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Predominance of small dense Low Density Lipoprotein (LDL) is associated with a two to threefold increase in risk for Coronary Heart Disease (CVD). Small, dense HDL (High Density Lipoprotein) particles protect small dense LDL from oxidative stress. Technological advancements have introduced an array of techniques for measuring diameters of LDL and HDL as well as estimating overall particle heterogeneity. However, there is lack of comparative studies between these techniques, and, hence, no conclusive evidence to establish the merits of one method relative to others. The primary purpose of this study was to compare Nondenaturing Gradient Gel Electrophoresis (NDGGE) and Dynamic Laser Light Scattering (DLLS) methods in determining LDL and HDL particle diameter. Our comparison entailed: 1) Evaluating the two methods in terms of their reproducibility 2) Correlating the two methods(in future studies method selection would be driven by time and cost considerations if the two methods correlate), and 3) Evaluating the two methods in terms of their ability to identify bi-modal samples. A secondary purpose of this research was to investigate the effect of refrigerated plasma storage on particle diameter. Reproducibility was measured as Coefficient of Variance (CV). Within and between runs, CV for LDL and HDL for NDGGE were <6% and <15%, respectively and for DLLS, CV within runs were <3% and <5.5%, respectively. No correlation was observed between LDL diameter from the two methods. NDGGE showed two bands for 157 HDL samples of which only 24 samples showed bimodal peaks in DLLS. In order to study the effect of storage, three sample sets of LDL and two sample sets of HDL were used. NDGGE showed a significant difference between mean diameter of fresh and stored LDL and HDL sample for all sets, whereas DLLS showed a significant difference in only one LDL sample set and none for HDL sample sets. We conclude that DLLS may be a better method for measuring LDL diameter because NDGGE overestimated LDL diameter. However, NDGGE was able to resolve subpopulation better in an HDL sample than DLLS. Thus, NDGGE may be a better choice for measuring HDL diameter than DLLS.

An array of analytical methods including density gradient ultracentrifugation, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were utilized to analyze serum high density lipoprotein (HDL) subfractions from two cohorts of normolipidemic individuals, which included subjects with diagnosed coronary artery disease (CAD), and angiographically proven non-CAD controls. These methods collectively provided characteristic information about the two populations of individuals including composition, electrophoretic mobilities, molecular weights, isoforms, and post-translational modifications of HDL apolipoproteins. This information proved useful in identifying potential biomarkers for CAD risk, and understanding the biological functions of a novel atherogenic HDL phenotype in individuals with CAD. Through the implementation of the aforementioned methodologies, new isoforms of apoC-I were identified. MALDI-MS, detected a shifting of approximately 90 Da in the mass to charge ratios corresponding to apoC-I peaks in the serum subfractions from all CAD cohort patients. This shifting was not observed in the non-CAD cohort, which displayed apoC-I peaks in accordance with the known mass of this protein. In addition to the shifting observed in the CAD cohort, some CAD patients showed further modifications of apoC-I that were indicative of oxidative processes. Interestingly, one patient, who has not been diagnosed with CAD, and has a family history of the disease, contained the apoC-I isoforms. This feature could underlie this subject’s known family history of CAD, and serve as an initial screening that could indicate the future development of CAD in this individual. Through collaborative work with Johns Hopkins University, it was initially observed that apoC-I enriched HDL induced apoptosis of aortic smooth muscle cells. Conversely, apoC-I depleted HDL induced minimal to no apoptosis, which led to the hypothesis that apoC-I is a contributor to atherogenic HDL and is a potential risk factor for CAD. Further collaborative work with Johns Hopkins assessed the apoptosis levels induced by HDL from both cohorts of patients. A distinct difference in apoptosis was identified between the two cohorts. High density lipoprotein subfractions from subjects in the CAD cohort, all of which contained the apoC-I isoforms, induced marked apoptosis compared to the non-CAD controls. These results further supported the hypothesis that apoC-I compromises the functionality of HDL and showed that through the induction of apoptosis, apoC-I can contribute to the destabilization of atherosclerotic plaque and the acceleration of CAD.

Background: Kawasaki disease (KD) affects mainly children younger than five years of age, leading to coronary artery lesions, and even to life-threatening myocardial infarctions. Since 1976, Kawasaki disease has occurred among thousands of children in Taiwan. Evidence suggests that inflammation plays a key role in the pathogenesis of atherosclerosis. Significant determinants of high sensitivity C-reactive protein (hs-CRP), which is a sensitive indicator of inflammation, as well as white blood cell (WBC) count, and high-density lipoprotein cholesterol (HDLc) and coronary artery lesion were identified. The relationships between these factors¡¦ concentration and arterial lesion were likewise investigated and had reported. The aim of this study was to determine the serum levels of the hs-CRP, WBC count, and plasma HDLc levels in patients with later phase of KD. Methods and Materials: From July 2005 to June 2006, 97 children with Kawasaki disease at least 1 year after diagnosis were recruited in this study. These participated children had been diagnosed as KD and collected at the interval of 2001 to 2004. Diagnosis was based on the 1984 revised by the KD Research Committee in Japan. The participants were grouped into 45 patients with KD and coronary aneurysms (Group I), 52 patients with KD and normal coronary arteries (Group II), and 50 healthy age-matched children (Control Group III). Their WBC count, systemic and diastolic blood pressures, body mass index, age, sex, fasting total cholesterol concentrations, triglyceride, high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol, serum hs-CRP levels, and coronary artery lesion by cardioechography were recorded and compared. The analytical differences between hs-CRP, WBC count, and plasma HDLc levels and the coronary artery events in KD were examined. Results: Serum hs-CRP levels of Group I patients (mean 0.264 mg/dl) was significantly greater than that of Group II (mean 0.155 mg/dl, p=0.006) and Group III patients (mean 0.116 mg/dl, p =0.017). Similarly, the WBC count of Group I patients (mean 6,543.11/mm3) was significantly greater than that of Group II (mean 5,720.19/mm3, p=0.029), and Group III patients (mean 5,611.27/mm3, p =0.012). However, plasma HDLc levels of Group I patients (mean 41.42 mg/dl) was significantly lesser than that of Group II (mean 44.79 mg/dl, p=0.035), and Control Group III patients (mean 46.58 mg/dl, p=0.027). There was a positive association between hs-CRP and WBC count levels (r = 0.641, p < 0.05), but none between hs-CRP and plasma HDLc levels. Conclusions: There is the possibility of ongoing low-grade inflammation late after the convalescent phase of Kawasaki disease in children with coronary aneurysms, which may have a role in increasing coronary artery dysfunction. These results also suggest that hs-CRP, WBC count, and plasma HDLc levels are useful parameters for predicting formation of coronary artery lesion even in children after onset of KD.

BACKGROUND & AIMS - Metabolic syndrome (MetS), characterized by abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, and insulin resistance is a major public health concern in the United States. The effect of Apolipoprotein E (Apo E) polymorphism has been relatively well studied in relation to cardiovascular disease; however, its effects on MetS are not well established. METHODS - We conducted a cross-sectional study consisting of 1,551 participants from the National Heart, Lung, and Blood Institute (NHLBI) Family Heart Study to assess the relation of Apo E polymorphism with the prevalence of MetS. Information on the different Apo E genotypes was extracted from the database and we defined MetS according to the AHA-NHLBI-IDF-WHO Harmonized Criteria. We used generalized estimating equations to estimate adjusted odds ratios for prevalent MetS and the Bonferroni correction to account for multiple testing in the secondary analysis. RESULTS – Our study population had a mean age (SD) of 56.5 (11.0) years and 49.7% had MetS. There was no association between the Apo E genotypes and MetS. The multivariable adjusted ORs (95% CI) were 1.00 (reference), 1.26 (0.31-5.21), 0.89 (0.62- 1.29), 1.13 (0.61-2.10), 1.13 (0.88-1.47) and 1.87 (0.91-3.85) for the *e3/e3, *e2/e2, *e2/e3, *e2/e4, *e3/e4 and *e4/e4 genotype respectively. In a secondary analysis, the *e2/e3 genotype was associated with lower HDL levels, with the multivariable adjusted ORs (95% CI) of 0.59 (0.36-0.95) when compared to the reference *e3/e3 genotype. CONCLUSIONS - Our findings do not support an association between Apo E polymorphism and MetS in a multi-center population based study of predominantly white US men and women. The *e2/e3 genotype was associated with lower HDL levels as compared to the *e3/e3 genotype. KEY WORDS: Apolipoprotein E (Apo E) polymorphism, metabolic syndrome, blood pressure, glucose, waist circumference, triglycerides, high-density lipoprotein cholesterol

Abstract Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol. Ethanol causes changes in the concentration and composition of plasma lipoproteins and it also influences the enzymes and transfer proteins that modify lipoproteins in plasma. PEth might be one of these changes brought on by ethanol in the circulation. The present study was designed to investigate whether qualitative changes in high density lipoprotein (HDL) phospholipids caused by ethanol can mediate the beneficial effects of alcohol on atherosclerosis, and to investigate the transfer of PEth between lipoproteins and the effects of PEth on the charge of lipoprotein particles. PEth was shown to be transferred from low density lipoproteins (LDL) to HDL particles mainly by transfer proteins other than cholesteryl ester transfer protein (CETP). The transfer of PEth between lipoproteins enables the redistribution of PEth between lipoproteins in plasma. The results of this study provide evidence that PEth in HDL particles stimulates the vascular endothelial growth factor (VEGF) secretion from vascular wall cells. The increase in the secretion was mediated through protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signalling pathways. PEth-containing HDL particles were able to increase the VEGF secretion in rats in vivo. Similar effects were also observed when rats were given HDL particles isolated from the plasma of alcoholics. The PEth-induced change in the electrical charge of lipoproteins may affect the binding of lipoproteins to their receptors and binding proteins. The effects of PEth on the secretion of VEGF from the endothelial cells were shown to be mediated through HDL receptor. The changes in HDL particles caused by phosphatidylethanol may modify the metabolism of lipoproteins and lipid-mediated signalling pathways regulating VEGF in vascular wall cells.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17905号 / 農博第2028号 / 新制||農||1018(附属図書館) / 学位論文||H25||N4801(農学部図書室) / 30725 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 加納 健司, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20801号 / 医博第4301号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 山下 潤, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM