A modificação oxidativa da LDL possui um papel crucial na patogênese da aterosclerose que é uma das principais causas de mortalidade nos pacientes renais crônicos. Uma subfração de LDL modificada in vivo, denominada LDL eletronegativa (LDL-), é formada a partir de modificações da parte protéica (ApoB100) e lipídica (fosfolípides, triglicérides e colesterol) da LDL induzidas por diversos mecanismos. A LDL (-) tem menor afinidade pelos receptores da LDL, é citotóxica para células endoteliais e apresenta atividade pró-inflamatória, quando comparada à LDL nativa. Com o objetivo de investigar as alterações do estado nutricional relacionadas à formação da LDL(-) nos pacientes renais crônicos, analisou-se neste estudo as concentrações plasmáticas de LDL(-), anticorpos IgG anti-LDL(-) e seus imunocomplexos em pacientes sob hemodiálise (HD, n=25) ou sob diálise peritoneal (DP, n=11) e indivíduos saudáveis (grupo controle, n=10), relacionando-as ao perfil lipídico e às concentrações plasmáticas de α-tocoferol e ascorbato. Os resultados mostraram que a concentração de LDL(-) foi maior (p<0.01) nos pacientes hemodialisados (575,6±233,1µg/mL) quando comparados aos pacientes submetidos à diálise peritoneal (223,4±117,5 µg/mL) e aos controles (54,9±33,3µg/mL). Os níveis de anticorpos IgG anti-LDL(-) foram mais elevados (p<0,00001) nos controles (O,36±0,09µg/mL), quando comparados aos pacientes DP (0,28±0,12µg/mL) e HD (0,2±0,1 µg/mL). As concentrações dos imunocomplexos no grupo controle (0,35±0,20µg/mL) foram significativamente maiores comparadas às dos grupos HD (0,15±0,07µg/mL) e DP (0,22±0,07µg/mL). Não houve diferença das concentrações plasmáticas de ascorbato e de alfa-tocoferol (normalizada pela concentração de colesterol) nos grupos estudados. A maioria da população estudada estava eutrófica, segundo o índice de massa corpórea (IMG). Conclui-se que as concentração de LDL(-) nos pacientes HD e DP foram significativamente mais elevadas, enquanto os níveis de anticorpos IgG anti-LDL(-) foram menores, nos pacientes HD e DP comparados ao grupo controle. As análises de correlação demonstraram que os valores de prega cutânea tricipital (PCT) se correlacionaram diretamente com as concentrações plasmáticas dos imunocomlexos (r= 0,37; p= 0,01) e inversamente com as concentrações plasmáticas de LDL(-) (r= - 0.37; p= 0,018). As concentrações plasmáticas dos anticorpos anti-LDL(-) se correlacionaram diretamente com os valores do IMC (r= 0,83 p=0,00001) e da circunferência da cintura (r= 0,75 p= 0,0001). / A minimally modified form of LDL, with structural ApoB100 modification and lower affinity by LDL receptors, has been described in blood plasma. This circulating modified form of LDL, named electronegative LDL, LDL(-), has increased negative charge, higher cytotoxicity and pro-inflammatory activity as compared to the native LDL. This LDL-is poorly described in hemodialysis and there is no study in peritoneal dialysis patients. Thus, the purpose of this study was to evaluate the relation of the nutritional status with the amount of electronegative LDL (LDL-), its autoantibodies and immune complexes (IC) in dialysed patients. LDL(-), its autoantibodies and IC were determined by ELISA in chronic kidney disease (CKD) patients undergoing hemodialysis (HD) or peritoneal dialysis (PD) and compared to subjects without CKD (controls). Nutritional status, lipid profile and plasma concentrations of alpha-tocopherol, ascorbate and immune complexes (IC) were also evaluated. Results are expressed as median of LDL-(µg/mL) and anti-LDL(-) IgG (OD405 nm). The concentrations of LDL(-) were higher in HD patients (575.6±233.1 µg/mL) as compared to PD (223.4±117.5µg/mL) and control groups (54.9±33.3µg/mL) (p<0.01). The anti-LDL(-) IgG auto-antibodies were elevated in controls (0.36±0.09µg/mL) in relation to PD patients (0.28±0.12µg/mL) and HD patients (0.2±0.1 µg/mL) , (p<0.00001). A negative correlation was observed between anti¬-LDL(-)lgG and LDL(-) levels (r = -0.43; P = 0.003) in the studied groups. The concentrations of le in the control group (0.35±0.20µg/mL) were higher compared with HD (0.15±0.07µg/mL) and PD (0.22±0.07µg/mL) groups. No differences were found for the plasma levels of ascorbate and alpha-tocopherol (normalized by cholesterol concentration) among the studied groups The body mass index (BMI) was normal in the majority of the studied subjects. The highest LDL(-) concentrations were found in HD patients, and for the first time, we showed that PD patients also have high levels of LDL(-) when compared with non-CKD subjects. The levels of anti-LDL(-) IgG in CKD patients were lower compared to controls. The correlation analysis showed that the values for triceps skin fold were positively correlated with blood plasma concentrations of IC (r= 0.37; p=0.01) and negatively correlated with LDL(-) concentrations (r= - 0.37; P 0.018). The concentrations of anti-LDL(-) autoantibodies were directly ·correlated with BMI (r= 0.83 p=0.00001) and waist circunference (r= 0.75 p= 0.0001).

Diversos estudos têm evidenciado que as doenças inflamatórias estão fortemente ligadas à condição de estresse oxidativo. Dentre elas, uma das mais estudadas é a aterosclerose. A aterosclerose é uma doença de grande importância mundial, devido à alta mortalidade resultante de condições clínicas decorrentes da doença. Estudos demonstram que a modificação da LDL é um fator importante no desenvolvimento desta doença. Por isso, a determinação da LDLox plasmática é essencial, não apenas para investigar sua relevância para as doenças cardiovasculares, mas, também, como um auxiliar no diagnóstico destas doenças. A oxidação da lipoproteína de baixa densidade (LDLox) induz à formação de epítopos imunogênicos na molécula. Contudo, o papel desses anticorpos na fisiopatologia da aterosclerose e o seu significado clínico permanecem indefinidos. O objetivo deste estudo foi produzir anticorpos anti-LDLox para uso em diagnóstico laboratorial de risco cardiovascular e também avaliar a associação entre a LDLox e o complexo formado entre o LDLox e o anticorpos anti-LDL na resposta inflamatória de macrófagos pela ativação da NADPH oxidase associada a proteína dissulfeto isomerase (PDI). A ativação da NADPH oxidase de macrófagos requer o acoplamento das subunidades citosólicas aos componentes de grânulos e translocação do complexo à membrana do fagossoma. A PDI é uma chaperona envolvida no tráfego protéico celular, sendo encontrada na superfície de diversas células procarióticas e eucarióticas. Trabalhos prévios sugeriram que PDI pode ser um dos componentes responsáveis pela montagem de Nox2, facilitando o enovelamento correto das subunidades do complexo e/ou auxiliando o trânsito das subunidades até à membrana do fagossoma. Neste trabalho testou-se a atividade de Nox2 de macrófagos inflamatórios correlacionada à atividade redutase de PDI de membrana. Macrófagos dormentes apresentaram baixa atividade de PDI e não foi detectado consumo de oxigênio associado ao sistema Nox2. Sob estímulo de LDLox e do complexo LDLox/ anti-LDLox, os fagócitos consumiram quantidade significativa de oxigênio e apresentaram atividade de PDI, maior que as células dormentes. Testes com inibidores padrões de PDI (bacitracina e ácido ditionitrobenzóico- DTNB) confirmaram que a inibição de PDI determina decréscimo da atividade de Nox2, resultados obtidos através de ensaios espectrofotométricos (redução de citocromo c, 550 nm). Em conjunto, os resultados apontam que, simultaneamente à atividade de Nox2 em macrófagos estimulados, ocorre atividade redutase de PDI, confirmando que essa chaperona está diretamente associada à ativação e/ou manutenção da atividade da oxidase fagocitária. / Inflammatory disorders are strongly associated with the condition of oxidative stress, and atherosclerosis is one of the most studied condition. Atherosclerosis is a disease of great world-wide importance due to the high mortality of decurrently clinical conditions of the illness. Studies have demonstrated that the modification of the LDL is an important factor in the development of the illness. Therefore, the determination of the plasma LDL-ox is essential not only to investigate its relevance for the atherosclerotic diseases, but also to contribute in the diagnosis of these illnesses. The oxidation of low-density lipoprotein (oxLDL) induces the formation of immunogenic epitopes in molecules. However, the role of these antibodies in the pathophysiology of atherosclerosis and their clinical significance remain undefined. Objective this study was produce antibodies anti-LDLox in the diagnosis of atherosclerotic diseases and evaluate the association between LDLox and complex LDLox/anti-LDLox in activation the regulatory activity of PDI interconnected to Nox2 in inflammatory macrophages. Protein disulfide isomerase is an ubiquitously expressed enzyme that catalyses the rearrangement of disulfide bonds in target proteins. Previous studies suggest that PDI, which is a chaperone involved in protein trafficking and translocates to the cell surface, may regulate the phagocytic NADPH oxidase complex (Nox2). LDLox and complex LDLox/ anti-LDLox-triggered superoxide anion release was correlated with the PDI reductase activity detected in macrophages, as determined by oxygen consumption and cleavage of a fluorescent probe, respectively. Assays with the known PDI inhibitors bacitracin and dithionitrobenzoic acid were performed to confirm their ability to decrease the macrophages respiratory burst. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

by Chu Chi Yuen, Andrew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / ABSTRACT --- p.i / Chapter 1 --- INTRODUCTION --- p.3 / Chapter 1.1 --- Using Low density lipoprotein (LDL) as a drug carrier --- p.4 / Chapter 1.1.1 --- The structure of Low density lipoprotein (LDL) --- p.4 / Chapter 1.1.2 --- The metabolic pathway of LDL in human bodies --- p.4 / Chapter 1.1.3 --- The rationale for using LDL as a drug carrier --- p.7 / Chapter 1.1.4 --- Reconstitution of LDL with cytotoxic drugs --- p.9 / Chapter 1.1.5 --- Up and down regulation of LDL receptors --- p.11 / Chapter 1.2 --- Doxorubicin (DOX) --- p.12 / Chapter 1.2.1 --- Characteristics of DOX --- p.12 / Chapter 1.2.2 --- Drug actions of DOX --- p.14 / Chapter 1.2.3 --- The adverse side effects of DOX --- p.15 / Chapter 1.3 --- Multidrug resistance phenomenon in tumor cells --- p.17 / Chapter 1.3.1 --- The possible mechanisms of multidrug resistance --- p.19 / Chapter 1.3.2 --- The structure of P-glycoprotein --- p.20 / Chapter 1.3.3 --- The mechanisms of the P-glycoprotein --- p.22 / Chapter 1.3.4 --- Our aim in dealing with multidrug resistance --- p.22 / Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Animals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Culture media --- p.25 / Chapter 2.1.4 --- Chemicals --- p.26 / Chapter 2.1.5 --- Culture of cells --- p.27 / Chapter 2.2 --- Methods --- p.29 / Chapter 2.2.1 --- In vitro studies --- p.29 / Chapter 2.2.2 --- In vivo studies --- p.44 / Chapter 3 --- RESULTS --- p.51 / Chapter 3.1 --- In vitro studies --- p.51 / Chapter 3.1.1 --- Preparation of LDL-DOX --- p.51 / Chapter 3.1.2 --- Comparison of the cytotoxicity of DOX and LDL-DOX on HepG2 cells --- p.59 / Chapter 3.1.3 --- Modulation of LDL receptors on HepG2 cells and ECV304 cells… --- p.63 / Chapter 3.1.4 --- The effect of combined treatment of LDL-DOX and hyperthermia on HepG2 cells --- p.84 / Chapter 3.1.5 --- The effect of LDL-DOX on resistant cell line R-HepG2 cells --- p.90 / Chapter 3.2 --- In vivo studies --- p.105 / Chapter 3.2.1 --- The comparison of organ distribution of LDL-DOX and DOXin BALB-c mice after administration --- p.105 / Chapter 3.2.2 --- The comparison of organ distribution of LDL-DOX and DOX in nude mice bearing HepG2 cells after adminstration --- p.108 / Chapter 3.2.3 --- Histological studies of heart of nude mice bearing HepG2 cells treated with DOX and LDL-DOX --- p.111 / Chapter 3.2.4 --- Myocardial injury measured by Lactate dehydrogenase (LDH) activity in nude mice bearing HepG2 treated with DOX and LDL- DOX --- p.117 / Chapter 3.2.5 --- The comparison of DOX and LDL-DOX on reducing the tumor sizes and weight in nude mice bearing HepG2 cells --- p.119 / Chapter 4 --- DISCUSSION --- p.122 / Chapter 4.1 --- In vitro studies --- p.122 / Chapter 4.1.1 --- Preparation of LDL-DOX complex --- p.122 / Chapter 4.1.2 --- The cytotoxicity ofLDL-DOX --- p.125 / Chapter 4.1.3 --- The combined treatment of hyperthermia and LDL-DOX --- p.129 / Chapter 4.1.4 --- The ability of LDL-DOX to circumvent muiltidrug resistance --- p.131 / Chapter 4.2 --- In vivo studies --- p.134 / Chapter 5 --- CONCLUSION --- p.136 / Chapter 5.1 --- Conclusion --- p.136 / Chapter 5.2 --- Future pospective --- p.139 / BIBLIOGRAPHY --- p.140

Submitted by Ana Rosa Silva (arosa@ufpa.br) on 2012-07-31T14:53:18Z No. of bitstreams: 2 Tese_InvestighacaoEfeitoTerapeutico.pdf: 3174368 bytes, checksum: 3c98eae3552ae9b98de0472a212f7836 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-07-31T14:53:59Z (GMT) No. of bitstreams: 2 Tese_InvestighacaoEfeitoTerapeutico.pdf: 3174368 bytes, checksum: 3c98eae3552ae9b98de0472a212f7836 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2012-07-31T14:53:59Z (GMT). No. of bitstreams: 2 Tese_InvestighacaoEfeitoTerapeutico.pdf: 3174368 bytes, checksum: 3c98eae3552ae9b98de0472a212f7836 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2011 / Psyllium é uma fonte rica de fibra solúvel mucilaginosa e é considerado um suplemento dietético útil no tratamento de pacientes com hipercolesterolemia. O objetivo deste estudo foi avaliar a eficácia e a segurança da suplementação do psyllium na redução do perfil lipídico em crianças e adolescentes brasileiros dislipidêmicos. Cinqüenta e cinco sujeitos (6-19 anos) com hipercolesterolemia moderada foram avaliados em um estudo clínico, paralelo, duplo cego, controlado e randomizado, conduzido em 2 períodos. Inicialmente, todos participantes recrutados passaram por um estágio de adaptação à dieta restrita em gordura saturada (<7%) e colesterol (<200 mg/dia) que durou 6 semanas antes do tratamento. Após este período, os participantes elegíveis foram alocados aleatoriamente para 2 grupos (controle n=25 e psyllium n=30) usando uma seqüência numerada randomizada gerada por computador. Durante o período de 8 semanas do ensaio clínico, o grupo psyllium manteve a dieta restrita em gordura saturada e colesterol, suplementada diariamente com 7,0 g de psyllium , enquanto o grupo controle recebeu a mesma dieta adicionada com uma quantidade equivalente de celulose (placebo). No final do tratamento, quatro sujeitos foram excluídos após randomização (perdas no seguimento) totalizando 51 sujeitos (grupo controle=24; grupo psyllium n=27), que completaram o estudo. O grupo que recebeu psyllium apresentou um decréscimo significativo nas concentrações de colesterol total (CT) (4,1% [-0,20mmol/L]; p=0,01) e de LDL-colesterol (LDL-c) (7,2% [-0,24 mmol/L]; p<0,001) em comparação à linha de base. Reduções adicionais foram observadas quando comparadas com o grupo controle (CT:4,1% [0,20mmol/L]; p=0,002) e (LDL-c:7,8% [0,26mmol/L]; p=0,007). Nenhum dos participantes relatou aversão ao cheiro, sabor e textura do psyllium, nem a presença de efeitos adversos significativos. A terapia com psyllium se mostrou eficaz na redução das concentrações do LDL-c e demonstrou ser seguro e aceitável pela população do estudo. / Psyllium is one of the richest known sources of soluble mucilaginous dietary fibre, and is considered to be a useful supplement to dietary therapy for the treatment of patients with hypercholesterolemia. The aim of this study was to assess the efficacy and safety of psyllium as a dietary supplement for the reduction of the lipidic profile of dyslipidemic Brazilian children and adolescents. Fifty-five subjects (6-19y) with mild to moderate hypercholesterolemia were evaluated in a randomised, double-blind, placebo-controlled, parallel clinical trial, conducted in two periods. During the initial dietary adaptation phase, all subjects enrolled were treated with diet low in saturated fat (<7%) and cholesterol (<200mg/day) for 6-week to prior to the treatment phase. After this period, all eligible participants were allocated randomly to two groups (control n=25 and psyllium n=30) using a computer-generated random number sequence. Over an eight-week clinical trial period, one group (psyllium) were maintained a diet low in saturated fat and cholesterol supplemented daily with 7.0g of psyllium, while the control group received the same diet plus with an equivalent amount of cellulose (placebo). At the end of the treatment period, four subjects were excluded following randomisation (lost to follow up) leaving 51 subjects (control group n= 24; psyllium group n=27, who completed the study. At the end of experiment, the psyllium group presented a significant decrease in the concentrations of total cholesterol, TC (4.1% [-0.20 mmol/L]; p=0.01) and LDL-cholesterol, LDL-c (7.2% [-0.24 mmol/L]; p<0,001). Additional reductions were observed in comparison with the control group (TC: 4.1% [-0.20 mmol/L]; p=0.007) and LDL-c: 7.8% [-0.26 mmol/L]; p=0.002). None of the participants reported any aversion to the smell, taste, appearance or texture of the psyllium, and absence serious adverse effects. Psyllium therapy shows significant efficacy on lowering of the LDL-c. It also demonstrates to be safe and acceptable for pediatric population in the study.

by Chan Chi Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-137). / Chapter SECTION 1 --- INTRODUCTION / Chapter 1.1 --- Overview on lipids --- p.2 / Chapter 1.1.1 --- Definition and Classification of Lipids --- p.2 / Chapter 1.1.2 --- Lipoproteins and Apolipoproteins --- p.4 / Chapter 1.1.3 --- Outline of Lipoprotein Metabolism --- p.9 / Chapter 1.1.4 --- LDL Metabolism --- p.12 / Chapter 1.2 --- Dyslipidaemia and Cardiovascular Disease (CVD) --- p.16 / Chapter 1.2.1 --- Definition --- p.16 / Chapter 1.2.2 --- Dyslipidaemia and CAD --- p.16 / Chapter 1.2.3 --- Dyslipidaemia in Non-Insulin Dependent Diabetes Millitus Patients --- p.18 / Chapter 1.2.4 --- Claasification of Dyslipidaemia --- p.24 / Chapter 1.2.5 --- Causes of Hyperlipidaemia --- p.26 / Chapter 1.3 --- Dyslipidaemia and Atherosclerosis --- p.29 / Chapter 1.3.1 --- Pathogenesis of Atherosclerosis --- p.29 / Chapter 1.3.2 --- Mechanism of Atherogenesis --- p.31 / Chapter 1.3.3 --- Intrinsic Roles of LDL in Atherogenesis --- p.33 / Chapter (1) --- LDL Oxidizability --- p.33 / Chapter (2) --- LDL Particle Size Heterogeneity --- p.39 / Chapter 1.4 --- Management of Dyslipidaemia --- p.42 / Chapter 1.5 --- Aims of This Study --- p.49 / Chapter SECTION 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.52 / Chapter 2.1.1 --- Patients and Controls --- p.52 / Chapter 2.1.2 --- Drug Administration Trials --- p.54 / Chapter 2.1.3 --- Blood Samples --- p.55 / Chapter 2.1.4 --- Biochemicals --- p.56 / Chapter 2.1.5 --- Solutions and Buffers --- p.56 / Chapter 2.1.6 --- Apparatus and Equipment --- p.60 / Chapter 2.2 --- Methods --- p.62 / Chapter 2.2.1 --- General Clinical Biochemistry Tests --- p.62 / Chapter 2.2.2 --- Apolipoprotein Assays --- p.62 / Chapter 2.2.3 --- Ultracentrifugation of LDL Fraction --- p.63 / Chapter 2.2.4 --- De-Salting of LDL Fraction --- p.64 / Chapter 2.2.5 --- Qualitative Determination of LDL-Cholesterol and Protein Fractions --- p.64 / Chapter 2.2.6 --- In Vitro Assessment of LDL Oxidizability --- p.65 / Chapter 2.2.7 --- Electrophoretic Gel Pattern of LDL Fraction During In Vitro Oxidizability --- p.65 / Chapter 2.2.8 --- Study of LDL Particle Size --- p.66 / Chapter 2.2.9 --- Statistical Analysis --- p.67 / Chapter SECTION 3 --- RESULTS / Chapter 3.1 --- Quantitative Determination and Standardization of LDL Fractions --- p.69 / Chapter 3.2 --- In Vitro Assessment of LDL Oxidizability --- p.72 / Chapter 3.3 --- Electrophoretic Patterns of LDL during In Vitro Oxidizability --- p.72 / Chapter 3.4 --- LDL Sizing --- p.73 / Chapter 3.5 --- "Correlations of Triglycerides Concentration, LDL Particle Size and Oxidizability" --- p.76 / Chapter 3.6 --- Diabetes Millitus --- p.83 / Chapter 3.6.1 --- NIDDM Patients & Controls --- p.83 / Chapter 3.6.2 --- Effect of Drug Treatment on Serum Lipid-Lipoprotein- Apolipoprotein Profile --- p.86 / Chapter 3.7 --- Hypertriglyceridaemic Patients --- p.90 / Chapter 3.7.1 --- Patients & Controls --- p.90 / Chapter 3.7.2 --- Bezafibrate Treatment --- p.91 / Chapter 3.8 --- CAD Patients --- p.97 / Chapter 3.8.1 --- CAD Patients & Controls --- p.97 / Chapter SECTION 4 --- DISCUSSION / Chapter 4.1 --- Patients and Controls --- p.101 / Chapter 4.2 --- Ultracentrifugation of LDL Fractions --- p.102 / Chapter 4.3 --- In Vitro LDL Oxidizability --- p.103 / Chapter 4.4 --- "Association of TG, LDL Oxidizability and Particle Size" --- p.105 / Chapter 4.5 --- LDL Sizing --- p.106 / Chapter 4.6 --- Comparsion of Patients and Controls in Lipid-Lipoprotein- Apolipoprotein Profiles --- p.107 / Chapter 4.7 --- The Effect of Lovastatin and Acipimox on NIDDM Patients --- p.111 / Chapter 4.8 --- The Effect of Bezafibrate on Hypertriglyceridaemic Patients --- p.114 / Chapter SECTION 5 --- CONCLUSION --- p.116 / References --- p.119 / Appendices --- p.138

The purpose of this study was to resolve a critical gap in depression literature through assessment of the temporal relationship between depression and low LDL. A systematic review and meta-analysis was conducted to investigate the overall cross-sectional association between serum LDL and depression. Inconsistent findings suggest that more work must be done to clarify the link between LDL and depression. Next, Cox regression was used to explore the association between LDL and the subsequent onset of depressive symptoms within a subset of the Women’s Health Initiative cohort and evaluate the potential for effect modification by lipid-lowering medication use. This study provides evidence toward an association between low LDL and the subsequent onset of depressive symptoms, with increased risk confined to LDL below 100 mg/dL. Elevated risk was not associated with lipid-lowering medication use. The final study examined the differences in the magnitude and direction of change in serum LDL levels among individuals experiencing new-onset depression, and examines the potential for physical activity, energy intake, and total body weight to mediate the depression-LDL relationship. This study provides no evidence of an association between depression and subsequent serum LDL changes. Altogether, this data suggests that LDL that is below 100 mg/dL without the use of lipid-lowering medication may predispose individuals to a greater risk of depression, and also suggests that low LDL is not likely a state brought about by physiological or behavioral changes following the onset of depression.

In its current form, familial hypercholesterolemia (FH) is due to the presence at the heterozygous state of a mutant allele of the LDL receptor gene or of the APO gene. The patients with heterozygous familial hypercholesterolemia (HeFH) can only remove their LDL particles from the blood at about half the normal rate, leading to an approximate doubling of LDL-cholesterol (LDL-C) level, starting at birth and accumulating in many tissue resulting to tendon xanthomas, corneal arcus and early atherosclerosis (coronary heart diseases (CHD) occur typically at 35-55 years among men and at 55-75 years among women). In the early's 1990's, our interest in the field was stimulated by the fact that despite the impressive progress in the understanding of the pathophysiology and treatment of FH and a preferential r! egimen of reimbursement for statins in Belgium, many affected individuals were not diagnosed and adequately treated. In 1995, we initiated a project aimed to obtain objective information on the situation of subjects carrying familial hypercholesterolemia (FH) in Belgium and to develop better means for identifying these subjects amongst the population. During the period 1995-2006, 1305 suspected FH patients originating from 682 families were examined for a DNA-based diagnosis in our laboratory and amongst these, 544 patients (271 families) were confirmed to have FH. Seventy three mutations in the LDLR as well as the unique mutation in APOB (R3500Q) explained their FH, but amongst them, five mutations explained about 38% of FH in Wallonia and 4 mutations explained 29% of FH in Flanders. Such establishment of the spectrum of FH causing mutations and particularly, of the specific mutational spectrum by region can greatly contribute to facilitate the diagnosis of FH in the near future. In our region, we found that FH frequency is likely to be higher than the theoretical prevalence reported in most European countries (1/500). That means that, in Belgium, each GP should know at least 1 FH patient. There are several reasons that advocated for the need to precisely diagnose FH. One of which was clearly demonstrated by two of our studies: in the category of patients with very elevated cholesterol levels (VHC) and family history of early CVD (FHEC), FH patients have always more severe atherosclerosis than age-, sex-, cholesterol- and family history-matched non-FH-individuals, suggesting that the FH status is by itself a strong and independent risk factor. In this category of VHC and FHEC patients, we even found a “paradox of FH” in the fact that they have greater atherosclerosis despite relatively lower risk as predicted by the Framingham equation. Genetic test may also enhance the awareness of a greater risk than expected on the basis of other risk factors. One of the main interests of genetic tests is of course to facilitate the screening in FH family. We also developed some specific criteria in term of cholesterol levels, family history of early CHD and tendon thickening to facilitate the diagnosis of FH in Belgium. These criteria could be used to select patients susceptible to be confirmed by more expensive genetic tests. As shown by our experience in our lipid clinics, the current available lipid-lowering drugs can correct LDL-C to target in about 49% of the very high risk class of FH patients (including more than 80% FH patients of our co! hort), a real success compared to the early 1990's. In 2001, our work has contributed to the change of the reimbursement rules of statins in patients suspected of FH. Even if these new rules are not yet perfect, its positive point was that many more FH patients can now benefit of the special reimbursement compared to the situation before 2003. In conclusion, this works have illustrated many aspects regarding the existence, the frequency, the diagnostic problems and the clinical management of FH. Specially, our study suggest that FH remains underdiagnosed in despite of the fact that it is severe, can be diagnosed and effectively treated. For this reason, this work is not to be considered as a final point but rather as a “well fixed starting block” ready to support the beginning of more actions to improve the management of these FH patients at the higher level of public health.

A proteomic approach was applied to examine the protein composition of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in humans. LDL and HDL were isolated by density gradient ultracentrifugation, and proteins were separated with twodimensional gel electrophoresis (2-DE) and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and with amino acid sequencing using electrospray ionization tandem mass spectrometry. To improve the identification of low abundant proteins in silver stained 2-DE gels, 2,5-dihydroxybenzoic acid was used instead of α-cyano-4-hydroxycinnamic acid as matrix in the peptide mass fingerprinting procedure; this was demonstrated to give more matching peptide peaks, higher sequence coverage, and higher signal to noise ratio. Altogether 18 different proteins were demonstrated in LDL and/or HDL: three of these (calgranulin A, lysozyme C and transthyretin) have not been identified in LDL before. Apo C-II, apo C-III, apo E, apo A-I, apo A-IV, apo J, apo M, serum amyloid A-IV and α1-antitrypsin were found in both LDL and HDL, while apo B-100 (clone), calgranulin A, lysozyme C and transthyretin were found only in LDL, and apo A-II, apo C-I, and serum amyloid A only in HDL. Salivary α-amylase wass identified only in HDL2, and apo L and glycosylated apo A-II only in HDL3. Many of the proteins occurred in a number of isoforms: in all, 47 different isoform identities were demonstrated. A 2-DE mobility shift assay and deglycosylation experiments were used to demonstrate, for the first time, that apo M in LDL and HDL occurs in five isoforms; three that are both N-glycosylated and sialylated, one that is N-glycosylated but not sialylated and one that is neither N-glycosylated nor sialylated. LDL from obese subjects was found to contain more apo J, apo C-II, apo M, α1-antitrypsin and serum amyloid A-IV than LDL from controls,, and also more of an acidic isoform (pI/Mr; 5.2 / 23 100) of apo A-I. In addition, the new LDLassociated protein transthyretin, was found to be significantly more abundant in LDL from obese subjects. On the other hand, the amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3 / 23 100) were significantly less. Altogether, these findings (i) illustrate the power of 2-DE and mass spectrometry for detailed mapping of the proteins and their isoforms in human lipoproteins; (ii) demonstrate the presence of a number of new proteins in LDL (calgranulin A, lysozyme C and transthyretin); (iii) give precise biochemical clues to the polymorphism of apo M in LDL and HDL, and; (iv) indicate that obesity is associated with significant changes in the protein profile of LDL. It is concluded that new information on lipoproteins can easily be obtained through a proteomic approach, thus facilitating the development of a new proteomic field: lipoproteomics. Much further investigation in this field is warranted, particularly because newly discovered LDL and HDL proteins may play hitherto unknown role(s) in inflammatory reactions of the arterial wall and evolve as useful biomarkers in cardiovascular disease.

Compelling evidence indicated that major risk factors for atherosclerosis such as oxidatively modified low density lipoprotein (oxLDL), high glucose, and reactive oxygen species promote endothelial cell apoptosis and thereby may contribute to the initiation of atherosclerotic lesion formation. Using fast protein liquid chromatography (FPLC), plasma LDL from familial hypercholesterolemic (FH) patients were separated into five subfractions, L1¡VL5. Among them, L5 subfraction was highly electronegative and suppressed DNA synthesis in cultured bovine aortic endothelial cells (BAEC) and stimulated mononuclear cell adhesion to cultured endothelial cells in vitro. Because impaired angiogenesis plays an important role in the pathogenesis of atherosclerosis, the anti-angiogenic functions of LDL subfractions from FH subjects were examined. Subconfluent BAEC (6 to 10 passages) maintained in DMEM containing 10% serum were treated with LDL subfractions at a dose of 20 £gg/ml, and the effects on anti-angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, and secretion of matrix metalloproteinase (MMP) were determined. Similar to Cu2+ ox-LDL, FH-L4 and FH-L5 inhibited cell proliferation to 80.9¡Ó2.4% (p<0.05) and 58.5¡Ó4.3% of control (p<0.001), respectively, while the other FH (L1-L3) and all subfractions isolated from normocholesterolemic (N) subjects had negligible effects. Similarly, FH-L4 and -L5, but not FH-L1 to -L3, retarded cell migration to 326.9 ¡Ó 19.4 (p<0.05) and 215¡Ó16 cells (p<0.001 with the control values of 402¡Ó34 cells), respectively. FH-L5 induced almost 20% of BAEC to undergo apoptosis; FH-L4 caused very mild effects, and other subfractions did not affect apoptosis In addition, FH-L4 and -L5 perturbed tube formation by BAEC in culture (5.8¡Ó0.2 and 3.4¡Ó0.4, respectively, versus control 8.5¡Ó1.5 tubes). Finally, FH-L4 and -L5 inhibited secretion of MMP-2 by BAEC (72.7¡Ó6.9 and 18.9¡Ó4.8% of control, respectively). The results demonstrate that FH-L5 potently affects multiple processes that are vital to normal angiogenesis, FH-L4 had milder effects, and other FH and N subfractions had negligible effects. In turn, these effects in vitro on processes pivotal to angiogenesis are consistent with potential effects of ox-LDL on endothelial dysfunction during atherogenesis in vivo.

Hypercholesterolemia is a major risk factor for cardiovascular disease (CVD). Interestingly, the consumption of dairy products, namely milk, has been shown to lower cholesterol. The mechanism of action surrounding this observation has been attributed to the protein fraction of milk. While there have been many studies evaluating the effects of dietary protein sources on cholesterol concentrations, few studies have evaluated specific animal protein components and no human clinical studies regarding the effects of animal plasma protein fractions on cholesterol metabolism have been conducted. This study examined the effect of an oral serum bovine immunoglobulin protein fraction (bIg) derived from US Department of Agriculture approved beef (aged < 30 months) on lipid indices in hypercholesterolemic humans. Participants included men and women (aged 25 – 70 years) with mild hypercholesterolemia (5.44-6.99 mmol/L) who were not receiving cholesterol-lowering medication. Treatment consisted of the randomized, double blind, parallel group, placebo-controlled administration of 5 grams (g) bIg daily for 6 weeks (W) in 52 participants (n = 26 each in treatment and control groups). Mean (± SD) baseline treatment and placebo total cholesterol (TC) was 6.33 ± 0.1 mmol/L and 6.16 ± 0.1 mmol/L respectively. A repeated-measures multivariate analysis of covariance (MANCOVA) covaried for change in total energy and alcohol intake, and a Tukey posthoc examination of the data showed that the bIg-treated group demonstrated a significant reduction in TC at 3-week (W) (5.98 ± 0.5 mmol/L; P < 0.05) and 6-week (W) (5.97 ± 0.7 mmol/L; P > 0.05) intervals compared to baseline. The 6W concentration was significantly lower than the placebo group (P < 0.05). Additionally, study findings displayed no significant changes in the placebo group or in any other lipid indexes or markers associated with hepatorenal or cardiovascular health. Consumption of bIg appears to lower major lipid indexes associated with CVD.