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The role of oleic acid and cholesterol in neonatal diet Rioux, Marie-France 1994

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THE ROLE OF OLEIC ACID AND CHOLESTEROL IN NEONATAL DIET by MARIE-FRANCE MOUX B.Sc, University of Laval, 1986 M.Sc, University of Montreal, 1988 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (School of Family and Nutritional Sciences, Division of Human Nutrition) THE UNIVERSITY OF BRITISH COLUMBIA September 1993 ^ Marie-France Rioux, 1993 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 I^ Qmi ly a^J Ajv/fyili'^ fta ^ '^CnW j The University of British Columbia Vancouver, BC, Canada Date Oct 1'^ l^^l^ ABSTRACT Oleic acid (18:1) and cholesterol are not considered to be essential dietary nutrients for adults or infants. Human milk provides a significant amount of each of these nutrients. In contrast, most infant formulas contain relatively low 18:1 and cholesterol. The overall objective of this thesis was to determine the importance of 18:1 and cholesterol in natural milk to infant nutrition. Recent studies found reduced 18:1 in brain total lipid of piglets fed formula with 17% 18:1 rather than sow milk providing 37% 18:1. Oleic acid is a major fatty acid in brain myelin lipid and is rapidly deposited during myelination. It is important, therefore, to know if reduced 18:1 in brain total lipid reflects deposition of myelin lipid with reduced 18:1 and/or delayed myelination, or is related to changes in other brain membranes. The first part of this thesis determined the fatty acid composition of myelin total lipid, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and of plasma and liver phospholipid (PL) from piglets fed from birth to 15 d with formula containing low (17%) or high (38%) 18:1, or sow milk. Brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP'ase) activity and cerebrosides concentration were also determined and used as indicators of myelination. Piglets fed the low 18:1 formula had lower 18:1 in their plasma and liver PL than sow milk-fed piglets. Formula providing a similar level of 18:1 to sow milk resulted in higher 18:1 in piglet plasma and liver PL than in sow milk-fed piglets. Brain cerebrosides and CNP'ase activity and myelin 18:1 were similar in sow milk- and formula-fed piglets, irrespective of the formula 18:1 content. These studies suggest that supplying 18:1 in formula in a similar quantity to natural milk is not essential to normal accretion of 18:1 in brain myelin. Several studies have reported that plasma cholesterol and PL levels of 20:4n-6 are lower and PL levels of 18:2n-6 are higher in infants fed formula than in infants fed human milk. Plasma cholesterol and possibly the dietary intake of cholesterol, could be related to plasma PL n-6 fatty acid metabolism because plasma PL 18:2n-6 is usually used for esterification of plasma free cholesterol. Whether the low cholesterol content of infant formula compared to human milk is related to the difference in plasma n-6 fatty acids between infants fed human milk and formula is not known. The second part of this thesis determined the effect of feeding a formula containing low (0.05 mmol/L) or high (1.09 mmol/L) cholesterol content, or sow milk, on plasma, liver and bile lipid fatty acids and liver LDL receptor mass in piglets fed from birth to 18 days. Liver microsomal HMG CA reductase activity and plasma lathosterol were assayed as indices of liver and body cholesterol synthesis, respectively. Providing cholesterol in the formula did not correct the significantly lower plasma cholesterol, or plasma and liver PL 20:4n-6 associated with formula feeding. The liver total cholesterol and cholesterol esters, biliary bile acids and PL concentration were significantly higher and the liver HMG CoA reductase activity and plasma lathosterol:cholesterol ratio were significantly lower in piglets fed the formula with cholesterol than in piglets fed the formula without cholesterol. No evidence of lower hepatic LDL receptor mass was found in piglets fed sow milk compared to piglets fed formula. The results show marked differences in hepatic and bile n-6 fatty acid concentration between artificially and naturally fed piglets which do not seem to be explained by the difference in dietary cholesterol intake. Whether or not cholesterol and 18:1 should be added to infant formula in concentrations similar to that of natural milk is still unknown. Results from this thesis do not provide evidence that they should be added. I l l TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv ABBREVIATIONS ix LIST OF TABLES xii LIST OF FIGURES xiii ACKNOWLEDGEMENTS xiv 1 GENERAL INTRODUCTION 1 1.1 Milk and infant formula lipids and fatty acids 1 1.2. Fatty acid metabolism 4 1.3 Piglets as a model in perinatal nutrition research 7 2 PART 1: EFFECT OF MILK AND FORMULA 18:1 CONTENT ON MYELIN COMPOSITION AND MYELINATION 8 2.1 INTRODUCTION 8 2.1.1 Monoenoic fatty acids and neonatal nutrition 8 2.1.2 Characterization of central nervous system myelin 8 2.1.2.1 Process of CNS myelination 9 2.1.2.2 Characteristic composition of myelin 9 2.1.2.3 Metabolism of myelin 12 2.1.2.4 Changes in myelin lipid classes and fatty acid composition during development 13 2.1.2.5 Effect of diet on myelination 14 2.2 THESIS HYPOTHESIS AND OBJECTIVES 17 2.3 MATERIAL AND METHODS 17 IV 2.3.1 Chemicals 17 2.3.2 Formulas 17 2.3.3 Equipment 17 2.3.4 Immunoglobulin preparation 18 2.3.5 Animals and diets 19 2.3.6 Tissue preparation 20 2.3.6.1 Plasma 20 2.3.6.2 Liver 21 2.3.6.3 Cerebrum and myelin preparation 21 2.3.7 Tissue lipid extraction 22 2.3.7.1 Lipid extraction 22 2.3.7.2 Total cholesterol, free cholesterol and triglycerides 22 2.3.7.3 Lipid phosphorus 22 2.3.7.4 Cerebrosides 23 2.3.7.5 Fatty acid analysis 23 2.3.7.5.1 Lipid classes separation 23 2.3.7.5.2 Preparation of methyl esters and GLC 24 2.3.8 Brain CNP'ase 25 2.3.9 Protein 26 2.3.10 Statistical analysis 26 2.4 RESULTS 26 2.4.1 Animal body and brain weight 26 2.4.2 Plasma and liver lipid composition 27 2.4.4 Plasma and liver PL and CE fatty acid composition 28 2.4.5 Fatty acid composition of the myelin total lipid and PE and PC 29 2.5 DISCUSSION 36 2.5.1 Conclusive remarks and future directions 38 3 PART 2: EFFECT OF CHOLESTEROL ON PLASMA AND TISSUE N-6 FATTY ACIDS 42 3.1 INTRODUCTION 42 3.1.1 Cholesterol metabolism and transport 42 3.1.1.1 Cholesterol synthesis 42 3.1.1.2 Cholesterol catabolism 43 3.1.1.3 Cholesterol transport 44 3.1.1.4 LDL receptor and regulation of cholesterol metabolism 45 3.1.2 Plasma cholesterol and n-6 fatty acids in infants: Possible role of cholesterol in n-6 fatty acid metabolism 49 3.2 THESIS HYPOTHESIS AND OBJECTIVE 51 3.3 MATERIAL AND METHODS 51 3.3.1 Chemicals 51 3.3.2 Formulas 52 3.3.3 Equipment 52 3.3.4 Animal and diets 52 3.3.5 Tissue preparation 53 3.3.5.1 Plasma and plasma HDL 53 3.3.5.2 Bile 54 3.3.5.3 Liver 54 3.3.5.4 Microsomes and plasma membrane 54 3.3.6 Tissue lipid analysis 54 3.3.6.1 Lipid extraction 54 3.3.6.2 Total cholesterol, free cholesterol and triglycerides 54 VI 3.3.6.3 Lipid phosphorus 55 3.3.6.4 Biliary bile acids 55 3.3.6.5 Lathosterol 55 3.3.6.6 Fatty acid analysis 56 3.3.6.6.1 Lipid classes separation 56 3.3.6.6.2 Preparation of methyl esters and GLC 56 3.3.7 HMG CoA reductase activity 56 3.3.8 LDL receptor relative mass 57 3.3.8.1 /S VLDL preparation 57 3.3.8.2 Membrane solubilization 57 3.3.8.3 SDS-PAGE and electroelution 58 3.3.8.4 Gold-lipoprotein conjugation 58 3.3.8.5 Ligand blotting 58 3.3.8.6 Receptor identification and characterization 59 3.3.8.7 Linearity 59 3.3.9 Protein 59 3.3.10 Statistical analysis 59 3.4 RESULTS 60 3.4.1 Growth 60 3.4.2 Plasma lipid composition 60 3.4.3 Liver lipids, HMG CoA reductase activity, plasma lathosterol and LDL receptor mass 60 3.4.4 Bile lipid and fatty acid composition 61 3.4.5 Plasma and liver PL and CE fatty acid composition 63 3.5 DISCUSSION 69 vii 3.5.1 Conclusion and future directions 78 4 OVERALL CONCLUSION 81 5 REFERENCES 82 viu ABBREVIATIONS ACAT acyl-CoA: cholesterol acyltransferase 2'AMP 2'adenosine monophosphate 2'3'-cAMP 2'3'-cyclic adenosine monophosphate ANOVA analysis of variance dpo apoprotein BSA bovine serum albumin /SVLDL beta very low density lipoprotein "C degrees Celsius CE cholesterol esters a curie CNP'ase 2',3'-cyclic nucleotide 3'-phosphohydrolase CNS central nervous system d day decilitre dL DPM disintegrations per minute EDTA ethylenediamine tetraacetic acid g gram GLC gas liquid chromatography h hour HDL high density lipoprotein HMG CoA 3-hydroxy-3-methylglutaryl coenzyme A HPTLC high performance thin layer chromatography oe HSD oe hydroxysteroid dehydrogenase IX IDL kDa 1 LCAT LCP LDL MCT MBP mg min mmol mol M ml mRNA NAD? NADPH ng NTB P PC PE PI PL intermediate density lipoprotein kilodaltons litre lecithin cholesterol acyltransferase long chain polyenoic fatty acids low density lipoprotein Medium chain triglycerides myelin basic protein milligram minute mmol mole molar milliliter messenger ribonucleic acid nicotinamide adenine dinucleotide phosphate nicotinamide adenine dinucleotide phosphate, reduced form nanogram nitrotetrazolium statistical probability of mean differences not existing phosphatidylchol ine phosphatidylethanolamine phosphatidyl inositol phospholipid PLP PS pmol PMSF RBC SEC SEM SDS-PAGE SPH TBS TO TL TLC Tris VLDL fig timol vol wt proteolipid phosphatidylserine picomole phenylmethylsulfonyl fluoride red blood cells second standard erreur of the mean sodium dodecyl sulfate-polyacrylamide gel electrophoresis sphingomyelin Tris buffered saline triglycerides total lipid thin layer chromatography tris(hydroxymethyl)aminomethane very low density lipoprotein microgram micromole volume weight XI LIST OF TABLES 1.1 Fatty acid composition of human and sow milk and typical infant formulas 3 2.1 Composition of central nervous system myelin 10 2.2 Fatty acid composition of sow milk and formula differing in 18:1 content 20 2.3 Plasma and liver lipid concentrations in piglets fed sow milk or formula with different 18:1 content 27 2.4 Myelin lipid, brain cerebroside and 2',3' cyclic nucleotide 3'-phosphohydrolase (CNP'ase) activity of piglets fed sow milk and formula differing in 18:1 content 28 2.5 Fatty acid composition of plasma and liver phospholipid of piglets fed sow milk or formula differing in 18:1 content 31 2.6 Fatty acid composition of plasma and liver cholesterol esters of piglets fed sow milk or formula differing in 18:1 content 32 2.7 Fatty acid composition of myelin phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and total lipid (TL) of piglets fed sow milk or formula differing in 18:1 content . . . 34 3.1 Diet cholesterol content and fatty acid composition 53 3.2 Body and organ weights of piglets fed sow milk or formula with low or high cholesterol content 61 3.3 Plasma cholesterol and triglyceride concentrations of piglets fed sow milk or formula with low or high cholesterol content 62 3.4 Liver cholesterol and triglyceride concentrations of piglets fed sow milk or formula with low or high cholesterol content 62 3.5 Liver HMG CoA reductase activity, plasma lathosterol and the relative LDL receptor mass of piglets fed sow milk or formula with low or high cholesterol content 63 3.6 Composition of bile from piglets fed sow milk or formula with low or high cholesterol content 63 3.7 Major fatty acids of plasma and liver phospholipid (PL) of piglets fed sow milk or formula with low or high cholesterol content 65 3.8 Major fatty acids of plasma and liver cholesterol esters (CE) of piglets fed sow milk or formula with low or high cholesterol content 66 xu LIST OF FIGURES 1.1 Structure of linoleic acid 5 1.2 Schematic representation of the major pathways of n-9, n-6, n-3 fatty acid metabolism . . . 6 2.1 The effect of feeding sow milk and formula varying in 18:1 content of the proportion of 18:1 in piglet plasma PL and CE (A) and liver PL and CE (B) 33 2.2 The effect of feeding sow milk and formula varying in 18:1 content on the proportion of 18:1 in piglet myelin PE, PC and TL 35 3.1 Schematic representation of the exogenous and endogenous fat transport 47 3.2 Sequential steps in the LDL receptor pathways of mammalian cells 48 3.3 Representative example of results from the ligand blotting assay with 20 ng membrane protein for piglets f^ the formula low in cholesterol (lane 1), sow milk (lane 2) and formula high in cholesterol (lane 3) 67 3.4 The effect of feeding sow milk or formula with low or high cholesterol content on the major fatty acid components of piglet plasma and liver PL and CE 68 X l l l ACKNOWLEDGEMENTS First I would like to thank my research supervisor, to Dr. Sheila Innis, for her precious guidance throughout these years and for giving me the chance to prove myself that I was capable of accomplishing this task. I thank her also for her patience in dealing with my French accent and other language related problems. I would like to thank the members of my supervisory committee, Dr. Linda McCargar and Dr. Wayne Vogl for their helpful comments. I must also acknowledge the expertise and moral support of friends and very special people who worked beside me in Dr.Innis' laboratory. Especially I must thank Dr. Dianne Arbuckle, Dr. Jennifer Hamilton, Roger Dyer, Dr. Katharine Wall, Janette King, Laurie Nicol and Dr. Nancy Austead. I also grateftilly acknowledge the financial assistance from the B.C. Children's Hospital. I would also like to thank my parents, Marianne and Jacques, and my sisters and brother, Jacqueline, Monette, Chantale, and St6phane for their moral support; they all believed more stronger than I did in my potential. Thank you to my Mother for her frequent encouraging long distance calls. Special thanks to my husband, Jean-Louis who left his work in Montreal and agreed to follow me in Vancouver for this long adventure. I thank him for his understanding and his faith in me. Finally, to my daughter Marie-Eve, who was bom in the middle this chaotic period of my life, I thank her for her lovely smiles and for being such a good baby. XIV Chapter 1 GENERAL INTRODUCTION Over the last decade, the field of infant lipid nutrition has been the focus of extensive research. Specific emphasis has been given to the requirement for dietary essential n-6 and n-3 fatty acids for optimal growth and development. The long chain polyenoic fatty acids, fatty acids of carbon chain 20 or 22 with 3 or more double bonds (LCP) have been of particular interest because these fatty acids are present in human milk and are found in the structural lipids of all cell membranes. They are also believed to play an important role in central nervous system (CNS) function. Oleic acid (18:1), a monoenoic fatty acid, and cholesterol are not recognized as dietary essential nutrients for adults or infants but both are present in significant amounts in human milk. Most infant formulas, in contrast to human milk contain relatively low amounts of 18:1 and negligible amounts of cholesterol. Human milk is accepted as the ideal food for healthy infants born after term gestation. The metabolic role of many of the "non-essential" nutrients, such as oleic acid and cholesterol, provided by human milk, is not known. The objective of the projects described in this thesis was to define the possible nutritional and metabolic roles of oleic acid and cholesterol in the diet of the newborn using piglets as a suitable experimental model for the human infant. 1.1 Milk and infant formula lipids and fatty acids The lipid and fatty acid composition of human milk and infant formulas have been reviewed in detail (Jensen et al. 1978, Packard 1982, Innis 1992, Jensen and Jensen 1992). Fat represents 45 to 50% of energy in most human milk and infant formula. At least 98% of lipid is present as triglycerides. Phospholipids represent only 1.3% of the total lipid. Fat is dispersed in human milk in the fat globules. In infant formulas fat is maintained in dispersion with plant lecithins. The amount of cholesterol in human milk has been reported to range from 0.3-0.6 mmol/1 of milk. The cholesterol content of commercial formulas is usually below 0.1 mmol/1. Human milk contains a wide range of fatty acids including saturated, monounsaturated fatty acids, and the n-6 and n-3 series polyunsaturated fatty acids. The n-6 and n-3 fatty acids include the dietary essential fatty acids, linoleic (18:2n-6) and linolenic acids (18:3n-3) as well as small amounts of LCP fatty acids 1 such as arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). Levels of n-6 LCP usually represent about 1% and n-3 LCP about 0.6% of total fatty acids. Analysis of published data from nine studies of women in the United States who followed unrestricted diets have been compiled in a recent review by Innis (1992). The calculated mean levels of medium-chain saturated fatty acids (8:0-10:0) was 1.5%, the level of intermediate-chain length saturated fatty acids (12:0 and 14:0) was about 11%, palmitic acid (16:0) was 21%, and 18:1 was 35%. The mean levels of 18:2n-6 and 18:3n-3 were about 16% and 1 % respectively (Table 1.1). The variability in the fatty acids of mature human milk is possibly the result of differences in the mother's dietary fat intake (Innis 1992). The fat in infant formulas is a blend of one or more vegetable oils such as corn, soybean and coconut oils. Com and soybean oils contain about 50% to 60% linoleic acid; soybean oil also contains 6 to 9% linolenic acid. These vegetable oils contain low levels of oleic acid (18:1) and palmitic acid (16:0) compared to milk (Table 1.1). Coconut oil contains the intermediate chain length saturated fatty acids (14:0). Infant formulas made with these vegetable oils usually contain considerably more 18:2n-6 and less 18:1 and 16:0 than human milk. Oleo oils, derived from beef fat or saturated vegetable oils such as palm oil, contain palmitic acid (16:0) and are used in the products of some manufacturers. Formulas designed for preterm infants often contain medium chain triglycerides (MCT) oils containing the medium chain saturated fatty acids, 8:0 and 10:0. More recently, high oleic safflower oil has been included in some formula to provide higher concentration of oleic acid. Infant formula prepared with vegetable oils with or without oleo oils do not contain significant amount of n-6 or n-3 LCP. Table 1.1 Fatty acid composition of human and sow milk and typical infant formulas. Formulas and oil blends Fatty acids Human milk Pig milk Enfamil Soy,coconut Palm olein % Total fatty acids 3.0 14.1 25.0 3.8 35.0 16.0 5.9 Similac Coconut,com 7.8 41.4 10.3 2.8 15.2 22.5 3.6 SMA oleo,coconut. safflower.sov 3.3 20.5 12.8 7.4 38.9 13.1 1.1 u 8:0-10:0 12:0-14:0 16:0 16:1 18:0 18:1 18:2n-6 18:3n-3 20:4n-6 77-fin-^ 1.1-2.2 5.2-16.7 20-24 2.5-3.8 7.1-9.0 31-38 14.5-18.8 0.3-1.9 0.6-0.7 niMT, 0.1 3.5 30.5 9.0 4.4 37.5 11.1 1.1 0.5 0 9 Data given for human milk for the major saturated and monounsaturated fatty acids and linoleic and linolenic represent range of means (% total) for nine studies of women following unrestricted diets in the United States (Innis 1992). Data for human milk 20:4n-6 and 22:6n-3 represent range from women following unrestricted diets in the United State and Canada/BC (Innis 1992). Data given for sow milk represent mean of 2 samples. 1.2 Fatty acid metabolism Fatty acids are straight hydrocarbon chains terminating with a carboxylic acid group. They are usually components of complex lipids such as triglycerides, sterol esters, phospholipids, glycolipids. The length of the carbon chain ranges from 4 to about 26 carbon atoms and may be saturated, monounsaturated or polyunsaturated having no, one or more than one double bonds, respectively. Many notations have been established to identify the chain length and the number and position of any double bonds present. For example, the notation 18:2n-6 used to describe linoleic acid means the carbon chain length is equal to 18, the number after the colon, in this case 2, indicates the number of double bonds; the final n designation indicates the location of first double bond counting from the methyl end of the chain, in this case being at carbon 6 (Figure 1.1) (Hunt and Groff 1990). Mammalian cells can synthesize saturated fatty acids de novo starting from acetyl CoA by sequential addition of 2 carbon units, to a maximum of 16 carbons, i.e 16:0. Palmitic acid can then be elongated to stearic acid (18:0) and desaturated by the A 9 desaturase to 18:ln-9. Mammalian cells do not have the desaturase enzymes necessary to introduce an unsaturated bond at the n-6 and n-3 position of a fatty acid carbon chain. Therefore, a dietary source of linoleic acid (18:2n-6) and linolenic (18:3n-3) is essential for all animals (Innis 1992). The main dietary sources of 18:2n-6 are vegetable oils such as corn, safflower, soybean, cottonseed, sunflower, and peanuts oils. Vegetable oils rich in 18:3n-3 include linseed, soybean and canola oils. Linoleic acid and 18:3n-3 can be metabolized by desaturation and elongation to give a series of longer carbon chain fatty acids (Figure 1.2). The best known are 20:4n-6, 20:5n-3 and 22:6n-3. These fatty acids are usually found ester ified in the phospholipids which make up the structural matrix of cell membranes. Arachidonic acid, 20:3n-6 and 20:5n-3 are also substrates for biosynthesis of a complex series of potent cell regulators which include prostaglandins, thromboxanes and leukotrienes (Smith 1989). Desaturation of n-9, n-6 and n-3 fatty acids relies on the same enzymes. Therefore, competition among potential substrates for a given desaturase enzyme can occur. Preferential desaturation of 18 carbon fatty acids, however, occurs in the order 18:3n-3> 18:2n-6> 18:ln-9 (Brenner 1974). The balance, as well as absolute amounts of 18:2n-6 and 18:3n-3 and possibly 18:1, in milk and formula may, therefore, impact on the ability of the body to synthesize 20:4n-6 and 22:6n-3 from 18:2n-6 and 18:3n-3, respectively. 2 4 6 7 9 10 12 14 16 18 COOH 18:2n-6 Linoleic acid Figure 1.1 Structure of linoleic acid Acetyl CoA A 9 desaturation 16:0 8:0 ON / 24:1 18:1 18:2n-6 oleic acid A 6 linoleic acid If 20 \ desaturation \ elongation :2n-9 20:3n-6 I desaturation \ Diet Ae desaturation elongation A 5 desaturation 20:3n-9 20:4n-6 arachidonic acid * elongation 22:4n-6 1 22:5n-6 18:3n-3 linolenic acid \ 20:4n-3 1 20:5n-3 eicosapentaenoic acid \ 22:5n-3 1 24:5n-3 22:6n-3 = 0 docosahexaenoic acid V 24:6n-3 Figure 1.2. Schematic representation of the major pathways of n-9, n-6 and n-3 fatty acid metabolism. In addition, to desaturation and elongation pathways, 18:2n-6 and 18:3n-3 can be readily oxidized for energy. Linoleic acid can also be directly acylated into tissue triglycerides (TG), cholesterol esters (CE), and phospholipids (PL). Linolenic acid is also found in TG, CE, and PL but to a much lesser extent. In contrast to 18:2n-6 and 18:3n-3, 20:4n-6 and 22:6n-3 are less readly oxidized for energy and are rapidly acylated into tissue PL. Therefore, it is reasonable to assume that tissue assimilation of n-6 and n-3 fatty acids in growing infant tissues will depend on the balance and supply of dietary n-3 and n-6 fatty acids, activity of the desaturase enzyme and energy intake (Innis 1992). L3 Piglets as a model in perinatal nutrition research Piglets are often used in perinatal nutrition research studies because the perinatal timing of the brain growth spurt (Dobbing and Sands 1979), including the pattern of pre and postnatal myelination (Sweasey et al. 1976, Wiggins 1986) and the percentage of adult brain weight achieved at birth are similar to that of human (Dobbing and Sands 1979). Further, the postnatal exponential phase of myelination in pig is completed wifliin the first 3 wk of birth (Sweasey et al. 1976), occurring within the normal suckling duration of up to 25 d. Of importance for these studies, the monoenoic fatty acids and cholesterol content of sow milk is comparable to that of human milk (Hrboticky et al. 1989, Jones et al. 1990). Many similarities between the evolution of lipoproteins in newborn infants and growing piglets have been reported (Hollanders et al. 1985). The lipid profiles and physiochemical properties of the lipoproteins are also similar in growing pigs and human (Walsh Hentges et al. 1987). Of practical importance, the piglet unlike other commonly studied animals, is easly fed on artificial diets fi"om birth. Chapter 2 PART 1: EFFECT OF MILK AND FORMULA 18:1 CONTENT ON MYELIN COMPOSITION AND MYELINATION 2.1 INTRODUCTION 2.1.1 Monoenoic fatty acids and neonatal nutrition Many infant formulas containing com, soybean, or safflower as the predominant source of unsaturated fat, typically containing about 15% 18:1, which represent 50% less than in typical breast milic (Jensen et al 1978). Clinical studies have consistently demonstrated that plasma and/or RBC PL levels of 18:1 are lower in infants fed formula low in 18:1 than in infants fed human milk (Putnam et al 1982, Carlson et al 1986, Innis et al 1990). It is reasonable to assume that the lower circulating lipid 18:1 in infants fed formula low in 18:1 is related to their lower intake of 18:1. The provision of 18:1 in infant formula in quantities similar to those in human milk does not seem to have been considered important. This may be because 18:1 is not a dietary essential fatty acid as it can be synthesized in vivo by A 9 desaturation of 18:0 (Figure 1.2). The accretion of 18:1 in the brain total lipid from piglets fed exclusively from birth with a coconut-corn oil formula containing 17% 18:1, however, was lower than in piglets fed sow milk with 35% 18:1 (Hrboticky et al 1990), similar to that in human breast milk (Jensen et al 1978). Monoenoic fatty acids are major acyl components of central nervous system (CNS) myelin (Norton and Cammer 1984, Sastry 1985, Morell et al 1989). Further, 40% or more of the brain total lipid is in the lipids of the myelin membrane (Norton and Cammer 1984). It is reasonable, therefore, to ask if the decreased 18:1 in the brain total lipid of formula fed piglet was the result of changes in 18:1 in the myelin membranes. 2.1.2 Characterization of central nervous system myelin Myelin is a membrane characteristic of the peripheral and CNS which consists of numerous bilayers arranged spirally around the axon (Raine 1984, Ritchie 1984, Morell et al. 1989). The myelin sheath is an extension and modification of the plasma membrane of the oligodendrocytes in the CNS and of the Schwann cells m the peripheral nervous system (PNS). The myelin sheath has a protective role in shielding the axon from compression (Ritchie 1984). Importantly, myelin also functions as an insulating sheath; providing an envelope which insulates the conducting axon electrically from the external conducting medium permitting a more effective transmission. 2.1.2.1 Process of CNS myelination Myelination commences in the PNS at about the fifth month of gestation, then proceeds to the spinal cord and finally the brain. Brain myelination is most extensive after birth, following cell proliferation and is almost complete by two years of age (Yakovlev and Lecours 1967). Within the brain, different areas myelinate at different rates; the intracortical areas being the last to myelinate. CNS myelination is initiated when the axons to be myelinated reach a diameter of about 1 micron. A number of distinct stages can be observed in the formation of CNS myelin: a) oligodendrocyte proliferation and differentiation, b) oligodendrocyte process formation and contact with the axon forming loose cups around the axon, C) further extension of each cell process resulting in one lip of the cup (inner tongue) becoming insinuated beneath the other, D) subsequent development of myelin involving the rotation of this lip around the axon, and finally, E) active myelination and compaction of the myelin sheath around the axon (Raine 1984). 2.1.2.2 Characteristic composition of myelin The composition of myelin has been extensively reviewed by Norton and Cammer (1984) and Morell et al. (1989). Myelin has a water content of approximately 40% on a weight basis. The most outstanding feature of myelin, compared to other biological membranes is its high Upid:protein ratio. On a dry weight basis, myelin consists of 70-85% lipid and 15-30% protein. All of the lipids found in other brain membranes are present in myelin, and there are no lipids in myelin that are not also found in other subcellular fractions of brain. Although there are no lipids specific to myelin, the distribution of lipid classes in myelin is highly specific. For example, galactolipids (glycolipids) represent 27% (approximately 23% cerebroside and 4% sulfatide) of the total myelin lipids (Poduslo and Yang 1984) (Table 2.1). Table 2.1 Conqxisition of central nervous system myelin Lipids Total lipid (% of dry weight) Cholesterol Cerebrosides Sulfatides Total Phospholipids Ethanolamine Choline Serine Inositol Sphingomyelin Human 70.0 27.7 22.7 3.8 43.1 15.6 11.2 4.8 0.6 7.9 Myelin Bovine 75.3 28.1 24.0 3.6 43.0 17.4 10.9 6.5 0.8 7.1 Rat 70.5 27.3 23.7 7.1 44.0 16.7 11.3 7.0 1.2 3.2 All figure are averages obtained from analyses of adult CNS myelin. Source of data, Norton and Cammer 1984. I* individual lipids are expressed as weight percentage of total lipid. In addition to cerebrosides, the major lipids of myelin are cholesterol and phosphatidylethanolamine (PE). Phosphatidylcholine (PC) is also a major constituent. Mature myelin always has more PE than PC while the opposite is observed for all other brain membranes. Sphingomyelin (SPH) and phosphatidylserine (PS) are less abundant than PC and PE. Phosphatidyl inositol (PI), which is more concentrated in neuronal membranes, is a minor component of myelin (Table 2.1). There is a lack of documentation of pig myelin lipid expressed as % dry weight. Not only is the lipid class composition of myelin highly specific to the membrane, the fatty acid composition of the lipids is also characteristic. In general, myelin contains relatively high proportions of saturated fatty acids mainly, 16:0 and 18:0 and very high proportions of monounsaturated fatty acids, mostly 18:1. Proportions of n-6 and n-3 fatty acids are low. Myelin PE, however, contains a high proportion of 18:1, as well as considerable amounts of 20:4n-6 and 22:4n-6. The relative quantities of 10 20:4n-6 and 22:4n-6 differ depending on the species and stage of brain development (O'Brien and Sampson 1965, Sun 1972, Svennerholm et al. 1978, Huerther et al. 1986, Yeh 1988a). The major fatty acid of the CNS myelin cerebrosides and sulfatides are 24:0 and 24:1, amounts of 16:0, 18:0, 18:1, 22:0 and 26:1 vary depending on the species (O'Brien and Sampson 1965, Sweasy et al. 1976, Srinivasa 1977, Huerther et al. 1986, Yeh 1988a,). The protein composition of CNS myelin is simpler than in other brain membranes. The two major proteins, proteolipid (PLP) and myelin basic protein (MBP), account for 75% of the total myelin protein in most species (Norton and Cammer 1984, Morell et al 1989). PLP is a transmembrane protein with portions of the molecule in both the extracellular and cytoplasmic domains. MBP is a peripheral protein associated with the cytoplasmic surface of the bilayer (Ulmer 1988). In addition to PLP and MBP, the enzyme 2,3'-cyclic nucleotide 3'-phosphodiesterase (CNP'ase) is also present in myelin and accounts for approximately 4% by weight of the total protein. CNP'ase was the first enzyme to be characterized as a component of the myelin membrane; previously myelin was thought to be enzymatically inert (Vogel and Thompson 1988). CNP'ase activity is believed to be fairly specific to CNS myelin, despite is presence in oligodendroglial membranes (Morell et al. 1989). CNP'ase is very low in PNS myelin, where it is present at only 10% of the activity found in CNS myelin (Vogel and Thompson 1988). The enzyme is found at much higher specific activities in myelin than in whole brain homogenate or other brain cell or subcellular membranes. Studies on isolated myelin have shown approximately 60% of the total brain CNP'ase activity in the myelin membranes (Morell et al. 1989). Because of its specificity to myelin, CNP'ase has been used as a marker to estimate the purity of CNS myelin preparations (Prohaska et al. 1973). Studies on the developmental expression of CNP'ase indicate that CNP'ase appears early during myelination and its activity remains at a high level as myelin is produced (Sprinkle and McKhann 1978). The enzyme activity increases in brain during development in parallel with myelination. 11 2.1.2.3 Metabolism of myelin The metabolism of myelin had been extensively reviewed by Benjamins and Smith (1984), Sastry (1985) and Morell et al. (1989). The synthesis of myelin components and the possible mechanism for their transport into myelin sheath, however, are still questions of interest. Evidence supports the view that myelin components are synthesized within oligodendrocytes and enter the extented plasma membrane (Ledeen 1984, Sastry 1985). In vivo and in vitro metabolic studies have shown that oligodendrocytes contain the enzymes necessary for PL, galactocerebroside, and cholesterol synthesis (Sastry 1985, Koul et al. 1988). Oligodendrocytes also contain acetyl CoA carboxylase, the enzyme involved in the first step in formation of fatty acids (Tansey et al. 1988, Cammer 1991), as well as fatty acid synthase, an enzyme involved in the succeeding steps in the biosynthesis of fatty acids (Cammer 1991). It appears that elongation of n-3 and n-6, monounsaturated and saturated fatty acids and A 6 and 9 desaturation also occur in the oligodendroglial preparation isolated from 9 month fetal and adult bovine CNS white matter (Fewster et al. 1975). These investigators have shown that when labelled 14:0 was used as substrate, it was elongated to longer chain fatty acids. The pattern of elongation was 14:a-*16:0-»18:O*20:0. Labelled 16:1, 18:1, 20:1 was also found in the oligodendrocytes. In addition, the presence of a system for chain elongation of 18:1 to 24:1 was observed. These studies provide evidence that 18:1 and longer chain monounsaturated fatty acids characteristic of myelin can be synthesized by oligodendrocytes. However, little is known about the relative confribution of de novo synthesis of monounsaturated fatty acids by oligodendrocytes and uptake from the circulation across the blood-brain barrier. It has been suggested, however, that saturated fatty acids of exogenous origin are essential for the synthesis of brain myelin during the rapid phase of myelination in mice (Bourre et al. 1979). It could be speculated that myelin acquires 18:1 partly from saturated fatty acid precursors obtained from plasma and partly from in situ synthesis. Direct uptake of intravenous and oral 18:1 into adult rat brain has also been shown (Dhopeshwarkar and Mead 1970). This study, however, does not provide information as to whether the 18:1 taken up by brain from the 12 circulation was directed towards myelin lipid synthesis in oligodendrocytes. Only a few enzymes of lipid metabolism have been found to be associated with myelin (Ledeen 1984, Morell et al. 1989). These include cholesterol esterifying enzyme, UDP-galactose:ceramide galactosyltransferase, and enzymes involved in glycerophospholipid metabolism (CDP-choline:l,2-diracyl-sn-glycerol choline phosphotransferase, CDP-ethanolamine:l,2-diracyl-sn-glycerol ethanolamine phosphotransferase, CTP:ethanolamine phosphate cytidyltransferase, choline kinase, ethanolamine kinase). Acyl-CoA synthetase is also present in myelin suggesting the capacity to integrate free fatty acids into myelin lipid (Morell et al. 1989). The actual contribution to myelin lipid metabolism of these my el in-associated enzymes relative to that of enzymes found within oligodendrocytes is not clear. Furthermore, the source of substrate for these lipid-synthesizing enzymes found in myelin is not well known. It has been suggested that the axon, which is physically very close to myelin, could provide various substrates for the lipid-synthesizing enzymes of myelin (Ledeen 1984). The importance of this process, however, is not known. 2.1.2.4 Changes in myelin lipid classes and fatty acid composition during development. Major changes in the lipid class and fatty acid composition of CNS myelin occur during development (Norton and Cammer 1984, Morell et al. 1989). The proportion of cerebrosides increases by approximately 50% and PC decreases by a similar amount in human myelin in parallel with the rate of myelination (Svennerholm et al. 1978). These changes are most pronounced before 3 months of age. The measurement of cerebrosides in developing brain is considered a useful indicator of the progression of myelination (Norton and Poduslo 1973, Sweasey et al. 1976, Svennerholm et al. 1978). The proportion of cholesterol in myelin increases slightly or remains relatively constant during myelination. Similar changes to those in human are seen in myelin lipids of the developing domestic pig (Sweasey et al. 1976), mouse (Horrocks 1968) and rat (Norton and Poduslo 1973). Not only does the lipid class distribution undergo developmental changes, the fatty acid composition of these lipids also changes. Studies by Svennerholm et al. (1978) found that the greatest changes of the 13 myelin phosphoglycerides occured in PE. These developmental changes include an increase in proportion of monoenoic fatty acids with a concomittant decrease in saturated fatty acids, 16:0 and 18:0. Of the monoenoic fatty acids, 18:1 showed the greatest increase, from 25% to 48%. The porportion of 20:4n-6 decreases from birth to puberty, while 22:4n-6 increases from birth to 12 months, whereafter levels decrease to lower values found in adult myelin PE. The changes in saturated and monounsaturated fatty acid composition in PS and PC are similar to that of PE but of smaller magnitude. The fatty acid composition of SPH also changes considerably during maturation; the increase in monounsaturated fatty acids (mainly 24:1, 25:1, and 26:1) is pronounced (from 17% for newborn to 50% fatty acids in mature brain myelin SPH) (Svennerholm et al. 1978). The porportion of saturated fatty acids (C16-C22) decreases, but that of very long chain fatty acids (C22-C26) remains constant. A developmental increase in monoenoic fatty acids mainly 24:1 and a decrease in saturated fatty acids is observed in human myelin cerebrosides and sulfatides (Svennerholm et al. 1978), and in rat (Huerther et al. 1986). The activity of many enzymes involved in lipid metabolism and synthesis also increase in parallel with myelination (Benjamins and Smith 1984, Morell et al. 1989). The activity of the enzymes, ceramide galactosyltransferase, and ceramide sulfofransferase, involved in synthesis of cerebrosides and sulfatides, increase rapidly during myelination and then decrease markedly as the rate of myelination slows. Enzymes in the biosynthetic pathway for cholesterol also appear to increase in activity during myelination. Several enzymes associated with PL synthesis also increase and decrease in parallel with the rate of myelination; these include, glycerol phosphate dehydrogenase, enzymes involved in fatty acid synthesis, and enzymes in the pentose phosphate pathway which provides NADPH for lipid synthesis (Benjamins and Smith 1984, Morell et al. 1989). 2.1.2.5 Effect of diet on myelination. Any metabolic insult during the period of brain growth known as the "vulnerable" period may lead to permanent alteration of myelin. The vulnerable period include the phase of rapid deposition of myelin 14 and, more importantly the time of oligodendroglial cell proliferation. Once the normal numbers of oligodendroglia are reached, the period of rapid myelination is less vulnerable to dietary deprivation and in many cases responds to nutritional rdiabilitation (Smith and Benjamins 1984). Data are available to show that neonatal nutrition influences the process of myelination. Impairment of myelination has been observed both in animals and humans subjected to generalized undernutrition and to protein or essential fatty acid deficiency during early period of development (Smith and Benjamins 1984, Sastry 1985). Many studies have shown that myelin formation is particularly susceptible to nutritional deprivation imposed during early development (Srinivasa 1979, Martinez 1982, Yeh 1988a, Yeh 1988b). Nutritional deprivation can be produced in laboratory rodents by increasing litter number, limiting the time of access of the pups to their mothers for suckling and restricting the maternal food intake. In general, nutritional deprivation during perinatal brain development produces a reduction in the rate of myelin synthesis leading to a reduction in the quantity of myelin total lipid, cholesterol, PL and galactolipids in the human (Martinez 1982), and rat (Yeh 1988b). Changes in fatty acid composition, including a reduction of 24:0 and 24:1 in cerebrosides and sulfatides, also occurs. The reduction in longer chain fatty acids is accompanied by a proportional increase in 16:0 and 18:0 (Y^ 1988a). Most of the effects of undernutrition on myelination are also seen when protein deficiency is induced with sufficient calorie intake (Smith and Benjamins 1984). The lipid content of brain, mainly the myelin-associated lipids, of marasmic children of 10-16 weeks of age are much below those expected for adequately nourished children (Sastry 1985). A reduction of 24:0 and 24:1 in brain cerebrosides has been found in rats fed a protein deficient diet during the post-weaning period (Srinivasa 1979), similar to that described for brain of undernourished rodents (Yeh 1988a) Deficiency of essential fatty acids was found to result in lower brain weights (Smith and Benjamins 1984) and lower brain concentrations of galactolipids in mice (Mckenna and Campagnoni 1979). Sun (1972) reported that essential fatty acid deficiency induced in developing rats caused a marked change 15 in the fatty acid composition of the major myelin phospholipids PC, PE and SPH. The fatty acid changes included higher levels of 20:3n-9 and lower levels of 20:4n-6 and 22:4n-6 resulting in an elevated myelin triene/tetraene ratio. Synthesis of 20:3n-9 is the result of A9 desaturation of 18:1 and occurs during dietary deficiency of 18:2n-6 and 18:3n-3 (Brenner 1974). More recently, limited dietary intake of 18:3n-3 with sufficient 18:2n-6 was shown to cause a reduction in 22:6n-3 in rat myelin (Bourre et al. 1984, Youyou et al. 1986, Enslen et al. 1991). The reduction in 22:6n-3 was accompanied by increased 22:5n-6 (Bourre et al. 1984, Youyou et al. 1986, Enslen et al. 1991). This suggests a compensatory mcreased desaturation of n-6 fatty acids due to inadequate 22:6n-3 for incorporation into membrane lipids. Although much is now known about the biochemical changes in myelin due to protein or/and calorie malnutrition, or essential fatty acid deficiencies, little information is presently available concerning the effect of these deficiencies on the fiinctional properties of myelin. Studies in the rat have found lower performance in shock-avoidance tests due to dietary essential fatty acid deficiency, suggesting impairment of brain and possibly myelin function (Galli et al. 1975). However, biochemically abnormal peripheral myelin in essential fatty acid deficient rats was not accompanied by physiological, or morphological evidence of neuropathy (Evans et al. 1980). In contrast, a decrease in nerve conduction velocities has been reported for children with protein-calorie malnutrition (Kumar et al. 1977). These neurophysiological effects were associated with abnormal myelination because conduction velocity of a nerve impulse is a function of the fiber size and the state of myelination (Shah et al 1978, Shah and Salamy 1980). Whether or not the changes in the myelin membrane lipid composition are the underlying cause of the decreased conduction velocity, however, is not clear. In summary, published data are available to show that the total amount of myelin, its lipid quantity and composition is altered by undernutrition, protein malnutrition and essential fatty acid deficiency when the deficiencies are imposed during brain development. The effect of the dietary non-essential fatty acid supply, in particular oleic acid (18:1) on myelin composition and myelination has not been previously 16 investigated. The alteration of myelin fatty acid and lipid composition could conceivably alter the process of myelination as well as potentially influence myelin fiinction. 2.2 THESIS HYPOTHESIS AND OBJECTIVE Hypothesis: Feeding formula low in 18:1 results in reduced 18:1 in CNS myelin and alters the "normal" process of myelination in piglets. Objective: To determine the significance of the substantial quantities of oleic acid (35-40% fatty acids) in natural milk to the development of a normal brain myelin fatty acid composition, and to the rate of myelination, by comparison of CNS myelin from piglets fed formulas providing 17% or 38% 18:1, or fed sow milk with 38% 18:1. 2.3 MATERIAL AND METHODS 2.3.1 Chemicals All chemicals were purchased from Sigma Chemical Co., St Louis, MO., BDH Chemicals Canada Ltd., Vancouver, B.C. or Fisher Scientific, unless otherwise specified. Total cholesterol, and triglycerides were measured using the enzymatic kit from Diagnostic Chemical Ltd., Charlottetown, P.I. Free cholesterol was assayed using enzymatic kit reagents of Boehringer Mannheim Canada Ltd., Dorval, PQ. 2.3.2 Formulas Pig formulas were donated by Ross Laboratories, Columbus OH. 2.3.3 Equipment Low speed centrifugation (< 3000 g) was performed in a J-6B low speed centrifuge from Beckman 17 Instruments Canada Inc., Mississauga, ONT. Procedures requiring centrifiigation at 12,000 g were done in a B-20 high speed centrifiige International Equipment Company. High speed centrifiigation (75,000 g) was done using L8-55 or L7-55 Beckman ultracentrifuges. Gas chromatography was performed using a dual column Varian 6000 equipped with a Varian 654 data system or using a Varian 3400 equipped with an IBM computer system and Varian "STAR" software. Separation was achieved on 30m X 0.25mm glass cj^illary SP 2330 columns for all fatty acid methyl esters and a RTx-1, 25m x 0.25nMn capillary column from Restek, Corp., Bellefonte, PA. for plasma lathosterol. Spectophotometry was performed using a SP8-400 UVA I^S spectrophotometer. Pig immunoglobulins were concentrated using a Pelligan Ultrafiltration System, with a 100,000 nominal molecular weight limit filter, Millipore, Bedford, Massachusetts. Freeze drying was performed using a Dura Top Bulk Tray Drier and Dura-Dry condenser module fi-om FTS System Inc. Stone Ridge N.Y. Liver was coarsely homogenized in an electric blender from Philips Industries Ltd. Vancouver B.C. and then fiirther homogenized using Potter-Elvehem tissue grinders Wheaton from VWR, Scientific Canada Ltd. London, ONT, or a polytron Homogenizer from Janke and Kunkel GMBH and Co., Staufen, Germany. Brain tissue was homogenized in a Dounce tissue grinder Wheaton "200" using loose (A) and tight (B) pestles, from VWR, Scientific Canada LTD London, Ontario. 2.3.4 Immunoglobulin preparation Pig serum immunoglobulins were prepared according to Drew and Owen (1988). Approximately 120 1 of porcine abattoir blood was obtained. The blood was collected in 6 x 20 1 pails containing 25% (wt/vol) trisodium citrate as the anticoagulant (10 ml/1 blood) and allowed to settle overnight at 4°C. Throughout the preparation all materials were kept at 4°C. The following day, the blood was centrifuged at l(X)0g for 10 min, to remove red blood cells. Fibrin was precipitated by the addition of calcium chloride (4g/l plasma) and removed by centrifiigation (lOOOg for 10 min). Serum was fractionated using 100 ml/1 of a mixture of sodium polyphosphate glass (114.4 g/1) and sodium chloride (80g/l). After pH IS justment to 3.95 with 3N HCl, the mixture was stirred for 10 min, then allowed to equilibrate overnight. The next morning, the mixture was centrifuged at 3000g for 5 min to sediment the albumin. The immunoglobulin (IG) present in the supernatant was filtered through sand glass wool and concentrated by ultrafiltration. The concentrated fraction was stored at - 80°C and lyophilized for a minimun of 48 hrs prior to use. 2.3.5 Animals and diets. Male Yorkshire piglets of normal gestation (116-118 d) were obtained from Pitt Ineffable Growers, Pitt Meadows, BC Canada and from The University of British Columbia. Six, sow milk-fed piglets, each from a different litter, were kept on the farm and suckled by their mothers for 15 d. Piglets designated for formula feeding, six per group, were taken from the sow immediately after birth, before receiving colostrum. They were transferred to the Research Center, hand-fed every 3 h for 48 h, then every 3 h from 700-2300 h. The newborns were assigned at random to receive one of two formulas. The quantity of formula given was adjusted daily to achieve a growth rate of 200-250 g per day, equivalent to that of sow milk fed piglets. Littermates were not fed the same diet. Heating was provided with spot lamps attached above each cage. Passive immunity was provided to the formula fed animals by supplementation with pig serum-derived immunoglobulin (pig IG) to give an IgG content 20 mg/ml for the first 2 days and 5 mg/ml for the next 2 days. The piglets fed formula and those receiving sow milk all received 100 mg of an iron dextran complex (Pigtran 200) (intra muscular) on day 3 post partum. At 15 days post partum between 0900 to lOOOh (within 2 to 3 h after the last feed) blood was collected by cardiac puncture, with 150 g of EDTA/1 in 9 g NaCl/1 as the anticoagulant. The animals were then immediately killed by intracardiac injection of 10 ml of KCl (41 mmol). The composition of the formulas was based on infant formula but modified to resemble the macronutrient content of sow milk and to meet the nutrient requirements of growing piglets (NRC 1979). Formulas containing different amounts of 18:1 in the fat blend were achieved by using blends of different 19 unsaturated vegetable oils: 17% 18:1 formula, corn oil; 38% 18:1 formula, corn, soybean and high oleic-sunflower oils. The manipulation of 18:1 was at the expense of 18:2n-6. Saturated fatty acids were supplied by the inclusion of coconut oil at a similar level in the two formulas. The major fatty acid content of the sow milk and the formulas is shown in Table 2.2. Table 2.2 Fatty acid composition of sow milk and formula differing in 18:1 content. Fatty acids 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2n-6 18:3n-3 20:0 20:1 22:1 24:1 n-6LCP n-3 LCP Sow milk* ND 0.1 0.4 3.1 30.5 9.0 4.4 37.5 11.1 1.1 0.1 0.4 0.1 0.1 0.6 0.3 17 Formula % 18:1 % total fatty acids 3.7 2.9 22.9 9.4 10.4 ND 2.8 17.3 29.4 0.8 0.4 tr. tr. ND ND ND 38 3.1 2.4 20.3 8.3 7.8 0.1 3.3 38.4 15.5 0.6 0.2 0.1 tr. ND ND ND * means of two samples collected at 1 and 2 wk of lactation are given; ND, non detectable; tr., trace. 2.3.6 Tissue preparation 2.3.6.1 Plasma The plasma was separated by centrifugation of whole blood at 4 °C at 1000 g for 15 min. Plasma was separated from RBC aliquoted for analysis and stored at -°80 C. 20 2.3.6.2 Liver The liver was perfused through the portal vein with ice-cold saline to remove blood from the organ. The liver was then removed and weighed, the entire organ was homogenized with 50 ml of cold saline in an electrical blender and stored at -80°C. The thawed homogenate was further homogenized with a tissue grinder prior to aliquoting for assay of protein and lipids. 2.3.6.3 Cerebrum and myelin preparation The cerebrum was immediately removed, weighed and minced and homogenized for preparation of myelin according to Norton and Poduslo (1979). In brief, the brain was minced and homogenized with a Dounce glass hand-held homogenizer in 5 vol/wt of 0.32 mmol/1 sucrose, 15 mmol/1 Tris HCl (Inunol/l EDTA, lmmol/1 MgCl2 and 1.5 mmol/1 glutathione) by using 5 strokes of the loose pestle and 8 stokes of the tight pestie. An aliquot was taken for brain lipid extraction. The volume of brain homogenate utilised for myelin preparation corresponded to 5 g of brain tissue. The 5% homogenate was then diluted to 20% by addition of 15 ml homogenizing buffer per g/brain used. Thirteen ml of the homogenate was layered over 10 ml of 0.85 M sucrose in each of eight ultracentrifuge tubes and centrifuged at 75,000 g X 30 min. The layers of crude myelin occurring at the interface of the two sucrose solutions were collected by aspiration with blunt needle. The myelin layers were then suspended in water to a final volume of 180 ml, centrifuged at 75,000g x 15 min and the supernatant discarded. The myelin pellets were dispersed in water, homogenized and brought to a total volume of 180 ml again. The suspension was centrifiiged 12,000 g x 10 min and the supernatant again discarded. The pellets were dispersed in 180 ml water and the centrifiigation procedure repeated. These two osmotic shocks are necessary to release contaminating impurities, primarly microsomal membranes and axoplasm, trapped in the myelin vesicles during the homogenization procedure. The low-speed centrifiigation sediments the reforming myelin vesicles, while material of microsomal dimensions and impurities remain in the supernatant. The myelin pellets were combined and suspended in 100 ml of 0.32 M sucrose. This suspension was layered 21 over 0.85 M sucrose in 8 tubes and centrifuged at 75,000 g x 30 min. The final sucrose gradient is designed to remove any large material not removed in the first sucrose gradient. The purified myelin was removed from the interface with a blunt needle and suspended in 200 ml of suspension buffer, pH 7.4 (1 mM EDTA, 1 mM MgCl2, 15 mM Tris HCl and 1.5 mM glutathione) and centrifuged at 75,000 g X 5 min to remove most of the sucrose and concentrate the sample. The pellets were combined, homogenized and suspended in a final volume of 6 ml of suspension buffer. Aliquots of myelin were stored at -80°C until analysis. 2.3.7 Tissue lipid analysis 2.3.7.1 Lipid extraction Total lipids from plasma, liver, myelin and brain homogenate were extracted according to Folch et al. (1957) using chloroform:methanol:water 2:1:0.66 (volWolWol). 2.3.7.2 Total cholesterol, free cholesterol and triglycerides. Total, fi"ee cholesterol and triglycerides were measured enzymatically by using commercial kits. Appropriate standards were used to construct a standard curve for each assay. The lipid contents in liver (total and free cholesterol and triglycerides) and in myelin (total cholesterol) were measured in the lipid extract after reconstitution of 50 /il of lipid extract in isopropyl alcohol (Hrboticky et al. 1990) 2.3.7.3 Lipid phosphorus Lipid phosphorus was measured using 20 /il of the myelin lipid extract according to Chen et al. (1956). The assay is based on the color formed by the reduction of a phosphorus molybdate complex by the ascorbic acid. The samples were digested by adding 500 fii perchloric acid, marbles were placed on the top of each tube and placed in heating blocks for 1/2 h at 160 to 180 °C. After removing all the tubes from the heating blocks, 2.0 ml of water was added, then 0.5 ml of ammonium molybdate and 0.5 mL 22 of ascorbic acid. Samples were then vortexed and placed into a water bath at 60°C for 20 min to allow development of color. Absorbance was measured at 820 nm by spectrophotometry. 2.3.7.4 Cerebrosides Brain cerebrosides were measured according to Svennerholm (1956). In this method, the sugars contained in cerebrosides (galactose) are converted to furfural derivatives, in strong sulfuric acid, and are then coupled to a phenol (orcinol). The color developed is determined by absorbance. An aliquot of the lipid extract was transferred to a tube and evaporated under nitrogen. The residue was dissolved in 0.5 ml of ethanol and 2 ml 3N H2SO4 was added. The hydrolysis was performed in the closed tube for 2 h in a boiling-water bath. After cooling the tube in running cold water, the hydrolysate was filtered into a 10 ml cylinder. The hydrolysis tube was rinsed three times with 1 ml distilled water, which was then transferred to the filter. Distilled water was then added to the 10 ml mark. Three samples of 2 ml hydrolysate were pipetted into test-tubes placed in an ice bath. After 15 min, 4 ml of orcinol reagent (in strong H2SO4) was added to the sample and duplicate tubes. To the third tube, 4 ml of blank reagent was added. After 15 min, the solutions were throughly mixed and placed in a water bath at 80°C for 20 min. After heating, the tubes were chilled in the ice bath and the absorbance read at 505 nm. Known standards of 0, 50, 100 and 200 fig galactose were analyzed through all the steps. It is assumed that all of the lipid sugar is cerebroside sugar; the total amount of cerebroside is obtained by multiplying the measured amount of sugar by 4.6. 2.3.7.5 Fatty acid analysis 2.3.7.5.1 Lipid classes separation Myelin PE and PC were separated from the galactolipids and other PL classes by plate chromatography on silica gel 60, 10 x 20 cm high performance thin layer chromatography (HPTLC) using methylacetate:/i-propanol: chloroform: methanol:0.25% KCl (25:25:25:10:9, vol/vol/vol/vol/vol) (Vitiello 23 and Zanetta 1978) and chIoroform:methanol:acetic acid:H20 (50:30:8:4 vol/vol/vol/vol) (Skipski et al. 1964), respectively as the solvent system. The myelin PL bands were identified by brief exposure to iodine v^K)rs (15 sec). A pilot study was done to confirm a published report (Srinivasa et al. 1977) that exposure to iodine vapors not exceeding 15 sec has no detectable effect on the concentration of any fatty acid when determined by GLC. Plasma and liver PL and CE were separated from other lipid classes by thin layer chromatography using petroleum ether-diethyl ether-acetic acid (85:15:3 vol/vol/vol) (Innis and Clandinin 1981) on silica gel F-254, 20 X 20 cm pre-coated TLC plates. The separated PL and CE were visualized in U.V. light after spraying with 2'7' dichlorofluorescein. All lipid classes from myelin, plasma and liver were eluted from the silica with chloroform, methanol 2:1 (vol/vol) and dried down under nitrogen. 2.3.7.5.2 Preparation of methyl esters and GLC The recovered myelin PE and PC fatty acids were converted to methyl esters in 1 ml boron trifluoride reagent at 100°C for 30 min and 10 min, respectively. Myelin total lipids were transmethylated directly from the myelin lipid extract in 1 ml of methanol: HCl (5:1 vol/vol) at 100°C for 90 min (Hrboticky et al. 1989). The recovered plasma and liver PL were transmethylated in methanol:HCl (5:1 vol/vol) at 100 °C for 5 min. Methyl esters of the fatty acids from CE were prepared using 1 ml of boron trifluoride methanol-benzene-methanol (1:0.86:1 vol/vol/vol), at 100°C for 45 min. All fatty acid methyl esters were partitioned twice with 3 ml of 0.9% NaCl and 4 ml pentane. The pooled pentane layers were dried under nitrogen. Fatty acid methyl esters were separated, identified and quantitified by gas liquid chromatography. Helium was used as the carrier gas at a column flow of 1 ml/min. The samples were injected with the inlet split set at 10:1. Samples were injected at 80°C and the oven temperature programmed to remain at 80°C for 2 min. The column temperature was then increased to 170°C at a rate of 20°C/min and stabilized for 25 min, and then raised to 195°C (20°C/min) and stabilized for 18 min. The column was then heated to 245°C at 20°C/min and stabilized for 20 min prior to subsequent 24 analyses. Fatty acid methyl esters were identified by comparison of retention times with those of authentic standards. Milk total fatty acids were methylated using a one step direct transesterification method (Lepage and Roy 1986). In brief, an internal standard of C17 (50 ^ 1) was dissolved in 2 ml of methanol-benzene (4:1 vol/vol) and added to 100 /il of bile or milk. 200 fil of acetyl chloride was slowly added over a period of 1 min. Tubes were tightly closed with teflon-lined caps and subjected to methanolysis at 100°C for 1 h. After the tubes had been cooled in water, 5 ml of 6 % K2CO3 solution was slowly added to stop the reaction and neutralize the mixture. The tubes were then shaken and centriftiged (2000 g x 10 min) and an aliquot of the benzene upper phase was injected into the chromatograph. The equipment for GLC was as described below. 2.3.8 Brain CNP'ase Brain CNP'ase was measured according to Prohaska et al. (1973). The brain homogenate was first solubilized with deoxycholate and CNP'ase activity was measured as inorganic phosphate released from 2'AMP, by alkaline phosphatase. The solubilization procedure consisted of adding 0.1 ml of 0.2 M Tris-HCl, pH 7.5, to 0.2 ml of the 10% (wt/vol) homogenate, then adding 0.2 ml of a 1% (wt/vol) sodium deoxycholate solution. After 10 min at 0-4 °C, the samples were diluted to a protein concentration of 0.1 mg/ml and homogenizated (five strokes). Five to 25 ng of protein was added to 7.5 mM 2'3'-cAMP, 50 mM Tris-maleate buffer, pH 6.2 in a final volume of 0.2 ml. The substrate reaction was carried out at 30°C for 10 min. A sample containing substrate and buffer without protein served as the reagent blank. The reaction was terminated by placing the tubes in a boiling water bath for 30 sec. The tubes were returned to the 30°C water bath where 0.1 ml of 0.3 M Tris-HCl containing 21 mM MgClj, pH 9.0, was added along with 60 tig (0.72 units) of £. coli alkaline phosphatase (EC 3.1.3.1), resulting in a final pH 8.5. The inorganic phosphate which was liberated was determined in the same test tube directly as the phosphomolybdic acid complex and the absorbance measured at 410 nm. To the reaction mixture, 1.5 25 ml of isobutanol:benzene was added to extract the yellow chromophore and 1.5 ml of ammonium molybdate, 1.5% (wt/vol) in 0.5 N H2SO4. was then added to the solution and vigorously mixed. The yellow upper layer was read against the isobutanol:benzene. Standard phosphate was carried through this procedure. One unit of enzyme activity is defined as that amount which produces 1 /tmole of 2'AMP from 2'3'-c AMP/min under these conditions. 2.3.9 Protein The liver, brain and myelin protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. 2.3.10 Statistical analysis One way ANOVA was used to determine significant differences in the mean values of each variable among formula and sow milk groups. Within a fatty acid, analysis of contrasts (comparisons) was examimed only if the overall F statistic P value was < 0.05. Bonferroni corrections were used to determine which differences were significant; the level of significance was set at P < 0.017. Formal tests of differences among groups utilized Fisher's Least Square Difference and were based on least squares means and standard errors calculated from the ANOVA. Outliers detected by residual analysis methods were removed and the analysis was repeated. All calculations were performed using the GLM procedure in the Number Cruncher Statistical System, version 5.1 (Kaysville, Utah) 2.4 RESULTS 2.4.1 Animal body and brain weight Body weights at 15 d of age, after feeding from birth, were similar among the diet groups (means + SEM, 4.1 ± 0.4, 4.3 + 0.4, 4.3 ± 0.3 kg for piglets fed sow milk, and the formula with 17% 18:1 and 38% 18:1, respectively). Brain weights were also similar among the sow milk and formula-fed piglets 26 (means ± SEM, 35.0 ± 1.0, 36.3 ± 0.9, 34.1 ± 0.9 g for piglets fed sow milk, the formula with low and high 18:1, respectively). After 15 days, the piglets formula intake was not significantly different between both groups (10.7 + 0.9 1 and 9.7 + 0.9 1 for piglets fed the formula with high and low 18:1, respectively). 2.4.2 Plasma and liver lipid composition. Plasma and liver total and free cholesterol concentrations were significanlty lower in all the piglets fed formula than in the piglets fed sow milk, with the exception of liver free cholesterol which was not different between piglets fed the formula with high 18:1 and piglets fed sow milk. Feeding the formula with high compared to low 18:1 did not result in any significant differences in plasma or liver total or free cholesterol. The plasma and liver triglyceride concentrations were not different among the piglets fed the formulas and those fed sow-milk (Table 2.3). Table 2.3 Plasma and liver lipid concentrations in piglets fed sow milk or formula with different 18:1 content. Plasma Liver Formula % 18:1 Formula % 18:1 Sow milk 17 38 Sow milk 17 38 mmol/L iig/mg protein Total cholesterol 5.8 ± 0.9 2.7 ± 0.0* 2.8 ± 0.3* 33 ± 3 20 ± 1* 23 ± 2* Free cholesterol 2.1 ± 0.4 0.9 ± 0.1* 1.2 ± 0 . 1 * 26 ± 2 19 ± 2* 22 ± 2 Triglycerides 1.7 ± 0.1 1.3 ± 0.2 1.3 ± 0.3 64 ± 8 59 ± 9 44 ± 11 Mean ± SEM (n=6/group) * indicates values significantly different from sow milk group,(p<0.05). There were no statistically significant differences between the groups of piglets fed the formulas. 2.4.3 Myelin lipid composition, brain cerebroside and CNP'ase activity. Artificial feeding with formula rather than natural milk feeding, or the vegetable oil blend providing high or low intakes of 18:1 in formulas did not influence the myelin cholesterol or lipid phosphorus concentration or the brain CNP'ase activity and cerebroside concentration (Table 2.4). 27 2.4.4 Plasma and Liver PL and CE fatty acid composition. Plasma and liver PL and CE fatty acid composition is shown in Table 2.5 and 2.6 respectively. The % 16:0 in plasma PL and CE and liver PL was significantly lower in the piglets fed formula than in piglets fed sow milk, with the exception of the liver PL, which although lower in piglets fed the formula with 38% 18:1 was not significantly different. No significant differences were found between groups of piglets fed formula. The % 16:1 in plasma and liver PL and CE was consistently higher in naturally-fed compared to artificially-fed piglets. No significant differences were found in Table 2.4 Myelin lipid, brain cerebroside and 2',3' cyclic nucleotide 3'-phosphohydrolase (CNP'ase) activity of piglets fed sow milk or formula differing in 18:1 content. Formula % 18:1 Sow milk 17 38 Cholesterol 1.67 ± 0.05 1.60 ± 0.13 1.60 ± 0.11 ^mol/mg protein Phospholipids 1.68 ± 0.04 1.74 ± 0.12 1.76 ± 0.19 /tmol/mg protein Cerebrosides 0.12 ± 0.00 0.12 ± 0.01 0.13 ± 0.02 /imol/mg protein CNP'ase 1.16 ± 0.02 1.17 ± 0.03 1.16 ± 0.04 jumol/min.mg protein Data are means ± SEM (n=5-7). No significant differences were observed among groups. plasma PL or CE or liver CE 16:1 between the two groups of formula fed piglets. The proportion 16:1 was significantly higher in the liver PL of piglets fed the high 18:1 formula than in those fed the low 18:1 formula. The formula with low 18:1 (17%) resulted in a significantly lower proportion of 18:1 in the piglet plasma and liver PL (Table 2.5, Figure 2.1 A, B) and CE (Table 2.6, Figure 2.1 A, B) than feeding sow milk or the formula with 38% 18:1. Levels of 18:1, however, were significantly higher in the plasma and liver PL of piglets fed the formula with 38% 18:1 than in piglets fed sow milk containing 37.5 % of 18:1. In contrast, feeding the formula with 38% did not result in a higher plasma or liver CE % 18:1 compared to feeding sow milk. The sum of monounsaturated fatty acids in plasma and liver PL 28 and CE was also lower in piglets fed the formula with low 18:1 compared to those fed the formula containing 38% 18:1 or sow milk. As found for 18:1, the sum of monounsaturated fatty acids was higher in plasma and liver PL, but not in the CE fractions in piglets fed the formula with 38% 18:1 when compared to piglets fed sow milk. The % 18:2n-6 in plasma and liver PL and CE, was consistently higher in both groups of piglets fed formula than in the group fed sow milk. An exception was the liver CE of piglets fed the formula with high 18:1, for which the 18:2n-6 levels, although higher, were not significantly different from the piglets fed sow milk (P<0.03). Feeding the formula with low 18:1 and containing high 18:2n-6 (30%) resulted in higher plasma and liver PL and CE 18:2n-6 than feeding the formula with high 18:1 and containing only 16% 18:2n-6 (Table 2.5, 2.6). The % 20:4n-6 was significantly lower in plasma PL of piglets fed the formula with high 18:1, and in liver CE of both groups of piglets fed formula than in the group fed sow milk. Piglets fed the formulas also had significantly lower % 22:6n-3 in their plasma and liver PL than piglets fed sow milk. 2.4.5 Fatty acid composition of the myelin total lipid and PE and PC. The fatty acid composition of the myelin total lipid and isolated myelin PE and PC is shown in Table 2.7. Results for the myelin PE and PC fractions were studied because they represent the major phospholipid classes in CNS myelin, are especially rich in 18:1, and the PE also shows the largest fatty acid compositional change during development (Svennerholm et al. 1978). In contrast to liver and plasma, the proportion of 18:1 and total monoenoic fatty acids in myelin PE and PC and total lipid was generally lower in both groups of piglets fed formula than the group fed sow milk. These differences, however, were not statistically different (Fig 2.2, Table 2.7). No significant differences were found in the myelin PE or PC or myelin total lipid 18:1 between the groups of piglets fed the low (17%) and those fed the high 18:1 (38%) formula. No significant differences in the % of the any fatty acid was found in the myelin PE and PC among the three dietary groups. A few significant 29 differences were, however, observed in myelin total lipid between naturally and artificially fed piglets. The proportion of myelin total lipid 16:0 was significantly higher in the piglets fed the formulas than in the piglets fed sow milk (P<0.017, Table 2.7). The proportion of 18:2n-6 was higher in myelin total lipid from piglets fed the formula with low 18:1 and containing 29.4% 18:2n-6 than in piglets fed sow milk containing 11% 18:2n-6. No differences were found in the levels of any fatty acids in the myelin total lipid between the two groups of piglets fed formula, with the exception that the % 18:2n-6 was higher in piglets fed the formula containing low 18:1 and high 18:2n-6 (29.4%) than in piglets fed the formula with high 18:1 and low 18:2n-6 (16%). 30 Table 2.5 Fatty acid composition of plasma and liver phospholipid of piglets fed sow milk or formula differing in 18:1 contoit. 04 Fatty acids 16:1 18:1 20:1 22:1 24:1 £ Monos 16:0 18:0 22:0 24:0 £ Saturates 18:2(n-6) 20:4(n-6) 22:4(n-6) 22:5(n-6) 18:3(n-3) 20:5(n-3) 22:6('n-3) Sow milk 1.3 ± 0.1 14.9 ± 0.2 0.2 ± 0.0 tr. 0.3 ± 0.1 16.5 ± 0.3 23.6 ± 0.8 20.4 ± 0.4 0.2 ± 0.1 0.1 ± 0.0 45.2 ± 0.7 20.7 ± 0.1 11.1 ± 0.5 0.2 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.4 ± 0.0 2.6 + 0.2 Plasma phospholipid Formula % 18:1 17 0.1 ± 0.0* 8.8 ± 0.2** 0.1 ± 0.0*" tr. 0.2 ± 0.1 9.3 ± 0.2*« 19.0 ± 0.8* 24.0 ± 0.8* 1.0 ± 0.4* 0.6 ± 0.2 46.1 ± 0.9« 31.5 ± 1.2*« 9.5 ± 0.6 0.6 ± 0.1* 0.8 ± 0.1* 0.2 ± 0.0 0.0 ± 0.0* 0.9 + 0.1* 38 0.1 ± 0.0* 20.8 ± 0.5* 0.2 ± 0.0 tr. 0.2 ± 0.1 21.3 ± 0.5* 16.8 ± 0.4* 22.5 ± 0.5 0.6 ± 0.2* 0.4 ± 0.1 41.4 ± 0.7* 25.3 ± 1.1* 8.4 ± 0.6* 0.4 ± 0.1 0.6 ± 0.1* 0.3 ± 0.0 0.0 ± 0.0* 1.3 + 0.2* Sow milk 1.2 ± 0.1 11.2 ± 0.3 0.1 ± 0.0 tr. 0.0 ± 0.0 12.6 ± 0.3 17.4 ± 0.7 26.5 ± 1.0 0.2 ± 0.1 0.4 ± 0.0 45.4 ± 1.7 12.9 ± 0.6 18.7 ± 0.5 0.6 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 0.4 ± 0.1 5.5 + 0.5 Liver phospholipid Formula % 18:1 17 0.1 ± 0.0** 6.7 ± 0.1*» 0.1 ± 0.0 tr. 0.1 ± 0.0 7.0 ± O.I** 15.5 ± 0.6* 28.9 ± 0.5 0.1 ± 0.0 0.2 ± 0.0* 45.7 ± 0.3 22.2 ± 0.2** 18.8 ± 0.5 1.1 ± 0.1** 1.6 ± 0.1** 0.1 ± 0.0 0.0 ± 0.0* 1.5 + 0.1* 38 0.3 ± 0.0* 15.1 ± 0.6* 0.2 ± 0.0 tr. 0.2 ± O.I 15.8 ± 0.5* 15.9 ± 0.5 27.7 ± I.O O.I ± 0.0 0.2 ± 0.0* 44.7 ± 0.8 16.4 ± 0.7* 17.1 ± 0.6 0.7 ± O.I 1.0 ± 0.2* O.I ± 0.0 0.0 ± 0.0* 2.0 + 0.3* Data represent mean ± SEM (n= 17% 18:1 different from value for 5-7/group). * Indicates value significantly different piglets fed the formula with 38% 18:1 (p <0.017); from sow milk (p < 0.005), * indicates value for piglets fed the formula with tr; trace. 31 Table 2.6 Fatty acid composition of plasma and liver cholesterol esters of piglets fed sow milk or formula differing in 18:1 content U4 Fatty acids 16:1 18:1 20:1 22:1 24:1 £ Monos 16:0 18:0 22:0 24:0 L Saturates 18:2(n-6) 20:4{n-6) 22:4(n-6) 22:5(n-6) 18:3(n-3) 20:5(n-3) 22:6(n-3) Milk 6.4 ± 0.4 27.5 ± 2.6 nd tr. nd. 34.0 ± 2.2 23.0 ± 1.6 3.9 ± 0.3 tr. 0.1 ± 0.0 27.5 ± 1.6 31.9 ± 1.0 3.6 ± 0.2 0.3 ± 0.1 nd. 1.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 Plasma CE Formula % 18:1 17 1.0 ± 0.1* 13.2 ± 0.4** nd. tr. nd. 14.4 ± 0.5** 11.3 ± 0.5* 2.0 ± 0.2* tr. 0.2 ± 0.0 15.6 ± 0.9* 63.9 ± 1.3*« 4.2 ± 0.3 0.1 ± 0.0 nd. 0.3 ± 0.0"* 0.1 ± 0.0 0.3 ± 0.1 38 % total fatty 1.2 ± 0.1* 30.4 ± 0.3 nd. tr. nd. 31.7 ± 0.3 11.4 ± 0.3* 2.0 ± 0.2* tr. 0.2 ± 0.0 15.3 ± 0.5* 47.3 ± 1.1* 3.5 ± 0.4 0.1 ± 0.1 nd. 0.6 ± 0.0* 0.1 ± 0.0 0.4 ± 0.1 Milk acids 6.2 ± 0.5 34.8 ± 2.7 nd. 0.3 ± 0.1 nd. 41.8 ± 2.4 21.0 ± 1.5 8.3 ± 1.7 0.2 ± 0.0 0.1 ± 0.0 31.5 ± 2.1 21.0 ± 2.1 4.1 ± 0.3 0.4 ± 0.1 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 Liver CE Formula % 18:1 17 1.6 ± 0.2* 18.4 ± 0.9*« nd. 0.4 ± 0.1 nd. 21.1 ± 1.0** 18.6 ± 1.1 8.3 ± 0.4 0.6 ± 0.1 0.2 ± 0.1 30.7 ± 1.9 42.0 ±1.8** 2.3 ± 0.2* 0.2 ± 0.0 0.0 ± 0.0 0.2 ± 0.1 0.1 ± 0.0 0.4 ± 0.0 38 2.1 ± 0.6* 32.8 ± 2.6 nd. 0.5 ± 0.1 nd. 36.1 ± 1.9 17.3 ± 1.0 8.7 ± 1.0 0.6 ± 0.1 0.4 ± 0.2 30.5 ± 2.0 27.6 ± 1.9 2.2 ± 0.3* 0.2 ± 0.1 0.1 ± 0.0 0.3 ± 0.2 0.1 ± 0.1 0.4 ± 0.1 Data represent mean ± SEM (n=5-7/group) 18:1 different from value for piglets fed the . * Indicates value significantly different from sow milk (p < 0.005), * indicates value for piglets fed the formula with 17 % formula with 38 % 18:1 (p <0.017); tr., trace; ND, non-detectable. 32 Plasma 18:1 o (0 •+-> o 6^ B 40 Liver 18:1 30 -20 -10 -0 PL Figure 2.1. The effect of sow milk and formula varying in 18:1 content on the proportion of 18:1 in piglet plasma PL and CE (2.1 A) and liver PL and CE (2.1 B). The diets are represented as: sow milk, solid bars; formula with 17% 18:1, bars with diagnonal lines; formua with 38% 18:1, open bars. Values are means + SEM (n=6-7). ^ indicates value for 17% 18:1 different from the 38% 18:1 formula group, p< 0.002, * indicates values for formula group different from sow milk group (p< 0.017) 33 Table 2.7 Fatty acid composition of myelin phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and total lipid (TL) of piglets fed sow milk or formula differing in 18:1 content. Fatty acids 16:1 18:1 20:1 22:1 24:1 £ Monos 16:0 ^ 18:0 22:0 24:0 £Sats 18:2(n-*) 20:4(n-6) 22:4(n-6) 22:5(n-6) 18:3(n-3) 20:5(n-3) 22:6(n-3) Phosphatidylethanolamine Sow milk 1.6 ± 0.0 40.7 ± 1.4 3.1 ± 0.3 0.5 ± 0.1 tr. 46.1 ± 1.3 10.7 ± 1.1 11.1 ± 0.4 0.9 ± 0.3 0.5 ± 0.1 25.6 ± 1.7 1.8 ± 0.1 12.7 ± 0.1 7.5 ± 0.7 0.6 ± 0.1 0.1 ± 0.1 0.4 ± 0.1 2.6 ± 0.2 Formula % 18:1 17 1.1 ± 0.2 39.9 ± 1.9 2.7 ± 0.2 0.5 ± 0.0 tr. 44.4 ± 1.7 10.6 ± 1.0 10.3 ± 0.3 1.5 ± 0.5 0.3 ± 0.1 24.7 ± 0.9 2.5 ± 0.3 13.8 ± 0.3 8.2 ± 1.1 1.0 ± 0.3 0.1 ± 0.0 0.3 ± 0.1 2.5 ± 0.5 38 1.3 ± 0.1 39.0 ± 1.0 3.0 ± 0.2 0.5 ± 0.1 tr. 43.9 ± 0.9 12.2 ± 1.1 10.7 ± 0.2 1.8 ± 0.4 0.7 ± 0.2 27.3 ± 1.4 1.8 ± 0.1 12.6 ± 0.5 7.6 ± 0.7 0.8 ± 0.1 0.3 ± 0.1 0.5 ± 0.0 2.8 ± 0.4 Sow milk 1.0 ± 0.2 35.5 ± 0.5 0.5 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 37.7 ± 0.4 33.3 ± 1.3 19.5 ± 0.8 2.5 ± 0.3 1.1 ± 0.2 58.1 ± 0.2 1.8 ± 0.1 0.4 ± 0.0 0.0 ± 0.0 nd. nd. nd. 0.1 ± 0.0 I%osphatidylcholine Formula % 18:1 17 1.0 ± 0.1 34.4 ± 0.7 0.5 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 36.3 ± 0.7 36.1 ± 0.7 18.3 ± 0.3 2.1 ± 0.4 0.8 ± 0.1 59.7 ± 1.0 2.3 ± 0.2 0.4 ± 0.1 0.0 ± 0.0 nd. nd. nd. 0.2 ± 0.1 38 1.0 ± 0.1 32.1 ± 1.5 0.6 ± 0.1 0.1 ± 0.1 0.1 ± 0.0 33.1 ± 1.1 33.1 ± 1.4 20.7 ± 0.8 3.3 ± 0.5 1.2 ± 0.2 61.5 ± 1.2 1.9 ± 0.2 0.8 ± 0.4 0.2 ± 0.2 nd. nd. nd. 0.3 ± 0.1 Sow milk 1.5 ± 0.1 33.9 ± 0.5 1.5 ± 0.1 0.4 ± 0.1 1.7 ± 0.1 39.0 ± 0.6 13.6 ± 0.4 22.3 ± 0.3 2.8 ± 0.0 3.9 ± 0.1 45.1 ± 0.6 1.5 ± 0.1 6.5 ± 0.1 3.2 ± 0.2 0.3 ± 0.1 tr. 0.1 ± 0.0 1.7 ± 0.1 Total lipid Formula % 18:1 17 1.5 ± 0.1 32.8 ± 0.2 1.3 ± 0.1 0.4 ± 0.1 1.5 ± 0.1 37.6 ± 0.3 15.4 ± 0.5* 21.3 ± 0.3 2.8 ± 0.1 4.0 ± 0.2 46.2 ± 0.7 2.1 ± 0.2*' 6.6 ± 0.3 3.2 ± 0.3 0.5 ± 0.1 tr. 0.1 ± 0.0 1.5 ± 0.1 38 1.7 ± 0.1 33.1 ± 0.5 1.3 ± 0.1 0.3 ± 0.1 1.4 ± 0.1 38.1 ± 0.5 15.4 ± 0.5* 21.7 ± 0.4 2.7 ± 0.1 4.1 ± 0.2 46.5 ± 0.8 1.5 ± 0.1 6.3 ± 0.2 3.0 ± 0.3 0.4 ± 0.1 tr. 0.2 ± 0.0 1.6 ± 0.1 Data represent mean ± SEM (n=5-7/group). * indicates value significantly different from sow milk (p < fixjm value for the piglet fed the formula with 38% 18:1 (p <0.017); tr., trace, nd., not detected. 0.005), • indicates value for piglete fed the formula with 17% 18:1 different 34 OT 'o O >^  - 1 - ' o 1-4— o 30 2b 70 ^ Myelin 18:1 45 40 35 -15 -10 5 -0 Figure 2.2 The effect of feeding sow milk or formula varying in 18:1 content on the proportion of 18:1 in piglet myelin phosphatidylethanolamine (PE), phosphatidylcholine (PC), and total lipids (TL). The diets are represented as: sow milk, solid bars; formula with 17% 18:1, bars with diagnonal lines; formula with 38% 18:1, open bars Values are means ± SEM (n=5-7). No statistical differences between groups were found. 35 2.5 DISCUSSION I^iblished studies have reported significantly lower levels of monoenoic fatty acids in brain total lipid of piglets fed formula containing about 17% 18:1 than in piglets fed sow milk with 37% 18:1 (Hrboticky et al. 1990). Oleic acid (18:1) is a major fatty acid component of myelin PL, and its proportion in brain total lipid increases with progressive myelination (Svennerholm et al. 1978). This knowledge raises concern that the reduced 18:1 in the brains of piglets fed formula could reflect deposition of myelin with an abnormal fatty acid composition, or a delay in the progess of myelination. In my study, I wanted to determine if a low dietary intake of 18:1 during the postnatal exponential phase of myelination influences the proportional content of 18:1 in myelin lipid and/or the rate of myelination in piglets. In agreement with previous reports (Putnam et al. 1982, Hrboticky et al. 1990, Innis et al. 1990), the plasma and liver PL 18:1 was clearly influenced by the dietary supply of 18:1. The proportion of 18:1 was significantly lower in the plasma and liver PL of piglets fed formula with a fat blend providing 17% 18:1 compared to piglets fed sow milk or a formula with 38-40% 18:1. Similar findings were observed in plasma and liver CE fatty acids. Feeding the formula with 38% 18:1, however, resulted in a significantly higher % 18:1 in the plasma and liver PL than found in piglets fed sow milk. The extent of increase in 18:1 (Figure 2.1) does not seem to be readily explained by the small, 1-3% higher content of 18:1 in the formula than in the sow milk. Although the significance of these results is not clear they indicate that not only the level of 18:1, but also the level of other fatty acids or conceivably other non-lipid, or non-nutritive differences between natural milk and artificial infant feeds may be important to fatty acid metabolism and assimilation during development. As has been shown by others (Svennerholm et al. 1978), the analyses of piglet brain myelin PE, PC and TL, fatty acids demonstrated that 18:1 is the predominant fatty acid, representing more than 30-40% of the total fatty acids. The marked differences in plasma and liver PL and CE 18:1 among the piglets fed the different diets did not extend to brain myelin PE, PC or TL (Table 2.7, Figure 2.2). The results of the present investigation show that the fatty acid composition of the CNS myelin was not significantly 36 altered by formula providing 18:1 over the range of 17 to 38% fatty acids. Levels of 18:1 were high in the myelin PE, PC and TL irrespective of the substantial diet-induced decrease in the circulating lipid 18:1 of piglets fed the formula low in 18:1. The limited effect of the dietary and circulating lipid 18:1 supply on CNS myelin could possibly be explained by fatty acid synthesis and A 9 desaturation in the myelin-forming oligodendrocytes, rather than a dependence of uptake of plasma fatty acids for incorporation into newly formed myelin. These cells are known to be capable of synthesizing large amounts of lipid during the brain growth spurt (Sastry 1985) Of importance, feeding formula with a lower proportion of 18:3n-3 (0.6 or 0.8%, % total fatty acids, about 0.3% Kcal) did not alter the myelin n-3 fatty acid composition when compared to that of piglets fed sow milk. Previous studies have shown lower proportions of 22:6n-3 in CNS synaptic plasma membranes and retina, as well as other organs, (Hrboticky et al, 1989, Hrboticky et al. 1990, Arbuckle et al. 1991, Hrboticky et al. 1991, ) of piglets fed similar formula with 18:3n-3 at 0.3% diet Kcal. A number of studies in rodents have shown that severe dietary deprivation of 18:3n-3 fatty acids (0.02% diet Kcal) during development resulted in marked changes in the fatty acid composition of CNS myelin, including a reduction in the % 22:6n-3 (Youyou et al, 1986, Enslen et al. 1991,). The present study suggests that piglet brain myelin is relatively resistant to marginal dietary restriction of 18:3n-3 (0.3% Kcal) compared with other brain cell membrane fractions, such as synaptosomes (Hrboticky et al. 1989), or retina (Hrboticky et al. 1991). The % 16:0 in the myelin TL was significantly higher in both groups of piglets fed formula than in the sow milk-fed piglets. The reason why the formula diets increased the myelin % 16:0 is not known, particularly since both of the formulas had less than 1/3 the amount of 16:0 present in sow milk. The proportion of 18:2n-6 was higher in the myelin TL of piglets fed the formula containing 17% 18:1 with 29.4% 18:2n-6 than in the piglets fed sow milk or the formula containing 38% 18:1 and only 15.5% 18:2n-6 (Table 2.7). A similar diet-related increase in the brain % 18:2n-6 has been reported previously for piglets fed formula with high 18:2n-6 rather than milk (Hrboticky et al. 1990). The possible 37 physiological implications of these minor fatty acid changes in myelin lipids are unknown. The formula 18:1 content did not alter the myelin cholesterol or lipid phosphorus content, the brain CNP'ase activity or the brain cerebroside concentration (Table 2.4). It seems likely, therefore, that the "normal" process of myelination was not altered by the formulas fed to these piglets. In sununary, the studies described in this thesis have demonstrated that neonatal piglet plasma and liver PL and CE 18:1 levels are affected by the dietary 18:1 content. The changes in myelin fatty acids in piglets fed formula rather than milk were small, and did not seem to be directly related to the formula supply of 18:1. These results suggest that the amount of 18:1 in infant formula in the range of 17-38% fatty acids has no effect on the normal accretion of monoenoic fatty acids in cerebrum myelin lipids. These results are not in conflict with previous studies which found a reduced accretion of 18:1 in the brain total lipid of piglets fed formula containing low 18:1 (Hrboticky et al. 1990), but now suggest that brain membrane lipids other than myelin may be more sensitive to the dietary, and presumably plasma, fatty acid supply. A further important finding from the present study is that formula supplying 0.3% of energy as 18:3n-3 seems to support normal fatty acid patterns in myelin, despite clear deficiency in brain total lipid (Hrboticky et al. 1990), synaptic plasma membrane (Hrboticky et al. 1989) and retina (Hrboticky et al. 1991). These results may have important implications for understanding requirements and effects of diet fat on fatty acid accretion and metabolism by the oligodendrocytes as compared with other CNS cells and subcellular fractions. 2.5.1 Conclusive remarks and future directions. One of the major findings of this investigation is that the levels of 18:1 in myelin isolated from developing piglet cerebrum does not depend on dietary 18:1 supply. The myelin PE, PC and TL 18:1 composition, and rate of myelination was not affected by the lower levels of plasma 18:1 in piglets fed the formula providing only half as much 18:1 as found in milk. In previous studies (Hrboticky et al. 38 1990), the proportion of 18:1 was found to be reduced in brain of piglets fed the same formula containing only 17% 18:1. Therefore, the question still unanswered is : what cell and/or subcellular fractions, other than myelin, are sensitive to the dietary 18:1 supply ? This could be addressed experimentally by determining the fatty acid composition of various cells such as astrocytes, neurons, olidendocytes and cell and subcellular membranes such as synaptic plasma membrane, mitochondrial and microsomal membranes from brain of piglets fed different amount of 18:1. These studies would allow identification of the structures within the brain that are more dependent of circulating 18:1. Studies with piglets fed formula containing low 18:1 have already shown reduced monoene content, especially 18:1, in the synaptic plasma membrane compared to piglet fed sow milk (Hrboticky et al. 1989). Some other questions also arise from these studies. First, is the early postnatal period in piglets (0 -15 days), representive of the period for which myelin is most vulnerable to nutritional fatty acid deficiency ? It is well known that once formed, myelin is relatively stable (Wiggins 1982). Therefore, the period at which nutritional deficiency occurs is of great importance. This period of vulnerability has been defined to include the proliferation and development of the myelin forming cells, and the cellular mechanism underlying myelin initiation (Wiggins 1982). The period of actual myelin synthesis has not been identified to be particularly vulnerable to dietary deprivation (Wiggins 1982). In contrast, Davison and Dobbing (1966) defined the vulnerable period of brain growth to include both the proliferation of myelin forming cells (oligodendrocytes) and the actual synthesis of myelin. Because these sequential events occur pre- and postnatally in pig as well as in human, it would be of interest to investigate the effect of dietary 18:1 supply by using species where these events occur exclusively postnatally. If dietary deprivation of 18:1 is investigated after birth, it may be more appropriate to look specifically at regions of the brain which develop in the postnatal rather than prenatal period. Although total myelin accumulation is most rapid in humans and pigs after birth (Sweasey et al. 1976, Wiggins 1982), different regions in brain myelinate at different times and rates (Gilles et al. 1983). For example, the optic tract begins to myelinate in the human at 25 weeks gestation. The cerebellum begins to myelinate at the 39 beginning of the third trimester from which time the hemispheral portions of cerebellum contain microscopic myelin. The corpus callosum contains only microscopic myelin at the end of gestation. Myelination of this region is almost completely a postnatal event (Gilles et al. 1983). Presumably, portions of the brain which myelinate first are less vulnerable, and structures which mature late are more vulnerable to postnatal nutritional deprivation. Due to the late maturation of specific region of the brain such as the corpus callosum, postnatal nutritional deficiency may have a greater impact on myelin isolated from this region than myelin isolated from the entire cerebrum. If dietary 18:1 restriction is found to alter myelin fatty acid composition in specific regions, it would be of interest to study if the biochemical changes are associated with neurophysiological function, such as nerve conduction velocity. Nerve conduction velocity has been associated with the fimctional state of myelination in brain of mice deficient in myelin (Shah and Salamy 1980) and in children with protein-calorie malnutrition (Kumar et al. 1977). A logical next step then, would be to determine if the biochemical and neurophysiological changes can be reversed later on by adequate supply of dietary 18:1. A fiindamental question which arises from the current studies is what is the origin of the 18:1 in newly formed myelin ? Little is known about the relative contribution of de novo synthesis of monounsaturated fatty acid by the oligodendrocytes and the uptake of these fatty acids from the circulation. It would be of interest to undertake kinetic studies to compare the contribution of uptake of 18:1 through the blood brain barrier and fransfer to the oligodendrocytes with synthesis 18:1 by these cells during active myelin deposition. The results of the studies here seem to suggest that desaturation of saturated precursors in oligodendrocytes is possibly the most important source of 18:1 for myelin synthesis during development. It is also possible that the oligodendrocytes may compensate for the low dietary and circulating 18:1 by synthesizing more 18:1 from its precursors in order to regulate and maintain the supply of 18:1 directed towards myelin lipid synthesis. Formation of 18:1 from 16:0 and 18:0 has been found to be a significant biosynthetic process in brain homogenate (Cook and Spence 1973). Dhopeshwarkar and collaborators (1970) showed direct uptake by adult rat brain of labeled 18:1 given intravenously or orally, but the 40 relative importance of the contribution of circulating 18:1 in not clear. It has been also shown that after subcutaneous injection to 18 day old mice, labeled 18:0 was transported into brain myelin and incorporated into lipids suggesting that blood fatty acids are important for the synthesis of myelin lipid in mice brain (Golzan-Devillierre et al. 1978). In contrast, a recent study by Marbois et al. (1992) reported that labeled 16:0 did not enter the brain, suggesting that developing brain produces all required 16:0 by de novo synthesis. Clearly more research is needed to understand the mechanism by which myelin acquires its saturated, monounsaturated, n-6 and n-3 fatty acids during brain growth. 41 Chapter 3 PART 2: EFFECT OF FORMULA CHOLESTEROL ON PLASMA AND TISSUE N-6 FATTY ACmS 3.1 INTRODUCTION 3.1.1 Cholesterol metabolism and transport Cholesterol is a steroid molecule characterized by a four ring core structure and is present as a structural component of animal cell membranes. Cholesterol may be present either as the unesterified sterol or esterified with a fatty acid. Cholesterol also serves an essential role as the precursor for synthesis of many important steroids such as bile acids, sex hormones (estrogens, androgens, progesterone), the adrenocortical hormones and vitamin D. Cholesterol may be synthesized in the body de novo from acetyl CoA. Varying amounts of cholesterol may also be obtained from the diet. Cholesterol catabolism and excretion is almost exclusively through the biliary system, either as cholesterol itself or as its bile acids products (Hunt and Groff 1990, Russel 1992). 3.1.1.1 Cholesterol Synthesis Almost all tissues in the body are capable of synthesizing cholesterol from acetyl CoA. The liver and intestinal mucosal cells are, however, some of the most active synthesizing organs and contribute over 90% of endogenous cholesterol synthesis. At least 26 steps are involved in formation of cholesterol from acetyl CoA. The synthesis of cholesterol occurs in three major steps: (1) a cytoplasmic sequence by which 3 hydroxy-3 methyl glutaryl CoA (HMG CoA) is formed from 3 mol of acetyl CoA, (2) the conversion of HMG CoA to mevalonate by the enzyme HMG CoA reductase, representing the important rate-limiting step of cholesterol synthesis, (3) the conversion of mevalonate to squalene, squalene to lanosterol and lanosterol to demosterol which is then converted to cholesterol. An additional route can occur; lathosterol, an intermediate compound of this pathway, can also be produced from lanosterol and then converted to cholesterol (Hunt and Groff 1990) 42 Rates of cholesterol synthesis can be assayed by a variety of techniques. In laboratory animals, the activity of HMG CoA reductase in isolated microsomes is commonly used as an index of cholesterol synthesis. A criticism of the in vitro assay of HMG CoA reductase is that the method does not yield data from which to calculate the actual rate of cholesterol synthesis under in vivo conditions. Several methods of estimating rates of cholesterol synthesis from analysis of blood lipids are also available. These include assay of the incorporation of [^ H] water into plasma cholesterol and measurement of plasma mevalonate or lathosterol concentrations by GLC. The lathosterol:choIesterol ratio in plasma has been shown to correlate with the rate of hepatic HMG CoA reductase activity (Bjorklem et al. 1987) and other measures of whole body cholesterol synthesis (Kempen et al. 1988) in human. Plasma lathosterol levels in humans increase with administration of cholestyramine, a resin which is known to stimulate hepatic HMG CoA reductase activity (Bjorklem et al. 1987). Unlike methodology based on [^ H] water incorporation, measurement of lathosterol requires only single point blood sampling and also offers an advantage of requiring very small plasma samples. 3.1.1.2 Cholesterol catabolism The most important pathway for the metabolism and excretion of cholesterol in mammals is the formation of bile acids. The conversion of cholesterol into bile acids involves more than 15 enzymes; of importance are the hydroxylases, oxidoreductase and conjugating system (Bjorkhem 1992). The conversion of cholesterol to bile acids in mammals begins with a modification of the ring structure involving the introduction of a hydroxyl group at position C-7 of cholesterol (Russel and Setchell 1992). This reaction is catalyzed by a unique cytochrome P-450 enzyme, 7 a-hydroxylase. This enzyme is believed to catalyze the rate limiting step in bile acid synthesis. One of the last steps of bile acid synthesis involves the addition of a carboxylic acid group at position C-17. Cholic acid, one of the major primary bile acid in humans has hydroxyl groups at C-7 and C-12 in addition to C-3 hydroxyl of the native cholesterol (Hunt and Groff 1990). The other bile acids differ from cholic acid only in the number of 43 hydroxyl groups attached to the ring. Quantitatively, 98% of the secreted bile acids are conjugated with glycine or taurine, which attach through the carboxyl group of the steroid (Russell and Setchell 1992). Conservation of the bile acid pool is facilitated by the efficient reabsortion of bile acids from the intestine. The major site for reabsorption for bile acids is the terminal ileum (Russell and Setchell 1992). The bile acids returning to the liver from the intestine down-regulate the activity of the 7a-hydroxylase, thereby reducing the formation of bile acids from cholesterol. In a situation where bile acids would be prevented from returning to the liver, the activity of this enzyme is up-regulated, stimulating the conversion of cholesterol and its excretion (Hunt and Groff 1990). One example of this is the use of resins for the freatment of hypercholesterolemia. The resins bind bile acids in the intestinal lumen, this prevents their return to the liver, increasing the synthesis of bile acids from cholesterol (Hunt and Groff 1990). 3.1.1.3 Cholesterol transport Because of its low solubility in water, cholesterol is carried in the plasma in lipoproteins. Lipoproteins are complexes of proteins (apoproteins) and lipids (free and esterified cholesterol, PL and TG). The five major classes of lipoprotein are chylomicrons, low density lipoprotein (LDL), intermediate density lipoprotein (IDL), very low density lipoprotein (VLDL) and high density lipoprotein (HDL) (Brown and Goldstein 1986, Hunt and Groff 1990) Cholesterol transport can be divided into two pathways; the exogenous and endogenous pathways (Brown and Goldstein 1986). In the exogenous pathway, dietary cholesterol is absorbed through the intestine and secreted along with triglycerides in chylomicrons. Secretion of chylomicrons into the lymph continues for several hours after consumption of a meal rich in fat (Hunt and Groff 1990). In the capillaries of the adipose tissue and muscle, the sn 1 and 3 ester bonds of the triglycerides are cleaved by the enzyme lipoprotein lipase, and the fatty acids and monoglyceride products are taken up by the tissues (Brown and Golstein 1986). The resultant cholesterol-rich chylomicron remnants are cleared by the remnant receptors in the liver. The cholesterol is then either further metabolized to bile acids and 44 secreted into the bile, secreted in bile as cholesterol, stored in the liver as cholesterol esters following acylation by the enzyme acyl cholesterol acyl transferase (ACAT), or packaged with triglycerides into VLDL particles and secreted into the circulation, inaugurating the endogenous pathway, (Figure 3.1). The cholesterol-rich particles remaining after hydrolysis of VLDL triglycerides known as IDL may in part be cleared by the liver LDL receptors or undefined scavenger receptors. The remaining IDL is further metabolized to LDL. Most of the LDL binds to LDL receptors on hepatic or extrahepatic cells and is removed from circulation. HDL is synthesized by both the liver and the intestine. The cholesterol contained in HDL is esterified in plasma in a reaction catalyzed by a plasma enzyme known as lecithin: cholesterol acyltransferase (LCAT), using mainly 18:2n-6 from the sn-2 position of PC (Sgoutas 1972). The enzyme is activated by apolipoprotein A-I, which is a major apoprotein component of HDL. An important fimction of HDL lies in its capacity to transfer free cholesterol from peripheral tissues to the liver, in a process often referred as reverse cholesterol transport. The cholesterol esters produced by the LCAT reaction in turn may be transferred from HDL to LDL. This allows the LDL fraction to return esterified cholesterol acquired from HDL to the liver. 3.1.1.4 LDL receptor and regulation of cholesterol metabolism. The LDL receptor was described in 1973 by Goldstein and Brown 1984. They were in search of the molecular basis for the clinical manifestations of familial hypercholesterolemia through study of cultured human skin fibroblasts. They found LDL binds to normal fibroblasts (and also other cells, particularly hepatocytes and cells of the adrenal gland) with high affinity and attributed this binding to the presence of highly specific receptor molecules that bind LDL and related lipoproteins (Goldstein and Brown 1984, Brown and Goldstein 1986, Hunt and Groff 1990). LDL receptors are synthesized on the endoplasmic reticulum of the cell. In the golgi apparatus they are targeted for their final destination, on the plasma membrane. It is the apoprotein B-lOO, carried on 45 the surface of LDL and apoprotein E, carried on chylomicron, IDL and VLDL that are recognized by the LDL receptor. The LDL binds the receptor and is then internalized into the cell by receptor-mediated endocytosis. The LDL is then separated from the teceptor and delivered to a lysosome which degrades the lipoprotein and hydrolyses the lipid constituents. The liberated receptor returns to the surface of the cell where it is able to bind to LDL again. Cholesterol liberated from LDL controls intracellular cholesterol metabolism by several pathways. These involve suppressing synthesis of LDL receptor protein, reducing de novo cholesterol synthesis, decreasing the mRNA for HMG CoA reductase, increasing formation of cholesterol esters by activating acyl cholesterol acyl transferase (ACAT), and increasing the formation of bile acids from cholesterol by activating the 7 oe-hydroxylase (figure 3.2). These regulatory mechanisms serve to maintain intracellular and plasma cholesterol homeostasis at remarkably constant levels. When there is a high concentration of LDL cholesterol in the plasma, the LDL receptor is generally down-regulated, whereas low plasma LDL cholesterol levels are usually associated with increased numbers of LDL receptors on the cell surface. An increased demand for cholesterol by the cell is usually met by an increase in both receptor number and cholesterol synthesis, the opposite is true when abundant cholesterol is present. Any defect in LDL receptor number and function can be detrimental. For example, the absence of LDL receptor or defective receptor binding is responsible for homozygous familial hypercholesterolemia, which is characterized by very high blood cholesterol and death from coronary artery disease during childhood. Several nutrients in addition to cholesterol, affect cholesterol metabolism by regulating the LDL receptors. In general, dietary saturated fatty acids which are known to increase blood LDL cholesterol have been shown to suppress the LDL receptor activity, while unsaturated fatty acids known to decrease the plasma LDL cholesterol increased receptor-dependent LDL transport (Grundy and Denke 1990, Woollett et al. 1992). However, other studies have shown similar abundance of hepatic LDL receptor mRNA in Green African monkeys fed polyunsaturated and saturated fatty acids (Sorci-Thomas et al. 1989). 46 Dietary Capillaries LDL receptor Extrahepatic cells Figure 3.1. Schematic representation of the exgenous and endogenous fat transport. 47 LDL receptor Apo B-100 Cholesterol Tinoleate Oversupply of cholesterol jHMG COAreductase I LDL receptors Cholesterol f A C A T Cholestero ' oleate Cholesterol tZocOH^ Bile Adds Figure 3.2. Schematic rq>resentation of sequential steps in the LDL receptor pathways of mammalian cells. HMG CoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase; ACAT, acyl-CoA: cholesterol acyltransferase; 7« OH, 7oe hydroxylase. 48 3.1.2 Plasma cholesterol and n-6 fatty acids levels in infants: Possible role of cholesterol in n-6 fatty acid metabolism. Many studies have shown that infants fed human milk have higher serum total cholesterol (Friedman and Goldberg 1975, Ginsburg et al. 1980, Wagner and von Stockhausen 1988) and LDL cholesterol concentrations (Van Biervliet et al. 1986, Wagner and von Stockhausen 1988,) than infants fed formula. The effect of human milk compared to infant formula on serum cholesterol or cholesterol metabolism are not well understood. Differences in serum cholesterol between infants fed formula and infants fed human milk may reasonably be attributed to the differences in fatty acid composition between most commercial infant formula and breast milk (Jensen et al. 1978). Another possible reason for the higher plasma cholesterol in naturally compared to artificially fed infants could also logically be due to the differences in cholesterol content of milk and formulas. Formulas designed for preterm infants often contain MCT oils providing medium chain saturated fatty acids (8:0-10:0) and/or coconut oils with intermediate chain length saturated fatty acids (12:0, 14:0). In comparison to human milk, such formulas have high levels of 8:0-14:0, low levels of 16:0 and usually low 18:1 and high 18:2n-6. Human milk usually provides about 0.3-0.5 mraol cholesterol/L whereas infant formulas prepared with vegetable oils provide only about 0.05 mmol cholesterol/L. In addition to lower plasma cholesterol concentrations, infants fed formula have generally higher levels of 18:2n-6 and lower levels of 20:4n-6 in their RBC PL (Putnam et al. 1982, Carlson et al. 1986, De Lucchi et al. 1987) and plasma PL (Koletzko et al. 1989). Similar results have been found in piglets fed formulas containing coconut and corn oils when compared to piglets fed milk (Hrboticky et al. 1990). The higher plasma and RBC PL 18:2n-6 may in part be explained by the high content of 18:2n-6 in most formulas. However, infants fed with formula containing similar levels of 18:2n-6 to human milk still show higher RBC and plasma PL 18:2n-6 than infants fed human milk (Putnam et al. 1982). The lower level of 20:4n-6 in infants fed formula could be due the absence of 20:4n-6 in conventional infant formula compared to the small amount, usually 0.5 % total fatty acids, in human milk. Another possible reason 49 may be related to the interaction of plasma cholesterol and n-6 fatty acids. The esterification of plasma free cholesterol in human and pig is catalyzed by lecithin:cholesterol acyltransferase (LCAT) using 18:2n-6 from the sn-2 position of plasma PC (Sgoutas 1972, Glomset 1979, Pownall et al. 1985). Therefore, the plasma PL 18:2n-6 levels may depend to some extent on the utilisation of 18:2n-6 for esterification of free cholesterol derived from absorption and transport of dietary cholesterol. Conceivably, transfer of plasma PL 18:2n-6 to CE could influence the return of 18:2n-6 to hepatic tissues and, possibly increase availability for desaturation of 20:4n-6. Thus it seems reasonable to question whether the lower dietary cholesterol intake and lower plasma cholesterol concentrations of infants fed formula results in a lower turnover of plasma PL 18:2n-6 for esterification of plasma cholesterol than infant fed human milk. Several studies have considered the effect of dietary cholesterol on 18:2n-6 and 20:4n-6 metabolism. However, of those available most have been done in rodents (Garg et al. 1985, Garg et al. 1986, Garg and Sabine 1988, Takahashi and Horrobin 1988, Lee et al. 1991). In general, these studies found that addition of cholesterol to the diet resulted in increased 18:2n-6 and decreased 20:4n-6, or a reduced ratio of 20:3n-6 + 20:4n-6 to 18:2n-6 in plasma and tissue PL. These changes were observed when cholesterol was fed at levels varying from 0.2% - 2% by weight of the diet. These results were suggested to be the result of inhibition of desaturation of 18:2n-6 to 20:4n-6 by decreasing the activity of the A5 and A 6 desaturase enzymes (Garg et al. 1985, 1986). There are important species differences in CE and lipoprotein metabolism between rats and human or pigs. The predominant CE in rat plasma are 20:4n-6 but in the human, like the pig, cholesterol is esterified mainly with 18:2n-6 (Sgoutas 1972, Pownall et al. 1985). Because of the interrelation between plasma cholesterol and fatty acid metabolism, the possible role of cholesterol in infant formula as a modulator of plasma and tissue n-6 levels needs to be clarified. The effect of cholesterol added to formula in similar amounts to that in milk on plasma and liver PL and cholesteryl ester n-6 fatty acids was investigated in piglets. 50 3.2 THESIS HYPOTHESIS AND OBJECTIVES Hypothesis: Addition of cholesterol to formula will result in lower 18:2n-6 and higher 20:4n-6 levels in plasma and liver PL compared to piglets fed formula low in cholesterol. Objegtivg; To determine the importance of substantial amount of cholesterol in natural milk by comparison of the effect of formula providing negligible quantities of cholesterol or formula containing cholesterol in similar levels to that of milk with the effects of milk on: a) 18:2n-6 and 20:4n-6 levels in plasma and liver PL and CE. b) indices of hepatic cholesterol metabolism. 3.3 MATERIAL AND METHODS 3.3.1 Chemicals Chemicals were purchased as mentioned in section 2.3.1. The silylating reagents (hexamethyldisilane, trimethylchlorosilane and dimethylformide) for the analysis of lathosterol were purchase from Pierce, Rockford, IL. DL-Imethyl-^H] HMG CoA (10.9 Ci/mmol) and [2-''*C] mevalonic acid (50.1 mCi/mmol) for the HMG CoA reductase assay, were obtained from Dupont (Canada) Inc., Missisauga, ONT. Aqueous scintillation fluid was from Amersham Canada Limited, Oakville, ONT. Ligand blotting chemicals and supplies were purchased form Bio Rad Laboratories, Mississauga, ONT. 51 3.3.2 Formulas Pig formulas were donated by Mead Johnson, Belleville. ONT. 3.3.3 Equipment Low and high speed centrifugations were done using the equipment as described in section 2.3.3. Gas liquid chromatography was performed using the equipment as described in section 2.3.3. Electrophoresis and electroelution were performed on a Bio-Rad Mini-Protean II apparatus and transfer cell. The dialysis tubing (Spectra/Por, molecular weight cutoff 12,000-14,000) was from Baxter Canlab, Bumaby, B.C. Densitometry was performed on a video densitometer (model 620) from Bio-Rad. Liver was homogenized using the equipment described in section 2.3.3 3.3.4 Animal and diets Male Yorkshire piglets of normal gestation (116-118 d), six per treatment group, were obtained from Jansen Farm, Abbotsford, British Columbia. Sow milk fed piglets, each from a different litter, were kept on the farm and suckled by their mother until they were 18 days old. Piglets designated for formula feeding were taken from the sow within 24 h of birth and assigned at random to receive one of the two formula diets. Littermates were not fed the same formula. Procedures relating to the housing and feeding of the piglets were as described for in section 2.3.5. At 18 days post partum the animals were anaesthetized with ketamin/rompun (37.73.75 mg/kg, respectively) by intramuscular injection between 0900 and 1000 h after an overnight fast of about llh. The animals were sacrificed by intracardiac injection of 10 ml KCl (41 mmol) then blood was collected by cardiac puncture with 15% EDTA in 9 g NaCl/1 (wt/vol) as the anticoagulant. The composition of the formulas was based on preterm infant formula but modified to resemble the macronutrient content of sow milk, and to meet the essential nutrient requirements of growing piglets (NRC 1979). The two formula diets were identical except for their cholesterol content. One formula 52 contained 42 mg cholesterol/dl (10 mg free and 32 mg cholesterol-palmitate/dl), the other formula contained negligible amounts of cholesterol (2 mg cholesterol/dl). The formula fat was a 40:40:20 (vol/vol/vol) blend of MCT, soybean and coconut oils respectively. The fatty acid composition of sow milk and the formulas is in Table 3.1. Table 3.1 Diet cholesterol content and fatty acid composition. Sow milk* Cholesterol** 0.336 Formula low in cholesterol mmol/L 0.05 Formula high in cholesterol 1.09 8:0 - 12:0 14:0 16:0 16:1 18:0 18:1 18:2(n-6) 18:3(n-3) LCn-9 LCn-6 LCn-3 0.4 3.1 30.5 9.0 4.4 37.5 11.1 1.1 0.6 0.6 0.3 % Total Fatty Acids 51.9 3.6 5.9 -2.4 11.2 21.7 3.1 ---51.9 3.6 5.9 -2.4 11.2 21.7 3.1 --_ *, means of 2 milk samples collected at 1 and at 2 weeks of lactation. -, trace; LC, long chain fatty acids of carbon chain 20-24. ** Values for sow milk and formula cholesterol content are from Jones et al 1990 and Mead Johnson respectively. 3.3.5 Tissue preparation 3.3.5.1 Plasma and plasma HDL The plasma was separated as described in section 2.3.6.1. A separate aliquot of 100 /xl was taken for immediate preparation of HDL by precipitation of the apo B lipoproteins with 1 ml heparin (10,000 units/ml), 2 ml MnClj (2.0 M) and 1 ml NaCl (0.15 M) 53 3.3.5.2 Bile Bile was obtained from the gallbladder by aspiration using a syringe and 23 G needle. The bile was and rapidly frozen in liquid nitrogen and stored at -80°C. 3.3.5.3 Liver The liver was homogenized in 225 mM sucrose and 25 mM Tris buffer, pH 7.4, containing glutathione, leupeptin and ^rotinin to final concentration to 10 nunol/l, 1 fimol/l, 0.3 nmol/1 respectively using an electrical blender. Aliquots were used for preparation of microsomes and plasma membrane, and the remainder was stored at -80 "C 3.3.5.4 Microsomes and plasma membrane Liver microsomes were obtained by centrifiigation of liver homogenate for 20 min at 10,000g and the resulting supernatant centrifiiged for 1 h at 100,000g (Goodwin and Margolis 1976). The microsomal pellets were resuspended in 100 mM sucrose containing 50 mM KCl, 40 mM KH2PO4, 30 mM EDTA and 20 mM dithiothreitol, pH 7.2. The liver was further homogenized with a Polytron homogenizer at setting 10 for 10 sec for the plasma membrane preparation (Kovanen et al. 1979). The homogenate was centrifiiged for 15 min at 500 g and the resulting supernatant centrifiiged again for 15 min at 10,000 g. This final supernatant was then centifuged for 1 h at 100,000 g. The pellet was washed twice by resuspending in buffer and recentrifiiging, then stored at -70°C. 3.3.6 Tissue lipid analysis 3.3.6.1 Lipid extraction Total lipids from plasma and liver were extracted as described in section 2.3.7.1. Bile lipids were extracted using chloroformrmethanol 1:2 (vol/vol). 3.3.6.2 Total cholesterol, free cholesterol and triglycerides Total, free cholesterol and triglycerides were measured as described in section 2.3.7.2. Total cholesterol 54 was measured in the bile lipid extract after overnight exposure to fluorescent light to permit photodegradation of biliary pigment (Gurantz et al. 1981). The liver total and free cholesterol and triglycerides were measured in the lipid extract after reconstitution of 50 ^1 of lipid extract in isopropyl alcohol (Hrboticky et al. 1990) 3.3.6.3 Lipid phosphorus Lipid phosphorus was measured using 10 nl bile according to Chen et al. (1956) as described in section 2.3.7.3. 3.3.6.4 Biliary bile acids The biliary bile acids were measured using an enzymic-spectrophotometric method according to Mashige et al. (1981). The principle of this method is as follows: The bile acids are converted to 3-oxo bile acids with 3 a-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD"^  to NADH, the hydrogen generated is then transferred, with diaphorase (EC 1.6.4.3) catalysis, to nitrotetrazolium blue (NTB) to yield the diformazan, which is measured spectrophotometrically at 540 nm. In brief, 400 fd of deionized water was added to 100 /xl of bile extract, 0.5 ml of reagent 1 (66.35 mg of NAD, 20 mg NTB, 50 units of diaphorase (resuspend in O.OIM PO4 buffer pH 7.0-7.4), and the mixture brought to 50 ml with 0.2 mM Tris HCL buffer pH 7.5 (containing 10 g /I brij 96). Next, 0.5 ml of solution 2 (0.25 units of 3 a HSD in 25 ml of solution 1) was added to duplicate tubes. The mixture was then mixed and incubated 10 min at 37°C in water bath. To stop the reaction, 0.1 nJ H3PO4 (1.33 moles/1) was added. The absorbance at 540 nm was measured with an appropriate reagent blank. 3.3.6.5 Lathosterol Plasma lathosterol was measured as described previously (Bjorkhem et al., 1987, Hamilton et al. 1992). In brief, 5oe cholestane (internal standard) was added to 100 /il of pig plasma and saponified with 50% KOH and methanol (6:94, vol/vol) for 1 h at 80°C. The samples were extracted 3 times with petroleum ether, dried and silylated with hexamethyldisilane:trimethylchlorosilane:dimethylformamide, 20:2:4 55 vol/vol/vol, 5 min at room temperature. The sterols were analyzed by GLC. Samples were injected at SO^ C after remaining at this temperature for 1 min. The oven temperature was programmed to increase to 120''C (20°C/min), hold for 7 min, rise to 249°C (20''C/min), hold for 15 min, rise to 269°C (20°C/min) and hold for 20 min. The oven was then heated to 320°C (20°/min) for 5 min prior to subsequent analyses. The injector and detector were set at 300°C and 320°C, respectively. The helium flow rate (carrier gas) was 1.2S ml/min with the inlet splitter set at 100:1 and the relays programmed to come on at 0.7 min after the run start. The lathosterol peak identification was confirmed using an authentic standard. The plasma lathosterol was reported as the ratio of lathosterol: cholesterol. 3.3.6.6 Fatty acid analysis 3.3.6.6.1 Lipid classes separation Plasma and liver PL and CE were separated from other lipid classes as described in section 2.3.7.5.1 3.3.6.6.2 Preparation of methyl esters and GLC The recovered plasma and liver PL and CE were transmethylated as described in section 2.3.7.5.2. All fatty acid methyl esters were partitioned as described in section 2.3.7.5.2. Fatty acid methyl esters were separated, identified and quantified by GLC as described in section 2.3.7.5.2. Bile and milk total fatty acids were transmethylated using a one step direct transesterification (Lepage and Roy 1986). 3.3.7 HMG CoA reductase activity Microsomal HMG CoA reductase activity (EC 1.1.1.34) was measured as the rate (pmol/min) of formation of [^ H] mevalonate from [^ H] HMG CoA (Goodwin and Margolis 1976). Briefly 100 jiil of microsome suspension (0.1-0.5 mg/protein) was aliquoted into 20 ml srew-cap tubes containing 0.05 ml of resuspension buffer (0.1 M sucrose containing 0.05 M KCl, 0.04 M KH2PO4 and 0.03 M EDTA at pH 7.2). The mixture was preincubated for 20-30 min at 37°C in an oscillating water bath. The reaction was then initiated with the addition of 100 /tl of a substrate-cofactor mix (O.SmM HMG CoA, 120 mM glucose-6-phosphate, 8mM NADP, 7 lU of glucose-6-phosphate dehydrogenase and 1.5 /iCi/ml DL-56 [methyl-^H] HMG CoA). The reaction was stopped 20 min later by the addition of 50 /il of 12 N HCl. Glucose-6-phosphate dehydrogenase makes the conversion of pH] HMG CoA to [^ H] mevalonate irreversible and regenerates NADPH. Addition of HCI converts [^ H] mevalonate to [^ H] mevalonolactone which can be extracted. The mixture was then incubated for a further 30 min, removed from the water bath and approximately O.S g sodium sulfite added. Mevalonolactone was extracted from the reacted mixture with the addition of 10 ml toluene followed by 2-3 min of vigorous shaking on an Eberbach shaker. After allowing the tubes to stand at room temperature for 30 min, a portion of the toluene extract was aliquoted into scintillation vials. Aqueous scintillation fluid was then added and the toluene counted for the [^ H] content. Blanks were run as above with HCl added before the substrate cofactor mix. Recovery was determined from the percentage of ['^ C] mevalonic acid recovered in toluene extracts as mevalonolactone. 3.3.8 LDL receptor relative mass The detection of the LDL receptor was done by ligand blotting, using gold-labelled rabbit /S VLDL. In brief, plasma membrane proteins are separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose blot is then incubated with gold-tagged lipoproteins. Visualization of goId-/SVLDL/LDL receptor bands is then inhanced by silver staining. j8 VLDL, rather than LDL, was used as the ligand because of its high affinity for the receptor. j8 VLDL is a cholesteryl ester-rich VLDL remnant which accumulates in the plasma of rabbits in response to cholesterol feeding. 3.3.8.1 /3 VLDL preparation Male adult rabbits (New Zealand) were used to produce j8 VLDL. Rabbits were fed rabbit chow mixed with 100 g com oil and 20 g cholesterol/kg for 2 wk. /SVLDL (density = < 1.0006 g/ml) was isolated by ultracentrifiigation of rabbit plasma at 100,000g at 15°C for 2 h. 3.3.8.2 Membrane solubilization All procedures were performed on ice. The frozen plasma membrane pellet was resuspended in buffer 57 B (250 mM Tris-maleate) (pH 6.0), 2 mM CaCl2, 4 nM leupeptin, 12 /iM aprotinin, 2 mM phenylmethylsulfonyl fluoride (PMSF) using 18 and 21 gauge needles. Following this, NaCl, Triton and distilled water were added to achieve a final concentration of 125 mM Tris-maleate (pH 6.0), 1 mM CaCl2, 2 nM leupeptin, 6 fiM aprotinin, 1 mM PMSF, 160 mM NaCl, Ig/lOOml Triton X-100. Samples were mixed using a vortex over the next 10 min and centrifuged at 100,000 g for 1 h. The soluble protein (supernatant) was stored in aliquots at -70°C and the protein content determined according to Lowry et al. (1951). 3.3.8.3 SDS-PAGE and electroelution Membrane protein samples were randomly allocated to lanes and were separated by SDS-PAGE (Laemmli, 1970) on 1.5 mm thick, 6% acrylamide gels, under non-reducing conditions (55 min, 200 V). Proteins were electroeluted to 0.45 fi nitrocellulose, without methanol (2 h, 290 mA) and the blots were stored at -70°C. 3.3.8.4 Gold-lipoprotein conjugation Colloidal gold was dialysed overnight against 5 mM citrate phosphate (pH 5.5) at 4°C to adjust the pH for conjugation of the lipoprotein. Gold was then vigorously added to /S VLDL (diluted to 0.3 mg/ml in a glass tube) with a glass pipette to achieve a concentration of 15 fig /SVLDL protein/ml gold. This was mixed by inversion, and bovine serum albumin (BSA) added as a stabilizer to achieve a concentration fo 1 g/ 100ml. The mixture was then dialysed overnight against Tris-buffered saline (TBS;20 mM Tris (pH 8.0) and 90 mM NaCl) at 4°C and used within 12 h. 3.3.8.5 Ligand blotting Non-specific binding was blocked by incubating blots with a 5 % BSA (wt/vol) blocking solution (2 mM CaClj TBS) for 2 h at room temperature on a slowly shaking platform. The blocking solution was then replaced by the blotting solution (7.5 /ig/ml gold-jSVLDL, 2.5 % BSA (wt/vol), 2mM CaClj, TBS) and incubated at room temperature for 1.5 h with slow shaking. Subsequently, blots were washed as follows: 58 twice with BSA-free blocking solution, then 5 min with blocking solution containing 1 % BSA (wt/vol), for 10 min with BSA-free blocking solution and for 2 min in distilled water. Detection of the receptor bands was enhanced using a silver-based gold enhancement kit. A quality control lane was present on every gel and data on absorbance from densitometry expressed as sample/confrol absorbance. Analyses were performed twice using 20 ng of sample protein, based on studies on linearity and saturation of receptor binding as described in 3.3.8.7 3.3.8.6 Receptor identification and characterization Receptor band identity was verified by performing ligand blotting on hepatic membrane from 18 day old piglets in the presence of EDTA (20 mM with no added CaCy or sodium suramin (5mg/ml). The molecular weight of the receptor band was determined by comparison to high molecular weight standards run under reducing conditions (with 2-mercaptoethanol). 3.3.8.7 Linearity To ensure the validity of comparing receptor protein among dietary groups, saturation curves for the binding assay were done for all the diet groups using various amounts of membrane protein (10-50 ^ g). Subsequent comparison among the groups was made using 20 /ig protein, a concentration that was within the linear range of the binding assay for all the diet groups. 3.3.9 Protein The liver protein was measured as described in section 2.3.9. The protein content of the /? VLDL was measured according to Markwell et al. (1981). Because of the high triglyceride content of the samples, they were extracted (Folch et al. 1957) before spectrophotometry. 3.3.10 Statistical analysis Statistical analysis were done as described in section 2.3.10 59 3.4 RESULTS 3.4.1 Growth and formula intakes Body weights at 18 days of age were similar between the groups of piglets fed the formula (mean ± SEM, 3629 ± 193, and 3843 ± 330 g for piglets fed the formula without and with cholesterol, respectively) but were significantly lower than for the group of piglets fed sow milk sow fed (5085 ± 176). The piglets fed the formulas, however, had similar heart, brain, liver and kidney weights to piglets fed sow milk (Table 3.2). After 18 days, the piglets formula intake was not significantly different between both groups (8.5 ± 0.5 1 and 8.3 ± 0.7 1 for the piglets fed the low and high cholesterol content, respectively). 3.4.2 Plasma lipid composition. Plasma concentrations of total, free and esterified cholesterol were significantly lower in both groups of piglets fed formula than in the piglets fed sow milk. The inclusion of cholesterol in the formula has no significant effect on the plasma total, free or esterified cholesterol of the formula fed animals (Table 3.3). The plasma HDL cholesterol and TG concentrations were similar among the groups. The approximate two fold difference in fasting plasma cholesterol concentration between piglets fed sow milk and piglets fed formula was due to a difference in VLDL + LDL cholesterol (Table 3.3). 3.4.3 Liver lipids, HMG CoA reductase activity, plasma lathosterol and LDL receptor relative mass. Liver total cholesterol content was not different between the piglets fed sow milk and the piglets fed the formula with cholesterol; however, liver total cholesterol was significantly lower in piglets fed the formula low in cholesterol than in piglets fed sow milk or formula with cholesterol. The proportion of free and esterified cholesterol in the liver was also influenced by feeding the formula and by the formula cholesterol content. The liver free cholesterol concentration was significantly higher in both groups of piglets fed formula than in piglets fed sow milk. However, the liver cholesterol ester was lower in piglets fed formula than in piglets fed sow milk. The liver cholesterol ester, but not free cholesterol, was significantly increased by addition of cholesterol to the formula. The liver triglyceride concentration was 60 not different among piglets fed the formulas and piglets fed sow milk (Table 3.4). Table 3.2 Body and organ weights of piglets fed sow milk or formula with low or high cholesterol content. Sow milk Formula low in cholesterol high in cholesterol Body weight (g) 5085 ± 176 3629 ± 193* 3843 ± 330* Heart (g) 26.4 ± 1.3 22.3 ± 2.2 23.7 ± 1.0 Brain (g) 40.1 ± 1.0 39.0 ± 1.5 38.5 ± 0.9 Liver (g) 111 ± 5 122 ± 8 112 ± 11 Kidneys (g) 13.8 ± 0.6 14.4 ± 0.9 13.5 ± 1.0 Data are means ± SEM (n=6-9/group). *, indicates means significantly different from the sow milk group, (p<0.05). No significant differences were found between formula-fed groups. The liver microsomal HMG CoA reductase activity and the plasma lathosterol:cholesterol ratio were not different between the piglets fed the formula containing cholesterol and piglets fed sow milk. These values, however, were significantly lower than in piglets fed the formula with no added cholesterol (Table 3.5). Densitometric analyses of the gold-tagged 0 VLDL and piglet liver receptor protein complex revealed an easily detectable 140 kDA protein receptor (Figure 3.3). This receptor was not seen in the presence of EDTA or Suramin. The latter provides reasonable evidence that the receptor identified in the 140 KDA region is the LDL receptor. The density of the receptor bands increased linearly with up to 20 fig membrane protein in the assay for all the dietary groups. Evidence of saturation was found at higher concentrations. The relative absorbance of the bands using 20 fig membrane protein was not significantly different among the groups of piglets fed sow milk and formula (Table 3.5, Figure 3.3). 3.4.4 Bile lipid and fatty acid composition. Both groups of piglets fed formula had significantly lower concentrations of bile acid and cholesterol in bile than in piglets fed sow milk (Table 3.6). Piglets fed the formula without cholesterol, but not those fed the formula with cholesterol, had significantly lower bile PL concentrations than piglets fed sow milk. The addition of cholesterol to the formula significantly increased the bile acid and PL concentrations, but 61 not the cholesterol concentration of the bile. The bile fatty acid composition was remarkably different between piglets fed milk and those fed the formulas (Table 3.6). The % 16:0, 18:1 and 20:4n-6 was significantly lower and the % 18:0 and 18:2n-6 significantly higher in piglets fed the formulas than in piglets fed sow milk. Addition of cholesterol to the formula bad no significant effect on fatty acid composition of the piglet bile. Table 3.3 Plasma cholesterol and triglyceride concentrations of piglets fed sow milk or formula with low or high cholesterol content. Sow milk Formula low in cholesterol Formula high in cholesterol Total cholesterol Free cholesterol Cholesterol ester HDL-cholesterol VLDL+LDL Triglycerides 4.60 ± 0 . 1 8 1.34 ± 0.13 3.26 ± 0.10 1.71 ± 0.18 2.88 ± 0.28 0.81 ± 0.17 mmol/L 2.30 ± 0.28* 0.75 ± 0.08* 1.55 ± 0.21* 1.45 ± 0.18 0.84 ± 0.13* 0.91 ± 0.19 2.15 ± 0.10* 0.62 ± 0.05* 1.53 ± 0.10* 1.47 ± 0.08 0.66 ± 0.06* 0.64 ± 0.09 Data are means ± SEM (n=5-6/group). * indicates values significantly different from sow milk group, (p<0.05). No significant differences were found between formula-fed groups. Table 3.4 Liver cholesterol and triglyceride concentrations of piglets fed sow milk or formula with low or high cholesterol content. Sow milk Formula low in cholesterol Formula high in cholesterol Total cholesterol Free cholesterol Cholesterol esters Triglycerides 20.50 ± 0.67 15.79 ± 0.72 4.72 ± 0.78 46.1 ± 8. (jig/mg protein) 17.56 ± 0.32* 17.96 ± 0.37* 0.12 ± 0.06* 67.8 ± 5.5 20.13 ± 0.43* 18.32 ± 0.34* 1.82 ± 0.24** 55.9 ± 12.6 Values are means ± SEM; (n=5-6/group). *, indicates mean significantly different from the sow milk group, (p<0.05). *; indicates mean for group receiving formula high in cholesterol significantly different from the group receiving formula low in cholesterol, (p<0.05). 62 Table 3.5 Liver HMG CoA reductase activity, plasma lathosterol and the relative LDL receptor mass of piglets fed sow milk or formula with low or high cholesterol content. Sow milk Formula low in Formula high in cholesterol cholesterol Hepatic HMG CoA reductase G>moI/min.mg 2L1 ± 2.4 94.6 ± 22.3* 30.3 ± 7.1» protein) Plasma lathosterol.cholesterol 0.8 ± 0 . 1 1.1 ± 0.1* 0.7 ± 0.0* (jimohmnaol) Hepatic LDL receptor 0.5 ± 0 . 1 0.4 ± 0.0 0.3 ± 0 . 1 Data are mean ± SEM (n=5-7/group) for all groups except n=3/group for hepatic LDL receptor cholesterol. *, indicates mean significantly different from sow milk group, (p < 0.05). *; indicates mean for group receiving formula high in cholesterol significanty differ^it from the group receiving formula low in cholesterol, (p<0.05). Table 3.6 Composition of bile from piglets fed sow milk or formula with low or high cholesterol content. Sow milk Formula low in Formula high in cholesterol cholesterol Bile acids (r;mol//tl) 123.2 ± 7 71.6 ± 5* 98.2 ± 4*« Cholesterol (lymol/^l) 2.5 ± 0.2 1.0 ± 0.1* 1.5 ± 0.2* Phospholipid(i;mol//tI) 20.3 ± L6 7.8 ± 0.5* 15.6 ± 2.4' Major bile fatty acids (% total) 16:0 29.5 ± 6.1 22.0 ± 0.6* 21.5 ± 0.6* 18:0 12.4 ± 0.5 21.3 ± 0.8* 20.9 ± 0.6* 18:1 17.3 ± 0.6 8.5 ± 0.3* 9.3 ± 0.4* 18:2(n-6) 20.5 ± 0.6 35.2 ± 0.6* 34.7 ± 0.8* 20:4(n-6) 9.2 ± 0.3 6.4 ± 0.4* 6.7 ± 0.6* 22:6(n-3) 2.4 ± 0.2 1.9 ± 0.2 2.0 ± 0.2 Data are means ± SEM (n=5-6). *; indicates mean significantly different from sow milk group, (p<0.05), *; indicates mean for group receiving formula high in cholesterol significantly different from the group receiving formula low in cholesterol, (p<0.05). 3.4.5 Plasma and liver PL and CE fatty acid composition. Several studies have compared the fatty acid composition of plasma lipids and/or RBC from infants (Putnam et al. 1982, Carlson et al. 1986) and piglets (Hrboticky et al. 1989) fed formulas or milk. As found in my studies, the plasma PL levels of 18:1, 20:4n-6 and 22:6n-3 were significantly lower and 63 18:2n-6 was significantly higher in piglets fed formula rather than milk (Figure 3.4, Table 3.7). As in the plasma PL, the liver PL 20:4n-6 was significantly lower in piglets fed formula than in piglets fed sow milk. In contrast, the liver PL 22:6n-3 was similar among the diet groups. The focus of these studies was the possible effect of adding cholesterol to formula, in similar levels to that in milk, on the n-6 fatty acid composition of PL and CE. The % 18:2n-6 was consistently higher in plasma and liver PL and CE, and 20:4n-6 was significantly lower in plasma and liver PL of piglets fed formula, irrespective of the cholesterol content, when compared to the piglets fed sow milk (Figure 3.4, Table 3.7, 3.8). The inclusion of 1.09 mmol cholesterol/1 la the formula had no significant effect on the levels of 18:2n-6 or 20:4n-6 in the plasma or liver PL or CE. The plasma PL % 16:0, however, was significantly lower and the % 18:0 signficantly higher in piglets fed the formula containing cholesterol than in those fed the formula without cholesterol. The increase in liver CE content due to addition of cholesterol to the formula (Table 3.4) was accompanied by a significant increase in the liver CE % 18:1 (Table 3.8). No other significant differences were found in the major fatty acid components of plasma or liver PL and CE between the two groups of formula fed piglets. 64 Table 3.7 Major fatty acids of plasma and liver phospholipid (PL) of piglets fed sow milk or formula with low or high cholesterol cont^it. 9^  en Fatty acids 14:0 16:0 16:1 18:0 18:1 18:2(n-6) 18:3(n-3) 20:2(n-6) 20:3(n-6) 20:4(n-6) 20:5(n-3) 22:0 22:4(n-6) 22:5(n-3) 22:6(n-3) Sow milk 0.2 ± 0.0 22.1 ± 0.7 1.1 ± 0.1 21.8 ± 0.3 12.8 ± 0.5 18.6 ± 1.0 0.5 ± 0.0 0.4 ± 0.0 0.8 ± 0.1 13.0 ± 0.5 0.4 ± 0.1 0.5 ± 0.1 0.6 ± 0.1 2.6 ± 0.2 3.5 ± 0.3 Plasma PL Formula low in cholesterol 0.7 ± o.r* 20.9 ± 1.1" 0.1 ± 0.0* 25.4 ± 0.6*« 8.3 ± 0,1* 31.9 ± 1.0* 1.1 ± 0.0* 0.3 ± 0.0 0.4 ± o.r 6.6 ± 0.5' 0.3 ± 0.1 0.5 ± 0.2 0.3 ± O.r 0.9 ± 0.1* 1.5 ± 0.4* Formula high in cholesterol 0.5 ± 0.0* 18.5 ± 0.5* 0.0 ± 0.0* 29.6 ± 1,1* 7.6 ± 0.4* 30.5 ± 0.7* 1.0 ± 0.1* 0.3 ± 0.0 0.4 ± 0.0* 6.9 ± 0,4* 0.3 ± 0.0 0.5 ± 0.1 0.2 ± 0.0* 1.1 ± 0.0* 1,7 ± 0.2* Sow milk % total fatty acids 0.1 ± 0.0 14.6 ± 0.4 2.1 ± 0.2 24.9 ± 0.4 10.9 ± 0.6 13.1 ± 0.5 0.2 ± 0.0 0.3 ± 0.0 0.8 ± 0.1 20.4 ± 0.2 0,4 ± 0.1 0.2 ± 0.0 0.9 ± 0.1 2.9 ± 0.1 6.1 ± 0.3 Liver PL Formula low in cholesterol 0.3 ± 0.0* 10.2 ± 0.4* 0.1 ± 0.0* 31.0 ± 0.4* 5.8 ± 0.2* 22.6 ± 0.9* 0.5 ± 0.1* 0.8 ± 0.0* 0,8 ± 0.1 17,3 ± 0.7* 0.6 ± O.r 0.2 ± 0.0 0.5 ± 0.0' 2.1 ± 0.2* 5.3 ± 0.6 Formula high in cholesterol 0.3 ± 0.0* 9,6 ± 0.3* 0.1 ± 0.0* 30.8 ± 0.5* 6.4 ± 0.2* 22.4 ± 0.7* 0.5 ± 0.1* 0.8 ± 0.1* 0.7 ± 0.1 17.7 ± 0.6* 0,6 ± 0.0 0.2 ± 0.0 0.6 ± 0.1* 2.2 ± 0.1* 5.3 ± 0.5 Data represent mean ± SEM (n=5-9). *; indicates value for piglets fed formula signiiicantly different from piglets fed sow milk (p<0.017), •; indicates value for piglets fed the formula low in cholesterol significantly different from the group receiving formula with high cholesterol (p< 0.017). 65 Table 3.8. Major fatty acids of plasma and liver cholesterol esters (CE) of piglets fed sow milk or formula with low or high cholesterol content. §^  Fatty acids 14:0 16:0 16:1 18:0 18:1 18:2(n-6) 18:3(n-3) 20:2(n-6) 20:3(n-6) 20:4(n-6) 20:5(n-3) 22:0 22:4(n-6) 22:5(n-3) 22:6(n-3) Sow milk 0.5 ± 0.0 21.6 ± 1.0 6.1 ± 0.4 3.5 ± 0.6 22.1 ± 1.8 39.3 ± 1.5 1.6 ± 0.2 0.1 ± 0.0 0.1 ± 0.0 3.8 ± 0.2 0.2 ± 0.0 0.1 ± 0.0 0.0 ± 0.1 0.0 ± 0.0 0.2 ± 0.0 Plasma CE Formula low in cholesterol 1.4 ± 0.2' 11.1 ± 0.5' 0.5 ± 0.1* 2.3 ± 0.4 11.4 ± 0.9' 65.2 ± 1.7' 2.1 ± o.r 0.1 ± 0.0 0.1 ± 0.0 3.3 ± 0.2 0.3 ± 0.1 0.4 ± 0.2* 0.1 ± 0.0* 0.0 ± 0.0 0.2 ± 0.0 Formula high in cholesterol 1.3 ± 0.1* 11.5 ± 0.2* 0.5 ± 0.0* 2.4 ± 0.1 10.9 ± 0.2* 66.7 ± 0.3* 1.8 ± 0.1 0.0 ± 0.0 0.1 ± 0.0 3.0 ± 0.3* 0.1 ± 0,0 0.3 ± 0.0' 0.0 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 Sow milk % total fatty acids 0.1 ± 0.0 17.5 ± 1.7 5.5 ± 0.2 7.5 ± 0.9 38.5 ± 2.3 22.4 ± 1.1 1.1 ± 0.2 0.3 ± 0.1 0.9 ± 0.2 3.5 ± 0.4 0.2 ± 0.0 0.5 ± 0.1 0.5 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 Liver CE Formula low in cholesterol 0.5 ± 0.3 15.0 ± 1.5 0.2 ± O.r 9.3 ± 0.7 16.0 ± 0.8** 49.4 ± 2.7* 1.5 ± 0.0 0.4 ± 0.1 0.5 ± 0.2 3.0 ± 0.3 0.3 ± 0.0 1.0 ± 0.2 0.2 ± O.r 0.1 ± 0.0 0.3 ± 0.1 Formula high in cholesterol 0.7 ± 0.2 14.7 ± 0.6 0.3 ± 0.0' 8.2 ± 0.2 18.9 ± 0.3* 46.9 ± 0.8* 1.9 ± 0.2* 0.6 ± 0.1 0.5 ± 0.1 2.8 ± 0.2 0.3 ± 0.1 0.4 ± 0.3 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.1 Data represent mean ± SEM (n=5-9/group). *; indicates value for group of piglets fed formula significantly different from piglets fed sow milk (p< 0.017). *; indicates value for group receiving formula low in cholesterol significantly different from the group receiving formula with high cholesterol (p<0.017). 66 -205 -116 - 97 .4 Figure 3.3 Representative example of results from the ligand blotting assay obtained with 20 fig membrane protein for piglets fed the formula low in cholesterol (lane 1), sow milk (lane 2) and formula high in cholesterol (lane 3). The molecular weights (205, 116, 97.4) are expressed in KDa. 67 P l a s m a PL O K P l a s m a CE Liver CE 16.0 18:0 18:1 18:2n-6 20:4n-6 22:6n-3 Figure 3.4 The effect of feeding sow milk or formula with low or high cholesterol content on the major fatty acid components of piglet plasma and liver phospholipid (PL) and cholesterol esters (CE). The diet groups are represented as sow milk, solid bars; formula low in cholesterol, cross hatched bars; formula high in cholesterol, diagnonal hatched bars. Values are means ± SEM (n=5-6/group). * indicates mean for groups of piglets fed formula significantly different from group of piglets fed sow milk (p< 0.017), ' indicates mean for group of piglets fed the formula with high cholesterol significantly different from group of piglets fed the formula low in cholesterol group, (p< 0.017) 68 3.5 DISCUSSION Human milk contains higher concentrations of cholesterol and long chain polyunsaturated fatty acids such as 20:4n-6 than most infant formulas (Jensen et al. 1978). Infants fed formula generally have lower plasma cholesterol levels (Van Biervliet et al. 1986, Wagner and von Stockhausen 1988) with concomittant higher 18:2n-6 and lower 20:4n-6 in their RBC PL (Putnam et al. 1982) and plasma PL (Koletzko et al. 1989) than infants fed natural milk. The role of cholesterol and 20:4n-6 in natural milk in the nutrition of human infants is subject to speculation, and the precise benefits of raising the circulating lipids in infants fed formula to levels similar to those in infants fed milk are difficult to predict. A possible advantage of raising the low level of plasma PL 20:4n-6 associated with formula feeding is related to the fact that 20:4n-6 is a precursor of the eicosanoids. The eicosanoids are important modulators and mediators in a large spectrum of physiological and developmental processes, including platelet aggregation and function, renal function and cell growth (Seyberth and Kuhl 1988, Smith 1989). The objective of the second part of this thesis was to investigate if infant formula with a similar amount of cholesterol to milk would raise the plasma PL 20:4n-6 to levels similar to that of piglets fed sow milk. The rationale for investigating the relationship of dietary cholesterol to n-6 fatty acid metabolism comes from the fact that the esterification of cholesterol, catalyzed by LCAT, usually uses 18:2n-6 from plasma phosphatidylcholine (Sgoutas 1972). The lower dietary and plasma cholesterol concentrations in infants fed formula could, therefore, result in lower utilization of plasma PL 18:2n-6 for esterification of plasma cholesterol. The return of CE 18:2n-6 to liver and consequently its availability for desaturation to 20:4n-6 could be reduced. Piglets were fed formula with or without cholesterol and which contained MCT oil as the predominant source of saturated fatty acids for the first 18 days of life. The results show that differences in plasma PL n-6 fatty acids due to formula rather than milk feeding extend to liver and bile lipids. The changes include higher levels of 18:2n-6 in plasma and liver PL and CE and in bile lipid, and lower levels of 20:4n-6 in plasma and liver PL and bile lipid in piglets fed formula with or without cholesterol rather 69 than milk. Neither the plasma nor the liver PL and CE 18:2n-6 or 20:4n-6 were altered by addition of cholesterol to piglet formula. Very few studies have looked at the effect of adding cholesterol to infant formula on 18:2n-6 and 20:4n-6 metabolism. A recent study by Van Bervliet et al. (1992b) investigated the effect of breast feeding, standard formula or formula containing cholesterol (half the content present in human milk) or with 18:3n-6 (precursor of 20:4n-6) on plasma CE fatty acid levels in infants from birth to 30 days of life. The published results show that the breast-fed infants had a higher serum total cholesterol and proportion of CE 20:4n-6 than infants fed the standard infant formula. The proportion of 20:4n-6 in CE but not the plasma cholesterol, was increased by the addition of cholesterol or 18:3n-6 to formula. The study with infants suggest that exogenous cholesterol and 18:3n-6 contribute to the normalization of plasma CE 20:4n-6 in infant fed formula. Other investigators (Hayes et al. 1992) have found higher plasma CE 20:4n-6 in infant fed breast milk than in infants fed formulas. As hypothesized in this thesis, Hayes and collaborators (1992) suggested that the difference in circulating lipid 20:4n-6 between the infants fed formula and infants fed milk may be due to the differences in cholesterol concentration between milk and formulas. The rational for their hypothesis was derived from the comparison of the plasma CE fatty acid profile from the vegetarian and non- vegetarian adults. The percentage of 20:4n-6 in plasma CE of breast-fed infants is more like that of non-vegetarian adults with high cholesterol diets whereas the lower 20:4n-6 levels in infants fed formula is more similar to that of the vegetarian adults with low cholesterol diets. However, vegetarian and non-vegetarian adults have also a different 20:4n-6 in take. In contrast, the studies with piglets in this thesis found that, the lower levels of 20:4n-6 in piglets fed formula were limited to the PL and, no differences were observed in the CE. Furthermore, the results obtained do not support a role of dietary cholesterol in the metabolism of fatty acids in newborn piglets. However, and unexpectedly, providing cholesterol in the formula did not increase the piglet plasma cholesterol levels. Therefore, because feeding the formula high in cholesterol did not alter the circulating total, free or esterified cholesterol, it is likely that it had no effect on the rate of plasma cholesterol esterification. In order to fully test the hypothesis, an increase in the plasma 70 cholesterol, in response to cholesterol feeding may be necessary. Other studies in piglets have also found no significant differences in plasma cholesterol after feeding formula providing high cholesterol (145 mg cholesterol/dl) or low cholesterol (<2mg/dl) for 2 weeks (Engelhardt et al. 1991). Studies with low birth weight infants have similarly found no increase in plasma cholesterol concentration after supplementation of the formula with cholesterol and taurine in amounts comparable to that in human milk (Rassin et al. 1983). The mechanism by which the serum cholesterol concentration remained high in piglets fed natural milk compared to those fed a formula providing cholesterol in similar amounts to that of sow milk is unknown and intriguing. Possibly, it may be the result of the difference in fatty acid composition between the formulas and milk, rather than differences in cholesterol content or absorption. It is well known that different fatty acids differ in their effect on plasma cholesterol. Dietary influences on serum lipids and lipoproteins have been reviewed by Grundy and Denke 1990. Dietary unsaturated fatty acids, mainly 18:2n-6 as well as 18:1, have been associated with lower plasma cholesterol levels than saturated fatty acids. Saturated fatty acids, on the other hand, are well known to raise plasma cholesterol and LDL cholesterol. However, not all saturated fatty acids share this cholesterol-raising property. For example, medium chain saturated fatty acids (C8:0-C10:0) have not been associated with hypercholesterolemia. Similarly, 18:0 is not believed to raise plasma cholesterol. Palmitic acid (16:0) however, has been recognized to promote increased plasma cholesterol. Recent studies in non-human primates have demonstrated that myristic (14:0) + lauric acid (12:0) are more effective in increasing total cholesterol concentration than 16:0 (Hayes et al. 1991). Lauric acid (12:0) alone has been shown to raise total and LDL cholesterol, in human, but is not as potent as 16:0 for its hypercholesterolemic effect (Denke and Grundy 1992). Other studies using non-human primates, however, have shown that 16:0 is minimally hypercholesterolemic (Hayes et al. 1991), and suggest that that 16:0 is only hypercholesterolemic when cholesterol is fed to give a situation where the LDL receptors are down regulated (Khosla and Hayes 1992). In contrast with this, studies in humans have shown that 16:0 raises LDL cholesterol concentration even on a cholesterol-free diet (Denke and Grundy 1992). Sow 71 milk, like human milk, has high levels of both 16:0 and cholesterol. The formulas fed to the piglets in the studies in this thesis, as in many preterm infant formulae, contained 8:0 + 10:0 as the predominant saturated fatty acids rather than 16:0 as in milk. Possibly, the relative hypercholesterolemic effect of milk compared to formula could be due to the combination of high 16:0 and cholesterol. The effect of the fatty acid composition of milk and formula diets on cholesterol metabolism in infants is not well understood. It is known, however that the fatty acid composition of breast milk and formula does affect the serum cholesterol lipoprotein profiles in human infants (Ginsburg et al. 1980, Van Biervliet et al. 1981, Mize and Uauy 1991, Hayes et al. 1992). Van Biervliet et al. (1981) have shown that full term newborns fed from birth to 30 days with adapted formulas containing low cholesterol but similar levels of 14:0, 16:0, 18:1, 18:2n-6 to that in human milk, have plasma total cholesterol values similar to that of the breast-fed infants. When another group of infants were fed a formula containing higher 18:2n-6, total and HDL cholesterol were lower than in breast-fed infants. These results could suggest that the milk fatty acid profile rather than the cholesterol content determines the infants plasma cholesterol profiles. In contrast, results obtained by Wagner and von Stockhausen (1988) suggest that factors other than milk fatty acid content may influence the infant plasma cholesterol levels. They found that infants fed formula, from birth to 3 months, containing 12:0, 14:0, 16:0 and 18:2n-6 comparable to that in milk but containing lower levels of cholesterol have lower plasma total and LDL cholesterol than infants fed milk. More recently. Van Biervliet et al. (1992a) investigated the effect of feeding breast milk or an experimental formula containing cholesterol and fatty acid profile comparable to that in milk on plasma lipids and apoproteins of full term infants fed from birth to 90 days. The total cholesterol and apo B values were significantly higher in both the breast-fed infants and those who received the experimental formula compared to infants who received a formula containing low cholesterol and LCPs but comparable levels of 12:0, 14:0, 16:0, 18:1 and 18:2n-6. These results could suggest that the cholesterol rather than the fatty acid content of milk may be responsible for the higher plasma cholesterol in infants fed milk compared to infants fed formula. However, results from my thesis do not support this hypothesis, because 72 piglets fed the formula providing cholesterol comparable to that of sow milk did not have plasma cholesterol similar to that of piglets fed sow milk. An earlier study (Van Biervliet et al. 1986) has shown that infants fed a formula from birth to 30 days, containing similar 14:0, 16:0, 18:2n-6, 18:1 and cholesterol content to that of milk still have lower plasma cholesterol and LDL cholesterol than infants fed milk. These results could suggest that factors other than milk cholesterol and fatty acid content may be responsible for the regulation of cholesterol metabolism in infants fed milk (Van Biervliet et al. 1986). The discrepancies among the above studies may be attributed to differences in the type of formula fed, the amount of cholesterol added to the formula and the duration of the study. Clearly, more studies are needed to clarify the effect of milk and formula feeding on cholesterol metabolism in infants. Whether the absorption of cholesterol from formula is equivalent to the absorption of cholesterol from milk is not known. The experimental formula fed to the piglets, contained 50% fatty acid as medium chain fatty acids (MCFAs). The metabolism of MCFAs differs in many respects from that of long chain fatty acids (LCFAs). After absorption, LCFAs are incorporated into triglycerides and secreted in chylomicrons into the lymphatic system. In contrast MCFAs ^ 12:0 are mainly transported via the portal circulation as unesterifred fatty acids bound to serum albumin (Bach and Babayan 1982). Futhermore, MCFAs are more hydrophylic than LCFA and do not require solubulization in mixed micelles for absorption. Several studies have shown that the concentration of serum and liver cholesterol is lower in rats fed MCT than in rats fed long chain triglycerides (Senior 1968). This may support the suggestion that the absorption or mode of transport of MCFA may reduce sterol absorption (Takahashi and Underwood 1974). Cholesterol must be incorporated into mixed micelles to be absorbed, and must be incorporated into chylomicron or intestinal VLDL or LDL to be transported. However, results from other studies do not support an hypothesis that diets with MCT lower cholesterol absorption. For example, lymphatic absorption of cholesterol was not different among rats given intragastric doses of 3 ml of various fat emulsions containing structured triglycerides with 8:0, 10:0 and 18:2n-6 (Ikeda et al 1991). This study, however, does not exclude the possibility of long term effects of feeding MCFAs on 73 cholesterol absorption. The study of cholesterol metabolism in piglets fed milk and formula fed piglets in this thesis involved assay of HMG CoA reductase activity, plasma lathosterol, LDL receptor relative mass and liver and bile lipid composition. These analyses found that the liver HMG CoA reductase activity was similar in young piglets fed the formula containing cholesterol and sow milk, and lower than in piglets fed the formula with no cholesterol added. Previous studies by Jones et al. (1990) reported higher rates of cholesterol synthesis (as indicated by hepatic HMG CoA reductase activity) in S day old pigs fed low-cholesterol formula rather than sow milk. In general, endogenous cholesterol synthesis is reduced in response to increased dietary cholesterol intake in normal individuals (Quintao et al. 1971, Nestel and Poyser 1976, Whyte et al. 1977, Lin and Connor 1980, McNamara et al. 1987). In contrast, other investigators have not found this inverse relationhip between increased dietary cholesterol intake and cholesterol synthesis (Katan and Beynen 1987, Gylling and Miettinen 1992). Individual variation in the effects of dietary cholesterol on cholesterol synthesis have been reported by McNamara et al. (1987). Their results demonstrate that 31% of the studied subjects did not compensate for the increase of cholesterol intake by decreasing endogenous cholesterol synthesis. The lower HMG CoA reductase activity in piglets fed sow milk or the formula with cholesterol was accompanied by higher liver total and esterified cholesterol. These results suggest that the decrease in HMG CoA reductase activity in piglets fed the formula containing cholesterol was due to hepatic uptake of exogenous cholesterol from the formula. These findings suggest that at least some of the cholesterol provided by the formula was absorbed, entered the liver (probably as chylomicrons remnants), suppressed de novo synthesis of cholesterol, and was esterified by ACAT for storage as liver CE. Previous studies have shown that cholesterol-rich chylomicron remnants reduce the reductase activity (Lakshmanan et al. 1981). The formula with cholesterol also decreased the piglet plasma lathosterol:cholesterol ratio. Studies in human adults have shown that the plasma lathosterolxholesterol ratio is correlated with liver (Bjorkhem et al. 1987) as well as whole body cholesterol synthesis (Kempen et al. 1988). This information suggests that addition of cholesterol to the 74 piglet formula reduced cholesterol synthesis, possibly in extrahepatic organs such as the intestine, as well as the liver. I found no significant measurable differences in the hepatic LDL receptor relative mass among the piglets fed sow milk and piglets fed the formula diets. These results suggest that the higher plasma cholesterol following sow milk feeding cannot be explained by the difference in receptor-mediated uptake of LDL-choIesteroI. The expected inverse relationship between changes in plasma cholesterol levels and hepatic LDL receptor mass found in the adult human (Soutar et al 1986) was not present in newborn piglets. The reason why down regulation did not occur is not known. Although, these premilinary results represent only 3 piglets per group, and they are interesting and provocative. The apparent absence of differences in LDL receptor mass between the natural and formula fed piglets could be due to the methodology used. Because the LDL receptor protein was assayed in vitro, it is possible that the LDL-receptor binding affinity and/or LDL clearance was different in vivo. Using an vitro assay ignores possible in vivo modulators which may be present. For example, LDL binding to cultured adult rat hepatocyte receptors has been found to increase in the presence of insulin (Salter et al. 1987). Under most experimental conditions, HMG CoA reductase activity and the LDL receptor number are coupled (Goldstein and Brown 1984, Brown and Goldstein 1986). When there is a high concentration of plasma cholesterol, both the receptor number and cholesterol synthesis are usually reduced. The study with piglets infers that hepatic LDL receptor number and cholesterol synthesis measured by the HMG CoA activity and lathosterol are uncoupled during the suckling period. The reason for these results is unknown. Lack of concordance between cholesterol synthesis and LDL receptors, in which receptor number was changed but cholesterol synthesis rates were unchanged have been reported for adult guinea pigs fed diets rich in polyunsaturated or saturated fatty acids (Ibrahim and McNamara 1988). Because bile acid and cholesterol excretion through the bile is the principal route for cholesterol removal from the body, the difference in cholesterol metabolism observed between milk and formula-fed piglets could be related to a difference in bile formation. The studies in this thesis found higher biliary bile acid. 75 cholesterol and phospholipid concentrations in piglets fed sow milk compared to piglets fed formula. The higher biliary bile acid and cholesterol concentrations in piglets fed milk than in piglets fed formula could reflect an increased concentration of these lipids due to decreased bile flow. Reduced bile flow, leading to increased bile lipid concentrations, could reduce the excretion of cholesterol from liver as cholesterol and/or bile acids. This could explain the higher plasma cholesterol and hepatic cholesterol ester concenfrations in piglets fed sow milk rather than formula. However, the measure of bile lipid concentration does not give any information on bile lipid secretion or bile flow. The higher bile acid concentration in bile of piglets fed sow milk could also reflect higher reabsorption of bile acids through the enterohepatic circulation, or higher rates of synthesis and secretion with unaltered flow rates. There are data to show that feeding human milk is associated with a decreased fractional removal rate, with no difference in the rate of bile acid synthesis, when compared to feeding formula in preterm infants (Jarpenpaa et al. 1983). Term infants fed formula have also been shown to have higher faecal bile acid concentrations than infants fed breast milk (Hammons et al. 1988). Studies with premature infants bom at 31-36 weeks of gestation showed that feeding human milk resulted in higher duodenal concentration of bile acids (Watkins et al. 1983, Jarvenpaa et al. 1983) and a higher bile acid pool size (Watkins et al. 1983) than feeding formula at all ages. Long-term studies of the effects of breast- versus formula- feeding on bile acid metabolism have not been performed in humans. Studies in non-human primates have shown that breast and formula feeding in infancy have different, long-term effects on biliary lipid composition in adulthood (Mott et al. 1991). The bile cholesterol saturation index was found to be higher in adult baboons which were breast-fed as infants compared with those fed formulas as infants. These studies suggest a fundamental difference in cholesterol and bile metabolism originating from infancy. Whether or not the lower concentration of bile acid in bile of piglets fed formula is of physiological significance to fat absorption, reflects higher faecal bile acid excretion, or a reduced bile acid pool size is unknown. These studies with piglets, however, support published data from infants which suggest that 76 the type of infant feeding influences bile acid metabolism (Watkins et al. 1983, Jarvenpaa et al. 1983, Hammons et al. 1988). Addition of cholesterol to the formula fed to piglets in these studies significantly increased the biliary bile acid and phospholipid concentration, but did not have any effect on bile cholesterol concentration. Studies in premature infants did not find any apparent effect of cholesterol in infant formula (12.6 mg/dl) on fasting duodenal bile acid concentration and conjugation pattern (JarvenpaS et al 1983). Whether or not addition of cholesterol to formula is an important determinant of bile steroid secretion in infants is unknown and needs to be further investigated. Body weights were lower in the 18 day old piglets fed formula than in those fed sow milk. Organ weights were similar between the formula- and sow milk- fed piglets, therefore, it seems unlikely that the lower body weight in artificially fed animals was due to undernutrition. Of importance, however, the piglets had a higher daily formula intake in the studies in the first part of this thesis than the piglets in this experiment. Whether or not this has a potential confounding effect is unknown. In summary, the studies in this thesis have shown that feeding formula leads to substantial changes in cholesterol, bile acid and fatty acid metabolism. Providing cholesterol in the formula did not correct the significantly lower plasma cholesterol or plasma and liver PL 20:4n-6 associated with formula feeding. The reason for the lower plasma PL 20:4n-6 in piglets fed formula rather than milk remains unknown. The results from this thesis do not provide evidence to support an hypothesis that dietary cholesterol plays an important role in regulating the plasma PL 20:4n-6 in piglets fed formula. However, the results do not exclude the possibilty that dietary cholesterol in infant formula may still play a role in n-6 metabolism, if an increase in plasma cholesterol is attained Although the explanation is not known, the results suggest that the differences in plasma cholesterol concentration which result from feeding formula compared to milk may be due to factors other than cholesterol intake alone. No evidence was found to suggest that piglet hepatic LDL-receptor mass was lower following sow milk rather than formula feeding. The physiological significance of the difference 77 in bile lipid concentrations due to milk and formula feeding in relation to fat digestion and absorption and to body cholesterol homeostasis should be further studied. In addition, studies on the long term significance of milk and formula feedings on cholesterol and fatty acid metabolism is warranted. 3.5.1 Conclusion and future directions The findings of part 2 of this thesis were as follow: 1) Fundamental differences in cholesterol, fatty acid and bile acid metabolism exist between young piglets fed milk and those fed formula. a) Plasma total and LDL cholesterol were higher in piglets fed sow milk than in those fed formula, irrespective of the amount of cholesterol in the formula. b) Plasma and liver PL and CE and bile total lipid fatty acid composition were significantly altered by formula feeding. The differences included higher plasma and liver PL and bile 18:2n-6 and lower 20:4n-6 in piglets fed formula than in piglets fed sow milk. c) The bile acid, cholesterol, and phospholipid concentration of bile lipid was significantly higher in piglets fed sow milk than in piglets fed formula. 2) Formula providing cholesterol in similar amounts to that of milk: a) did not correct the significantly lower plasma cholesterol or plasma and liver PL 20:4n-6 associated with formula feeding. 78 b) resulted in higher total cholesterol and cholesterol ester concentrations in the liver, and higher bile acid and phospholipid concentrations in bile lipid than feeding formula with no cholesterol added. c) resulted in lower hepatic microsomal HMG CoA reductase activity and plasma lathosterol:cholesterol ratio than feeding the formula with no cholesterol added. d) did not have any effect on the relative LDL receptor mass. The reason why plasma and liver PL 20:4n-6 and plasma cholesterol concentrations are lower in piglets fed formula compared to milk is still unanswered. However, one possible reason to explain the low plasma and liver PL 20:4n-6 levels in piglets fed the formula supplemented with cholesterol is that plasma total and LDL cholesterol levels were unaffected by this formula diet. In order to test this hypothesis, it would necessary, in future studies, to design a formula that would raise the plasma total and LDL cholesterol to levels similar to that of sow milk-fed piglets and determine if the low plasma and liver PL 20:4n-6 levels increase. This requires an initial study as to why addition of cholesterol to the formula did not increase the piglet plasma LDL cholesterol. If the fatty acid composition of the formula, specifically the use of MCT as the source of saturated fat was responsible, formula with or without cholesterol with levels of 12:0, 14:0, 16:0, 18:2n-6 and 18:1 similar to that of milk should be studied. These experiments might allow study of whether the higher plasma cholesterol in piglets fed sow milk is due the milk fatty acid profile alone, a combined effect of milk fatty acid composition and cholesterol content, or neither. Milk usually contains 15 mg free cholesterol and 5 mg CE/dl, the piglet formula fed in the present investigation had 32 mg cholesteryl-16:0 and 10 mg free cholesterol. Whether or not the lower free cholesterol, higher CE had any effect on the availability of cholesterol for absorption in young piglets 79 is unknown. Therefore, it may be necessary to add cholesterol not only in similar amounts but also in similar form to that of milk. If this type of formula is found to increase the low plasma total and LDL cholesterol concentrations, to values similar to those found with milk feeding, experiments as done here could then be repeated to determine if transport of piglet plasma cholesterol plays a role in n-6 fatty acid metabolism. If no relation is found between formula cholesterol and 20:4n-6 levels, it may be necessary to consider the addition of 20:4n-6 in formula to increase the low plasma PL 20:4n-6. This would suggest that the enzyme system responsible for the desaturation of 18:2n-6 to 20:4n-6 is not adequate to achieve levels of 20:4n-6 found with natural milk feeding. Finally, it is also possible that the formula providing the same fatty acid profile and cholesterol content to milk will still result in differences in cholesterol metabolism. This would suggest that some other milk factors absent from formula would need to be considered. Other questions which arise from these studies include whether the cholesterol was completely absorbed when added to a formula containing 50% MCT as the source of saturated fat. Measurement of cholesterol absorption from formulas varying in saturated fatty acid chain length would be of interest. Further study on how milk and formula affect bile lipid content would be worthwhile. Future studies might include analysis of some factors involved in bile acid metabolism, such as the bile acid pool size, intraluminal concentration of bile acids, bile acid excretion in feces, bile flow and how these parameters relate to fat absorption. Further studies on difference in cholesterol metabolism between formula and milk fed piglets are also needed. For example, the data obtained by ligand blotting for the LDL receptor relative mass should be confirmed with other methods such as turnover of ['^ I] LDL in piglets fed milk or formula. This would give information about the clearance of LDL in vivo. 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