Lipid Diene Conjugation in Human Saliva
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LIPID DIENE CONJUGATION IN HUMAN SALIVA CHRISTOPHER YIK FAI FONG B.Sc. University College and Middlesex School of Medicine, London A Thesis submitted to the University of London for the degree of Doctor of Philosophy Spring 1992 1 ProQuest Number: 10609874 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609874 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT Diene conjugation (DC) has been widely recognised as a marker of free radical attack on polyunsaturated lipids. 0ctadeca-9cis,lltrans- dienoic acid (18:2(9c,lit)) which is a non-hydroxy, non-hydroperoxy isomer of linoleic acid (18:2(9,12)) accounts for over 95% of all DC in human tissues and tissue fluids studied. This isomer can be generated by free radical mechanisms. The % molar ratio (%MR) of 18:2(9c,llt)/18:2(9,12) has also been used as a free radical marker. The first objective of the work embodied in this thesis was to establish the presence of DC lipids, in particular 18:2(9c,lit), in both esterified and non-esterified lipid fractions of fresh human saliva. It was established that 18:2(9c,llt) and other specific DC products could be generated in vitro by incubating saliva. The biological variables affecting 18:2(9c,llt) generation were studied. The mechanism by which 18:2(9c,llt) and other DC products were generated in vitro and in vivo was investigated. Studies involving antibacterial agents and bacterial culture revealed that 18:2(9c,llt) and other conjugated dienes were generated enzymically by bacteria present in saliva. The possibility that this was, at least in part, a free-radical-mediated process could not be ruled out. Since the bacteria thought to be responsible are widely distributed in humans, the above new findings have direct relevance to the measurement and interpretation of 18:2(9c,llt) and %MR. A pilot study was carried out to investigate the effects of diets on the distribution of 18:2(9,11) in both human serum and saliva. Finally, a preliminary clinical study was carried out to assess the possible implication of some of the above findings on the salivary measurements of 18:2(9,11) and %MR. 2 CONTENTS TITLE ABSTRACT TABLE OF CONTENTS LISTS OF FIGURES & TABLES ABBREVIATIONS ACKNOWLEDGEMENTS DEDICATION SECTION ONE : INTRODUCTION 1.1. Introduction to lipid diene conjugation (DC) 1.1.1. Definition 1.1.2. A brief historical perspective of lipid DC 1.1.3. Methodology 1.1.3.1. Early methodology 1.1.3.2. Modification of methodology 1.1.3.3. Measurement of lipid DC by chromatographic methods 1.1.4. The biological significance of lipid DC 1.2. Introduction to octadeca-9,11-dienoic acid 1.2.1. Identification of octadeca-9,11-dienoic acid ... 41 1.2.2. Octadeca-9,11-dienoic acid in humans ... 42 1.2.3. Octadeca-9,11-dienoic acid in other sources 1.2.3.1. In bacteria ... 44 1.2.3.1.1. Biohydrogenation pathways of rumen bacteria ... 44 1.2.3.1.2. The linoleateAl2-cis, All-trans isomerase ... 45 1.2.3.1.3. Generation of 18:2(9c,llt) by other bacteria ... 46 1.2.3.2. In animals ... 47 1.2.3.3. In human diets ... 48 1.2.4. The biological significance of octadeca-9,11-dienoic acid 49 1.3. Lipid DC, free radicals and lipid peroxidation 1.3.1. Introduction to free radicals 1.3.1.1. Definition ... 54 1.3.1.2. Formation of free radicals ... 55 1.3.1.3. Reactions of free radicals ... 56 1.3.2. Introduction to lipid peroxidation ... 57 1.3.2.1. Non-enzyme-catalysed lipid peroxidation ... 57 1.3.2.2. Enzyme-catalysed lipid peroxidation ... 58 1.3.3. Measurement of free radical activity and lipid peroxidation 1.3.3.1. Introduction ... 59 1.3.3.2. TBA test 60 1.3.3.3. Total DC measurement ... 61 1.3.3.4. Octadeca-9,11-dienoic acid as a free radical marker in experimental and clinical pathology 62 1.3.3.5. Other methods ... 64 1.3.4. Biological significance of free radicals and lipid peroxidation 65 4 1.3.5. In-vivo protection against free radical damage and lipid peroxidation ... 66 1.4. Introduction to human saliva ... 73 1.4.1. General introduction ... 73 1.4.2. Lipids in human saliva ... 75 1.4.3. Micro-organisms in human saliva ... 76 1.4.4. Free radicals and human saliva ... 78 1.4.4.1. Hydrogen peroxide in human saliva ... 78 1.4.4.2. Defence against free radical damage in human saliva ... 79 1.5. Aims of this project ... 81 SECTION TWO : MATERIALS AND METHODS 2.1. Materials 2.1.1. Chemicals and biochemicals ... 82 2.1.2. Solvents ... 85 2.1.3. Gases ... 85 2.2. Methods 2.2.1. Measurement of free radical activities in biological and experimental systems 2.2.1.1. Measurement of total diene conjugation ... 86 2.2.1.2. Fluorimetric measurement of TBA reactive substances 2.2.1.2.1. Preparation of TBARS for fluorimetric measurement ... 87 2.2.1.2.2. Fluorescence measurements ... 88 2.2.1.2.3. Calculation of TBARS ... 88 2.2.2. Measurement of non-esterified UFAs and their conjugated dienes by HPLC 2.2.2.1. Introduction to HPLC ... 90 5 2.2.2.2. Reverse-phase HPLC system ... 90 2.2.2.3. Preparation of biological specimens for HPLC analysis 2.2.2.3.1. Preparation of serum lipids for HPLC ... 91 2.2.2.3.2. Preparation of salivary specimens for HPLC ... 92 2.2.2.4. Preparation of experimental specimens for HPLC analysis 2.2.2.4.1. Preparation of lipid specimens for HPLC ... 92 2.2.2.4.2. Preparation of dietary lipids for HPLC ... 93 2.2.2.5. Calculation of the concentrations of diene-conjugated and non-diene-conjugated UFAs in various specimens 2.2.2.5.1. Introduction ... 93 2.2.2.5.2. Consideration of the recovery of the sample ... 94 2.2.2.5.3. Consideration of sample size ... 95 2.2.2.5.4. Calculation for the extract volume of the sample . 96 2.2.2.5.5. The full calculation incorporating both the recovery factor and the sample size for the concentrations of UFAs ... 97 2.2.2.5.6. Improved formula for the calculation of the concentrations of UFAs in samples where 18:2(9t,llt) was present in significant concentrations and/or was generated during experiments ... 97 2.2.2.5.7. Calculation of the molar ratios of 18:2(9,11) to 18:2(9,12) ... 98 2.2.2.5.8. Calculation for UFAs in esterified lipids ... 99 2.2.2.5.9. Calculation of the initial rates of generation of 18:2(9,11) and other conjugated dienes ... 100 2.2.3. Hydrolysis of esterified lipids in biological and experimental systems 2.2.3.1. Introduction ... 102 2.2.3.2. In biological systems 2.2.3.2.1. In blood ... 102 2.2.3.2.2. In saliva ... 103 2.2.3.3. In experimental systems 2.2.3.3.1. In lipid specimens ... 104 6 2.2.3.3.2. In dietary lipids 104 2.2.4. Studies with salivary specimens 2.2.4.1. Introduction ... 105 2.2.4.2. Collection of fresh human "whole" saliva ... 105 2.2.4.3. Preparation of the supernatant and the salivary cell suspensions (resuspended salivary pellets) ... 105 2.2.4.4. Comparative study of whole saliva, salivary cell suspension and supernatant ... 106 2.2.5. In-vitro incubation studies with whole saliva, salivary cell suspensions, and supernatant 2.2.5.1. Introduction ... 107 2.2.5.2. Incubation procedures ... 107 2.2.6. Investigation of the effect of the addition of 18:2(9,12) on 18:2(9,11) generation by incubated salivary cell suspensions 2.2.6.1. Introduction ... 108 2.2.6.2. Preparation of non-esterified 18:2(9,12) ... 108 2.2.6.3. Incubation of salivary cell suspensions in the presence of a fixed concentration of added 18:2(9,12) ... 109 2.2.6.4. Investigation of the effect of varying the concentrations of salivary cell suspension and added 18:2(9,12) on 18:2(9,11) generation 2.2.6.4.1. Introduction ... 109 2.2.6.4.2. Experimental procedures ... 110 2.2.7. Investigation of the effect of the addition of various lipids other than non-esterified 18:2(9,12) on the salivary generation of 18:2(9,11) and other conjugated dienes 2.2.7.1. Introduction ... Ill 2.2.7.2. Investigation of the effect of the addition of phospholipid on salivary 18:2(9,11) generation ... Ill 2.2.7.3. Investigation of the effect of the addition of trilinolein on salivary 18:2(9,11) generation ... 112 7 2.2.7.4. Investigation of the effect of the addition of cholesteryl linoleate on salivary 18:2(9,11) generation ..