLASMA WATER DEUTERIUM ENRICHMENT IN BLOOD SAMPLE DRAWN FROM CONTROL AND TYPE III HLP SUBJECTS DURING EXPERIMENTAL TRIAL Plasma water 2 H / 1 H relative to SMOW 1 Subject by Group Baseline2 24 hr^ corrected 24 hr 4 (parts per thousand [%o]) Controls: CD -100 700.2 (7.0) 4775.9 MKa -100 647.5 (5.1) 4460.0 JG -100 680.6 (0.2) 4658.3 DD -100 542.3 3828.8 EM -100 595.2 (6.2) 4145.9 RS -100 562.7 (3.6) 3950.9 HD -100 649.1 4469.6 MW -100 820.5 5498.0 Type III HLPs: NN -100 662.9 (0.1) 4552.1 WJ -100 686.6 (9.3) 4694.6 ES -100 680.6 (2.5) 4658.3 JB -100 702.7 (8.1) 4791.2 AT -100 675.4 (0.7) 4627.4 MKu -100 770.5 (7.2) 5198.0 DP -100 777.6 (47.9) 5240.6 EK -100 749.5 (20.4) 5071.7 •'•Standard Mean Ocean Water; 2theoretical value based on Vancouver tap water deuterium enrichment; ^mean (SD) of duplicate samples except when single sample only available; \"^ correction for dilution of plasma with Vancouver tap water, and deuterium enrichment of the diluent (sample calculation below) Corrected plasma water deuterium enrichment sample calculation: Subject NN: -plasma dilution: 1 part plasma: 5 parts Vancouver tap water -plasma deuterium enrichment = 662.9 %o -Vancouver tap water deuterium enrichment = -100 %o -baseline plasma deuterium enrichment = -100 %o -to equate H20 content of plasma (95% H20, 5% solids) and diluent (100% water, 0% solids), 0.95 correction factor applied to diluent = (undiluted plasma °/oo) - (Vancouver tap water %o) - (baseline plasma %o) = (662.9 %o x 6) - ([-100 %o x 5] x 0.95) - (-100 %o) = 4552.1 94 APPENDIX THIRTEEN PLASMA FREE CHOLESTEROL DEUTERIUM ENRICHMENT IN BLOOD SAMPLES DRAWN FROM CONTROL AND TYPE III HLP SUBJECTS DURING EXPERIMENTAL TRIAL Plasma free cholesterol 2 H / 1 H relative to SMOW 1 Subject by Group 0 hr 2 24 hr 48 hr (parts per thousand [%o]) Controls: CD -290.6 (4.7) -174.4 (10.5) -194.6 (1.0) MKa -283.2 (2.4) -133.5 (1.2) -165.9 (1.9) JG -293.0 (5.9) -142.9 (1.1) -95.9 (0.1) DD -297.0 (1.1) -170.6 (8.3) -173.4 (6.6) EM -304.8 (5.0) -123.5 (2.6) -173.2 (0.2) RS -280.0 (16.4) -118.5 (2.4) -124.0 (3.3) HD -306.0 (1.8) -137.2 (0.3) -149.6 (0.6) MW -247.4 (87.4) -20.0 (28.7) 42.6 (2.5) rype III HLPs: NN -292.4 (26.4) -200.2 (2.9) -201.43 WJ -282.0 (28.9) -140.5 (0.1) -157.3 (1.9) ES -300.9 (2.9) -190.2 (1.5) -182.9 (3.2) JB -290.7 (14.1) -176.8 (2.2) -150.9 (2.3) AT -303.2 -155.8 (2.5) -175.1 (3.0) MKu -295.6 (3.4) -235.1 (2.5) -227.7.(9.8) DP -113.1 (4.9) -19.9 (9.3) 32.5 (28.0) EK -292.1 (15.5) 2.1 (28.5) 37.9 (2.2) •'•Standard Mean Ocean Water; 2numbers are mean (SD) of triplicate samples; ^duplicate samples identical Cholesterol FSR sample calculation: Subject NN, 0-24 hr interval: -cholesterol deuterium enrichment at hour 0= -292.4 %o -cholesterol deuterium enrichment at hour 24 = -200.2 °/oo -plasma water deuterium enrichment= 4552.1 °/oo [-200.2 %o - (-292.4)] FSR (per day) = 4552.1 %o x 0.81 2 H / C x 27C/46H = 0.042 per day 95 APPENDIX FOURTEEN PLASMA FREE CHOLESTEROL DEUTERIUM ENRICHMENT BLOOD SAMPLES DRAWN FROM CONTROL AND TYPE III HLP SUBJECTS AT 12 HOUR INTERVALS DURING EXPERIMENTAL TRIAL Plasma free cholesterol 2 H / 1 H relative to SMOW 1 Subject by Group 0 hr 2 12 hr 24 hr 36 hr 48 hr (parts per thousand [%o]) Controls: CD -290.6 (4.7) -251.2 (2.8) -174.4 (10.5) -186.2 (8.8) -194.6 (1.0) MKa -283.2 (2.4) -211.0 (3.0) -133.5 (1.2) -160.6 (1.1) -165.9 (1.9) JG -293.0 (5.9) -216.8 (2.7) -142.9 (1.1) -96.4 (0.1) -95.9 (0.1) DD -297.0 (1.1) -241.0 (0.3) -170.6 (8.3) -193.1 (3.5) -173.4 (6.6) EM -304.8 (5.0) -219.5 (0.4) -123.5 (2.6) -164.4 (3.9) -173.2 (0.2) RS -280.0 (16.4) -191.1 (1.0) -118.5 (2.4) -117.4 (1.0) -124.0 (3.3) HD -306.0 (1.8) -226.8 (9.7) -137.2 (0.3) -148.3 (1.5) -149.6 (0.6) MW -247.4 (87.4) -64.0 (3.8) -20.0 (28.7) 50.3 (5.8) 42.6 (2.5) Type III HLPs NN -292.4 (26.4) -266.4 (4.9) -200.2 (2.9) -201.6 (3.8) -201.43 WJ -282.0 (28.9) -232.1 (0.7) -140.5 (0.1) -160.0 (1.7) -157.3 (1.9) ES -300.9 (2.9) -241.0 (16.6) -190.2 (1.5) -191.03 (1.7) -182.9 (3.2) JB -290.7 (14.1) -205.2 (26.4) -176.8 (2.2) -186.1 (2.3) -150.9 (2.3) AT -303.2 -208.6 (51.1) -155.8 (2.5) -173.3 (1.6) -175.1 (3.0) MKu -295.6 (3.4) -281.7 (5.2) -235.1 (2.5) -236.2 (7.3) -227.7 (9.8) DP -113.1 (4.9) -85.4 (1.2) -19.9 (9.3) 41.7 (8.8) 32.5 (28.0) EK -292.1 (15.5) -155.3 (5.5) 2.1 (28.5) 5.5 (5.5) 37.9 (2.2) ^Standard Mean Ocean Water; 2numbers are mean (SD) of triplicate samples; ^duplicate samples identical 96 APPENDIX FIFTEEN CHOLESTEROL FRACTIONAL SYNTHETIC RATE IN CONTROL AND TYPE III HLP SUBJECTS OVER 12 HOUR TIME INTERVALS DURING EXPERIMENTAL TRIAL Subject by Fed State . Fasted State Group 0-12 hr 12-24 hr 0-24 hr 24-36 hr 36-48 hr 24-48 hr 0-48 hr (FSR1 [per day]) Controls: CD 0.035 0.067 0.051 -0.010 -0.007 -0.009 0.021 Mka 0.068 0.073 0.070 -0.025 -0.005 -0.015 0.028 JG 0.068 0.066 0.067 0.042 0.0004 0.021 0.044 DD 0.061 0.077 0.069 -0.025 0.022 -0.002 0.034 EM 0.086 0.097 0.092 -0.041 -0.009 -0.025 0.033 RS 0.094 0.077 0.086 0.001 -0.007 -0.003 0.041 HD 0.074 0.084 0.079 -0.010 -0.001 -0.006 0.037 MW 0.140 0.034 0.087 0.054 -0.006 0.024 0.055 Mean 0.078 0.072 0.075 -0.002 -0.002 -0.002 0.037 + SEM 0.011 0.006 0.005 0.012 0.004 0.006 0.004 Type III HLPs: NN 0.024 0.061 0.042 -0.001 0.0002 -0.001 0.021 WJ 0.045 0.082 0.063 -0.017 0.002 -0.008 0.028 ES 0.054 0.046 0.050 -0.001 0.007 0.003 0.027 JB 0.075 0.025 0.050 -0.008 0.031 0.011 0.031 AT 0.086 0.048 0.067 -0.016 -0.002 -0.009 0.029 MKu 0.011 0.038 0.024 -0.001 0.007 0.003 0.014 DP 0.022 0.052 0.037 0.049 -0.007 0.021 0.029 EK 0.113 0.130 0.121 0.003 0.027 0.015 0.068 Mean 0.054 0.060 0.057 0.001 0.008 0.004 0.031 ±SEM 0.013 0.012 0.010 0.007 0.005 0.004 0.006 All Subjects: Mean 0.066 0.066 0.066 -0.0004 0.003 0.001 0.034 ±SEM 0.009 0.007 0.006 0.007 0.003 0.004 0.003 •^ Fractional Synthetic Rate 97 APPENDIX SIXTEEN INDIVIDUAL FREE CHOLESTEROL NET SYNTHESIS PER DAY BASED ON INDIVIDUAL Ml (CENTRAL) TOTAL CHOLESTEROL POOL SIZE IN CONTROL AND TYPE III HLP SUBJECTS OVER 12 HOUR TIME INTERVALS DURING EXPERIMENTAL TRIAL Subject by Fed State Fasted State Group M l Pool1 0-12 hr 12-24 hr 0-24 hr 24-36 hr 36-48 hr 24-48 hr 0-48 hr (g free cholesterol synthesized per day)2 Controls: CD 25.1 0.351 0.673 0.512 -0.100 -0.070 -0.090 0.211 MKa 25.3 0.688 0.739 0.708 -0.253 -0.051 -0.152 0.283 JG 24.7 0.672 0.652 0.662 0.415 0.004 0.207 0.435 DD 27.0 0.659 0.832 0.745 -0.270 0.238 -0.022 0.367 EM 26.5 0.912 1.028 0.975 -0.435 -0.095 -0.265 0.350 RS 24.7 0.929 0.761 0.850 0.010 -0.069 -0.030 0.405 HD 21.6 0.639 0.726 0.683 -0.086 -0.009 -0.052 0.320 MW 24.6 1.378 0.335 0.856 0.531 -0.059 0.236 0.541 Mean 24.9 0.778 0.718 0.749 -0.024 -0.014 -0.021 0.364 ±SEM 0.6 0.106 0.069 0.050 0.119 0.038 0.060 0.035 Type III HLPs: NN 27.5 0.264 0.671 0.462 -0.011 0.002 -0.011 0.231 WJ 30.0 0.540 0.984 0.756 -0.204 0.024 -0.096 0.336 ES 26.8 0.579 0.493 0.536 -0.011 0.075 0.032 0.289 JB 25.3 0.759 0.253 0.506 -0.081 0.314 0.111 0.314 AT 28.3 0.974 0.543 0.758 -0.181 -0.023 -0.102 0.328 MKu 21.6 0.095 0.328 0.207 -0.009 0.060 0.026 0.121 DP 33.2 0.292 0.691 0.491 0.651 -0.093 0.279 0.385 EK 15.8 0.714 0.822 0.765 0.019 0.171 0.095 0.430 Mean (all subj.) 26.1 0.527 0.598 0.560 0.022 0.066 0.042 0.304 + SEM 1.9 0.104 0.086 0.068 0.095 0.045 0.044 0.034 Mean (no subj. EK)27.5 0.500 0.566 0.531 0.022 0.051 0.034 0.286 + SEM 1.4 0.116 0.093 0.071 0.109 0.049 0.050 0.033 All Subjects: Mean 25.5 0.653 0.658 0.655 -0.001 0.026 0.010 0.334 + SEM 1.0 0.079 0.056 0.048 0.074 0.030 0.037 0.025 1see Table 7;2free cholesterol net synthesis per day (g) = size of M l total cholesterol pool (g) x cholesterol FSR corresponding to time interval x 0.4. Factor 0.4 used as it is estimated that approximately 40% of the M l (central) total cholesterol pool is free cholesterol (Jones et al. 1988). 98 APPENDIX SEVENTEEN CHOLESTEROL FRACTIONAL SYNTHETIC RATE IN CONTROL AND TYPE III HLP SUBJECTS OVER 12 HOUR TIME INTERVALS DURING EXPERIMENTAL TRIAL 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 Mean Cholesterol FSR (per day) F E D STATE F A S T E D STATE 0-12 hr 12-24 hr 24-36 hr 36 -48 hr Time Interval (hours) Controls Type III HLPs 99 APPENDIX EIGHTEEN INDIVIDUAL FREE CHOLESTEROL NET SYNTHESIS PER DAY BASED ON INDIVIDUAL Ml (CENTRAL) TOTAL CHOLESTEROL POOL SIZE IN CONTROL AND TYPE III HLP SUBJECTS OVER 12 HOUR TIME INTERVALS DURING EXPERIMENTAL TRIAL Mean Net Cholesterol Synthesis (g/day) 1.0 i • 100 BIBLIOGRAPHY Andersen, J.M., Turley, S.D., Dietschy, J.M., Low and High Density Lipoproteins and Chylomicrons as Regulators of Rate of Cholesterol Synthesis in Rat Liver in Vivo. Proc. Natl. Acad. Sci. (USA) 76:165-169, 1979. 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