Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 14

Conjugated linoleic acid (CLA) has gained much attention recently due to its beneficial health and biological effects on animals and humans. However, the CLA-forming enzyme system has not been studied in details. Six strains of Lactobacillus acidophilus L11, L12, L14, L15, Lactobacillus fermentum and Lactobacillus reuteri were used to study the growth conditions and the production of CLA-forming enzyme in MRS media containing linoleic acid concentrations at 37°C. The purification and characterization of a CLA-forming enzyme were reported for the first time. The results showed that this enzyme has a molecular mass of 72 kDa, and is composed of two subunits. The optimal pH and temperature were 7.0 and 37°C, respectively. Kinetic study indicated that the enzyme has a high affinity for linoleic acid having a Km value of 1.49 x 10 -5 M and the Vmax was 17.1 muM/mg/min. The enzyme activity was inhibited by the metal chelators. (Abstract shortened by UMI.)

Thesis (M.Sc.). / Written for the School of Dietetics and Human Nutrition. Title from title page of PDF (viewed 2007/08/30). Includes bibliographical references.

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Lui Oi Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [216]-240). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / 撮要 --- p.x / PUBLICATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoietic Development --- p.1 / Chapter 1.1.2 --- Leukemia --- p.8 / Chapter 1.1.2.1 --- General Diagnostic Tests for Leukemia --- p.9 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.17 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.20 / Chapter 1.2 --- Conjugated Linoleic Acid --- p.23 / Chapter 1.2.1 --- Introduction: Historical Development and Occurrence of Conjugated Linoleic Acid --- p.23 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Conjugated Linoleic Acid --- p.24 / Chapter 1.2.2.1 --- Chemical Structures of Conjugated Linoleic Acid Isomers --- p.24 / Chapter 1.2.2.2 --- Biosynthesis of Conjugated Linoleic Acid --- p.26 / Chapter 1.2.2.3 --- Metabolism of Conjugated Linoleic Acid --- p.30 / Chapter 1.2.2.4 --- Mode of Entry and Tissue Incorporation of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.2.5 --- Toxicology of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.3 --- Physiological Activities of Conjugated Linoleic Acid: Reported Health Benefits --- p.35 / Chapter 1.2.3.1 --- Anti-adipogenesis / Chapter 1.2.3.2 --- Anti-diabetogenesis --- p.36 / Chapter 1.2.3.3 --- Anti-atherosclerosis --- p.38 / Chapter 1.2.3.4 --- Anti-carcinogenesis --- p.39 / Chapter 1.2.3.5 --- Anti-tumor Activity --- p.40 / Chapter 1.2.3.6 --- Effects of Conjugated Linoleic Acid on Lipid Metabolism --- p.44 / Chapter 1.2.3.6.1 --- Actions on Phospholipids by Conjugated Linoleic Acid --- p.45 / Chapter 1.2.3.6.2 --- Conjugated Linoleic Acid as a Ligand for the PPAR System --- p.47 / Chapter 1.2.3.7 --- Immunomodulation --- p.47 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.50 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Animals --- p.52 / Chapter 2.1.2 --- Cell Lines --- p.52 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.52 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.54 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.58 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.59 / Chapter 2.1.7 --- Cell Death Detection ELISAPLUS Kit --- p.63 / Chapter 2.1.8 --- Reagents for Measuring Caspase Activity --- p.65 / Chapter 2.1.9 --- Reagents for Total RNA Isolation --- p.66 / Chapter 2.1.10 --- Reagents and Buffers for RT-PCR --- p.69 / Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.74 / Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.75 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.80 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.81 / Chapter 2.2.3 --- Determination of Cell Viability --- p.82 / Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.83 / Chapter 2.2.5 --- In Vivo Tumorigenicity Study --- p.83 / Chapter 2.2.6 --- Analysis of Cell Cycle Profile / DNA Content by Flow Cytometry --- p.83 / Chapter 2.2.7 --- Measurement of Apoptosis --- p.84 / Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.86 / Chapter 2.2.9 --- Measurement of Caspase Activity --- p.87 / Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species (ROS) --- p.88 / Chapter 2.2.11 --- Study of the Scavenging Activity of Antioxidants --- p.88 / Chapter 2.2.12 --- Gene Expression Study --- p.89 / Chapter 2.2.13 --- Protein Expression Study --- p.92 / Chapter 2.2.14 --- Measurement of Cell Differentiation --- p.95 / Chapter 2.2.15 --- Statistical Analysis --- p.98 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTICITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction / Chapter 3.2 --- Results --- p.99 / Chapter 3.2.1 --- Anti-proliferative Activity of CLA-mix and CLA Isomers on Various Myeloid Leukemia Cell Lines In Vitro --- p.101 / Chapter 3.2.2 --- Cytotoxic Effect of CLA-mix on the WEHI-3B JCS Cells In Vitro --- p.109 / Chapter 3.2.3 --- Cytotoxic Effect of CLA-mix on Primary Murine Myeloid Cells In Vitro --- p.111 / Chapter 3.2.4 --- Kinetic and Reversibility Studies of the Anti-proliferative Activity of CLA-mix on the WEHI-3B JCS Cells --- p.113 / Chapter 3.2.5 --- Effect of CLA-mix and its isomers on the Cell Cycle Profiles of the WEHI-3B JCS Cells In Vitro --- p.116 / Chapter 3.2.6 --- Effect of CLA-mix and its isomer on the Expression of Cell Cycle-regulatory Genes in the WEHI-3B JCS Cells --- p.123 / Chapter 3.2.7 --- Effect of CLA-mix and its isomer on the In V Tumorigenicity of the WEHI-3B JCS Cells ivo --- p.128 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.141 / Chapter 4.2 --- Results --- p.141 / Chapter 4.2.1 --- Induction of Apoptosis in Both Murine and Human Myeloid Leukemia Cells by CLA --- p.144 / Chapter 4.2.2 --- Effect of CLA and its Isomer on the Mitochondrial Membrane Potential of the WEHI-3B JCS Cells --- p.151 / Chapter 4.2.3 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Genes of the Bcl-2 Family in the WEHI-3B JCS Cells --- p.154 / Chapter 4.2.4 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Proteins in the WEHI-3B JCS Cells --- p.158 / Chapter 4.2.5 --- Effect of CLA-mix and its Isomer on the Induction of Caspase Activity in the WEHI-3B JCS Cells --- p.161 / Chapter 4.2.6 --- Effect of CLA-mix and its Isomer on the Induction of ROS in the WEHI-3B JCS Cells --- p.170 / Chapter 4.2.7 --- Effect of Antioxidants on the Induction of ROS by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.173 / Chapter 4.2.8 --- Effect of Antioxidants on the Induction of Apoptosis by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.176 / Chapter 4.2 --- Discussion / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.187 / Chapter 5.2 --- Results --- p.190 / Chapter 5.2.1 --- Morphological Alterations in CLA-mix- and CLA isomer-treated WEHI-3B JCS Cells --- p.190 / Chapter 5.2.2 --- Effects of CLA-mix on the Cell Size and Granularity of WEHI-3B JCS Cells --- p.196 / Chapter 5.2.3 --- Studies of the Surface Phenotypic Changes in the CLA-mix-treated WEHI-3B JCS cells --- p.198 / Chapter 5.2.4 --- Studies on the Induction of Monocytic Serine Esterase (MSE) Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.200 / Chapter 5.2.5 --- Studies on the Induction of Endocytic Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.201 / Chapter 5.2.6 --- Studies on the Expression of the Differentiation-regulatory Cytokine Genes in the CLA-mix-treated WEHI-3B JCS Cells --- p.202 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES REFERENCES --- p.208 / REFERENCES --- p.217

Ho, Lai Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 213-238). / Abstract also in Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgements --- p.vii / List of abbreviations --- p.ix / Table of contents --- p.xiv / Chapter Chapter One: --- General Introduction_ --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- An overview on neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.4 / Chapter 1.1.3 --- Epidemiology of neuroblastoma --- p.7 / Chapter 1.1.4 --- Clinical manifestation of neuroblastoma --- p.10 / Chapter 1.1.5 --- Diagnosis of neuroblastoma --- p.10 / Chapter 1.1.6 --- Conventional therapy of neuroblastoma --- p.12 / Chapter 1.1.7 --- Novel treatments of neuroblastoma --- p.14 / Chapter 1.2 --- Conjugated linolenic acid (CLN) --- p.16 / Chapter 1.2.1 --- An overview of polyunsaturated fatty acids and conjugated fatty acids --- p.16 / Chapter 1.2.2 --- Chemical structure and physical properties of CLNs --- p.18 / Chapter 1.2.3 --- Natural occurrence of CLNs --- p.21 / Chapter 1.2.4 --- Synthesis of CLNs --- p.22 / Chapter 1.2.5 --- Metabolism and pharmacokinetics of CLNs --- p.24 / Chapter 1.2.6 --- Major biological and pharmacological activities of CLNs --- p.25 / Chapter 1.2.6.1 --- Hypolipidemic and anti-obese effects --- p.25 / Chapter 1.2.6.2 --- Anti-cancer effects --- p.27 / Chapter 1.2.6.2.1 --- Anti-proliferation --- p.27 / Chapter 1.2.6.2.2 --- Chemoprevention --- p.28 / Chapter 1.2.6.2.3 --- Apoptosis-inducing --- p.28 / Chapter 1.3 --- Aims and scope of the study --- p.31 / Chapter Chapter Two: --- Materials and Methods_ --- p.34 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Animals --- p.35 / Chapter 2.1.2 --- Cell lines --- p.35 / Chapter 2.1.3 --- "Cell culture medium, buffers and other reagents" --- p.37 / Chapter 2.1.4 --- Reagents for DNA extraction --- p.46 / Chapter 2.1.5 --- Reagents for gel electrophoresis of nucleic acids --- p.47 / Chapter 2.1.6 --- Reagents and buffers for flow cytometry --- p.49 / Chapter 2.1.7 --- Reagents and buffers for measuring caspase activity --- p.50 / Chapter 2.1.8 --- Reagents for Hoechst staining --- p.53 / Chapter 2.1.9 --- Reagents and buffers for RNA extraction --- p.53 / Chapter 2.1.10 --- Reagents and buffers for DNA digestion --- p.54 / Chapter 2.1.11 --- Reagents and buffers for reverse transcription --- p.55 / Chapter 2.1.12 --- Reagents for real-time polymerase chain reaction --- p.57 / Chapter 2.1.13 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Culture of cell lines --- p.64 / Chapter 2.2.2 --- Preparation of NIH-3T3 conditioned medium --- p.65 / Chapter 2.2.3 --- Determination of cell viability --- p.65 / Chapter 2.2.4 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.66 / Chapter 2.2.5 --- Cytotoxicity test of CLNs on murine peritoneal macrophages --- p.67 / Chapter 2.2.6 --- Cytotoxicity test of CLNs on murine bone marrow cells --- p.68 / Chapter 2.2.7 --- Cytotoxicity test of CLNs on murine splenocytes --- p.68 / Chapter 2.2.8 --- Cytotoxicity tests of CLNs on human peripheral blood mononuclear cells --- p.69 / Chapter 2.2.9 --- Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining analyzed by flow cytometry --- p.69 / Chapter 2.2.10 --- Determination of colony forming ability --- p.70 / Chapter 2.2.11 --- Determination of cell invasiveness --- p.70 / Chapter 2.2.12 --- In vivo tumorigenicity assay --- p.71 / Chapter 2.2.13 --- Analysis of cell cycle profile/ DNA content by flow cytometry --- p.72 / Chapter 2.1.14 --- Measurements of apoptosis --- p.72 / Chapter 2.1.15 --- Measurements of differentiation --- p.77 / Chapter 2.1.16 --- Gene expression study --- p.78 / Chapter 2.2.17 --- Protein expression study --- p.81 / Chapter 2.2.18 --- Statistical Analysis --- p.84 / Chapter Chapter Three: --- Anti-proliferative Effect of CLN Isomers on Human Neuroblastoma cells --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Results --- p.89 / Chapter 3.2.1 --- Anti-proliferative effect of CLN isomers on various human neuroblastoma cell lines in vitro --- p.89 / Chapter 3.2.2 --- Direct cytotoxic effect of jacaric acid on neuroblastoma LA-N-1 cells in vitro --- p.100 / Chapter 3.2.3 --- Cytotoxicity of jacaric acid on primary murine cells and human normal cell lines --- p.103 / Chapter 3.2.4 --- Kinetic and reversibility studies of the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Synergistic anti-proliferative effect of jacaric acid with daidzein and retinoic acid on LA-N-1 cells in vitro --- p.110 / Chapter 3.2.6 --- Modulatory effect of jacaric acid on the number of cell division in LA-N-1 cells --- p.113 / Chapter 3.2.7 --- Effect of jacaric acid on the cell cycle profile of LA-N-1 cells --- p.115 / Chapter 3.2.8 --- Effect of jacaric acid on the invasiveness of LA-N-1 cells --- p.118 / Chapter 3.2.9 --- Effect of jacaric acid on the colony forming ability of LA-N-1 cells in soft agar --- p.120 / Chapter 3.2.10 --- Effect of jacaric acid on the in vivo tumorigenicity of the LA-N-1 cells --- p.122 / Chapter 3.3 --- Discussion --- p.124 / Chapter Chapter Four: --- Apoptosis- and Differentiation-inducing Effects of Jacaric Acid on Human Neuroblastoma Cells --- p.133 / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.2 --- Results --- p.138 / Chapter 4.2.1 --- Induction of DNA fragmentation and apoptotic ultrastructural changes in LA-N-1 cells by jacaric acid --- p.138 / Chapter 4.2.2 --- Induction of phosphatidylserine externalization by jacaric acid in human neuroblastoma cells as detected by Annexin V-GFP/ PI dual staining --- p.142 / Chapter 4.2.3 --- Effect of jacaric acid on the mitochondrial membrane potential in human neuroblastoma cells --- p.146 / Chapter 4.2.4 --- Effect of jacaric acid on the caspase-3 activity in LA-N-1 cells --- p.150 / Chapter 4.2.5 --- Effect of jacaric acid on the reactive oxygen species (ROS) generation in human neuroblastoma cells --- p.153 / Chapter 4.2.6 --- Morphological changes induced by jacaric acid in SH-SY5Y cells --- p.158 / Chapter 4.3 --- Discussion --- p.162 / Chapter Chapter Five: --- Mechanistic Studies of Anti-proliferative Effect of Jacaric Acid on Human Neuroblastoma Cells --- p.171 / Chapter 5.1 --- Introduction --- p.172 / Chapter 5.2 --- Results --- p.178 / Chapter 5.2.1 --- Effect of antioxidant a-tocopherol on the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.178 / Chapter 5.2.2 --- Effect of caspase inhibitors on the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.180 / Chapter 5.2.3 --- Jacaric acid modulated the mRNA expression of N-myc and other related transcription factors in LA-N-1 cells --- p.182 / Chapter 5.2.4 --- Jacaric acid modulated the protein expression of N-myc --- p.186 / Chapter 5.2.5 --- Jacaric acid modulated the mRNA expression of apoptosis-associated genes --- p.188 / Chapter 5.3 --- Discussion --- p.191 / Chapter Chapter Six: --- Conclusions and Future Perspectives --- p.202 / References --- p.212

This comprehensive review critically evaluates whether supposed health benefits propounded upon human consumption of conjugated linoleic acids (CLAs) are clinically proven or not. With a general introduction on the chemistry of CLA, major clinical evidences pertaining to intervention strategies, body composition, cardio-vascular health, immunity, asthma, cancer and diabetes are evaluated. Supposed adverse effects such as oxidative stress, insulin resistance, irritation of intestinal tract and milk fat depression are also examined. It seems that no consistent result was observed even in similar studies conducted at different laboratories, this may be due to variations in age, gender, racial and geographical disparities, coupled with type and dose of CLA supplemented. Thus, supposed promising results reported in mechanistic and pre-clinical studies cannot be extrapolated with humans, mainly due to the lack of inconsistency in analyses, prolonged intervention studies, follow-up studies and international co-ordination of concerted studies. Briefly, clinical evidences accumulated thus far show that CLA is not eliciting significantly promising and consistent health effects so as to uphold it as neither a functional nor a medical food.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 193-206).

Structured phospholipids with functional ingredients like conjugated linoleic acid (CLA) and plant sterols to deliver their physiological effects in different food formulations were synthesized. The lipase and phospholipase A2 catalyzed enzymatic acidolysis reaction between phospholipids (PLs) and CLA was used for fatty acid modification, while the phospholipase D catalyzed transphosphatidylation reaction between PLs and sterol was used for head group modification. Enzymatic processes were an effective way to produce structured phospholipids. Screening of four lipases and immobilized phospholipase A2 and combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PLs. The maximum incorporation achieved by the latter enzyme was 16% with soy PLs in 72 h. The class of phospholipids had a significant effect on the rate of incorporation of CLA compare to source of PLs. A method capable of predicting the rate of incorporation of CLA into phospholipids was developed using response surface methodology. A three-level four-factor Central Composite Rotatable Design (CCRD) was used. The four factors selected were lipase dosage (Ed, wt.% of substrate), substrate ratio (Sr,mol%), reaction time (ti, h) and reaction temperature (Te,oC). The enzyme load and substrate ratio had a greater effect on the rate of incorporation than did reaction time and temperature. A polynomial regression equation was developed to predict the reaction rate. The new phosphatidyl derivative, phosphatidyl-sitosterol, was found to be synthesized by the transfer reaction of phosphatidyl residue from phosphatidylcholine to β-sitosterol by phospholipase D from Streptomyces sp. in biphasic medium. The novel phosphatidyl .sitosterol derivative was identified by MALDI-TOF mass spectrometry. Plant sterols were modified to a more polar lipid class by synthesizing phospholipid derivatives of them. When these structured phospholipids were added to a whey protein based oil-in-water emulsion, the CLA incorporated structured phospholipids (CLA-PL) had higher heat stability and oxidative stability compared to the controls.