Estudos anteriores em nosso laboratório demonstraram que pacientes portadores de DAC apresentam diferenças no metabolismo do CL e CE de uma nanoemulsão artificial rica em colesterol (LDE), nos quais o CL apresentou maior remoção plasmática e depósito arterial. Dando continuidade a esta linha de pesquisa, neste trabalho foram avaliadas a cinética plasmática, representada pela taxa fracional de remoção (TFR), e a captação do 3H-colesterol livre (3H - CL) e 14C - colesterol esterificado (14C - CE) da LDE por segmentos arteriais e por órgãos de coelhos normais (n=17) e coelhos submetidos à indução de aterosclerose por dieta rica em colesterol (1%) (n=13). Além disso, avaliou-se a captação in vitro do 3H CL e do 14C CE da LDE por células endoteliais aórticas de coelhos. Por último, foi avaliada a influência da inibição da enzima lecitina-colesterol aciltransferase (LCAT), e indiretamente, a esterificação do CL em ratos normais (n=9) e tratados com diazepam (n=9). Em coelhos que receberam dieta normal, não houve diferença entre a remoção plasmática do 3H - CL e do 14C - CE. Em coelhos que desenvolveram hiperlipidemia e aterosclerose através de dieta rica em colesterol, o 3H - CL foi removido mais rapidamente da circulação do que o 14C - CE (p<0,05), entretanto houve maior captação de 14C - CE do que de 3H - CL no arco aórtico (p<0,05). Em ambos os grupos, os principais órgãos captadores de colesterol da LDE foram fígado, pulmão, adrenais e baço (p<0,05). Tanto a TRF quanto a captação hepática de 3H - CL e 14C - CE foram menores no grupo que recebeu a dieta rica em colesterol. Em células endoteliais aórticas de coelhos, a captação de 3H - CL foi maior que a de 14C CE independente da massa de LDE incubada (p<0,01). Em ratos, não houve diferença entre a captação das duas formas de colesterol da LDE pela aorta no grupo controle, entretanto, quando a atividade da LCAT foi diminuída pelo tratamento com diazepam, a captação arterial de 3H - CL foi maior do que a de 14C - CE (p< 0,01). A hiperlipidemia e distúrbios no processo de estabilização do colesterol, favorecem a dissociação entre o CL e o CE das lipoproteínas, e podem elevar o risco de desenvolvimento da aterosclerose, assim como agravar o processo de aterogênese. / I n previously studies, it was shown that free cholesterol (FC) and cholesterol ester (CE) of a cholesterol-rich nanoemulsion (LDE) behaves differently in patients with coronary artery disease (CAD). The FC plasma clearance and arterial deposition is greater than CE. In the present study we evaluate the plasma kinetics, estimated by the fractional clearance rate (FCR), and the tissue uptake of 3H-free cholesterol (3H FC) and of 14C cholesterol ester (14C - CE) of LDE by arterial segments and organs of rabbits with (n=13) and without atherosclerosis (n=17). Furthermore, it was evaluated the in vitro uptake of 3H FC and 14C - CE by rabbit aortic endothelial cells. Finally, it was evaluated the inhibition of the enzyme lecithin-cholesterol acyltransferase (LCAT), and indirectly, the FC esterification in rats non-treated (n=9) and treated with diazepam (n=9). In rabbits without atherosclerosis that received an standard diet there was no difference between the plasma clearance of 3H FC and 14C CE. In rabbits with hyperlipidemia and atherosclerosis induced by the cholesterol-rich diet the 3H - FC was removed faster than 14C - CE (p<0.05), however the arch aortic uptake of 14C CE was greater than of 3H - FC (0p<0.05). In both groups, liver, lungs, adrenals and spleen were the principal sites of LDE cholesterol uptake. The FCR and tissue uptake were smaller in rabbits with than those without atherosclerosis. In rabbit aortic endothelial cells the 3H - FC uptake was greater than 14C CE independently of incubated LDE mass (p<0.01). In control rats there was no difference on the arterial uptake of both cholesterol forms of LDE, but when the LCAT activity was diminished by the diazepam treatment, the arterial uptake of 3H FC were greater than 14C CE (p< 0.01). The hyperlipidemia and cholesterol stability alterations may lead to dissociation between lipoproteins FC and CE. This dissociation may increase the risk for atherosclerosis and likewise enhance the severity of atherosclerosis.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. The enzyme catalyzes hydrolysis of triacylglycerols (TG) of chylomicrons and of very low density lipoproteins (VLDL). However, the mechanisms involved in the regulation of this protein are not fully understood yet. Therefore, the aim of the theses is to study selected aspects of LPL activity regulation. Recently discovered apolipoprotein A-V (apo A-V) substantially affects triglyceridemia and it is presumed that it may function as LPL activator. However, its concentration in the blood is extremely low and we therefore investigated whether most of apo A-V could be bound to the heparan sulfate proteoglycan (HSPG) of vascular wall similarly to LPL. Intravenous heparin application in healthy volunteers resulted in an expected increase in LPL activity but apo A-V concentration did not change. Our results do not support the hypothesis that most of apo A-V is bound to HSPG of the capillary endothelium. An alcohol consumption plays also a role in LPL regulation - the long-term moderate alcohol consumption is known to increase enzyme activity; on the contrary, it is presumed that LPL activity is inhibited immediately after alcohol consumption. However, the direct evidence for such a premise is missing. The other aim of the theses was to...

Cholesterol 7-hydroxylase (CYP7A1) is the rate limiting enzyme of the classical pathway of bile acid (BA) synthesis, which catabolizes approximately half of cholesterol in man. Determination of CYP7A enzymatic activity is a key subject of lipid metabolism research. Direct determination of CYP7A1 activity in hepatic biopsy is mostly not allowed for ethical reasons, so indirect methods are used with serum markers such as 7α-hydroxy-4-cholestene-3- one (C4). The first, methodical aim of the work was to convert the introduced HPLC method for the determination of C4 to LC-MS in order to increase the sensitivity. We focused on the solid phase extraction step, adjusting the composition and volumes of the washing and elution solution. By converting the method from HPLC to LC-MS, the sensitivity was increased approximately 7 times (LD = 1.39 ng/ml). In the second, clinical part of our work, we attempted to confirm the preliminary results of our laboratory on the distribution of C4 in lipoprotein fractions (LPP) in order to find parameter that would correlate with CYP7A1 activity better than C4 level itself. Preliminary results (performed in healthy individuals) showed that most of C4 is carried on HDL, and that the C4 distribution within LPP fractions is similar among examined subjects. We repeated the...

ABO blood type has been shown to be associated with both cardiovascular risk and toxicity‐preventing activity (TxPA) stress response in elderly males. Twenty middle‐aged, healthy males, 14 blood type A and six blood type O, were involved in this project. Volunteers completed a battery of psychological assessments, then gave blood and had several psychophysiological measures taken prior to, during and after two stressors. The stressors consisted of mental arithmetic tasks plus audiotapes of combat sounds and a baby crying. The anger‐out and hard‐driving scores of blood type O subjects were significantly higher than the blood type A means. TxPA decreased significantly as a function of stress and some suggestive blood type effects of TxPA were found. Plasma protein, microhematocrit, plasma cortisol, finger temperature, skin conductance, blood pressure and two facial electromyograph (EMG) variables were also significantly affected by stressors but not by the blood type factor. No significant differences of any kind were found for total cholesterol, high‐density lipoprotein or pulse variables. The importance of age and other individual subject characteristics was discussed.

Agrégation de l'enseignement supérieur, Orientation sciences / info:eu-repo/semantics/nonPublished

High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in arteries contributing to atherosclerosis. In hereditary amyloidosis (AApoAI), a potentially fatal disease, N-terminal fragments of variant apoA-I deposit in vital organs and damage them. There is no cure for apoA-I amyloidosis and its structural basis is unknown. Previously, AApoAI mutations were mapped on the crystal structure of the human C-terminally truncated Δ(185-243)apoA-I. The results suggested that the mutation-induced destabilization of the lipid-free protein initiates β-aggregation. Our biophysical studies showed that amyloidogenic mutations G26R, W50R, F71Y and L170P did not necessarily destabilize the native structure, prompting us to search for additional triggers of apoA-I misfolding. We mapped residue segments predicted to promote β-aggregation (termed amyloid hot spots) on the atomic structure of ∆(185-243)apoA-I. The results suggested that perturbed packing of these hot spots, particularly residues 14-22, triggers amyloidosis. This enabled us to propose the first molecular mechanism of apoA-I misfolding. To explore a potential mechanism, we combined structural, stability, dynamics and functional studies of several amyloidogenic mutants and a non-amyloidogenic control, L159R. All mutants reduced structural protection of the segment 14-22, supporting our hypothesis that increased dynamics of this segment triggers AApoAI. The non-amyloidogenic mutant showed helical unfolding near the mutation site indicating susceptibility to proteolysis. We propose that the major factors that make apoA-I amyloidogenic are reduced protection of the major amyloidogenic segments combined with the structural integrity of the four-helix bundle to facilitate protein aggregation. Together, our results suggest that the fate of apoA-I in vivo depends on the balance between its misfolding, proteolysis, and protective protein-lipid interactions. Our structural and bioinformatics analysis of other members of the apolipoprotein family (A-II, A-IV, A-V, B, C-I, C-II, C-III, E, SAA) showed that apolipoproteins’ propensity to form amyloid is rooted in the proteins’ hydrophobicity, which is key to the lipid binding ability. The overlap of functional and pathologic interfaces suggests competition between normal protein function and misfolding. Therefore, increasing apolipoprotein retention on the lipid surface provides a potential therapeutic strategy against amyloidosis.

No description available.

Thesis (MSc. Medical Science (Chemical Pathology)) -- University of Limpopo, 2017 / Obesity has been associated with dyslipidaemia (increased levels of triglycerides, total cholesterol and low levels of HDL-C together with small dense lipoprotein particles) in the absence of metabolic disorders such as, type 2 diabetes mellitus and inflammation. Since community based studies in South Africa reported that obesity is more common in women, and rural Africans have a more favourable lipid profile compared to their White counterparts, the current study investigated the association of obesity in women without metabolic disorders with lipid levels and changes in proportions of small and large LDL and HDL particles. Methods The present study was part of the project “Prevention, Control and Integrated Management of Chronic Disease in a rural area, South Africa”. A total of 521 women participated in the above project. After excluding people with diabetes mellitus, insulin resistance and inflammation, 308 women were left and of these 67 were obese. Sixty seven ages matched, randomly selected non-obese women served as controls. Anthropometry variables as well as systolic and diastolic blood pressures were measured and the WHO steps questionnaire was administered to collect information on medication, lifestyle and diseases. Fasting blood levels of total cholesterol, HDL C, triglyceride, adiponectin, CRP, glucose and insulin were measured. Proportions of small and large HDL and LDL particles were determined. Results There was no significant difference in TC, TG and LDL-C levels (p=0.558, 0.087 and 0.948) between obese and non-obese women or between women with increased waist circumference (WC) and those with normal WC. The HDL-C concentration was significantly lower in obese women compared to women with non- obese (p=0.001). The lipid ratios TC/HDL-C and Apo B-100/Apo A-I were significantly higher in obese women than those with non- obese (p=0.013 and p=0.006) respectively. The same phenomenon was observed in women with xv increased waist circumference (p=0.001** and p=0.025* respectively). Adiponectin levels were significantly lower in obese women compared to non-obese women (p=0.004**) and in women with increased waist circumference compared to those with normal waist circumference (p=0.016*). The proportions of small dense HDL and LDL lipoprotein particles were similar in obese and non-obese women. Both obese and abdominally obese women had significantly higher odds ratios of low levels of HDL-C and elevated Apo B-100/Apo A-I. Adiponectin was a significant predictor of elevated TC and TG in both obese and abdominally obese women while BMI was a significant predictor of low HDL-C in obese women. Waist circumference was a significant predictor of low HDL-C in abdominally obese women. Conclusion In the current study, obesity in women was significantly associated with lipid abnormalities such as low HDL-C levels, raised lipid ratios (TC/HDL-C and Apo B 100/Apo A-I) and low levels of adiponectin, after excluding metabolic disorders / VLIR

Cholesterol is a fat-like molecule found among lipids in animal (including human) tissues. It is needed for maintaining good health. However, health issues have been raised because of the strong correlation between high levels of cholesterol in the body and cardiovascular disease. An HPLC method for quantitative determination of cholesterol in foods is presented. This involves a C-18 stationary phase using a 70:30 methanol: 2-propanol mobile phase with an UV detector set at 212 nm. The method showed linearity in the range 5.0 to 100.0 μg/mL and also good reproducibility with relative standard deviation of 4.22%, 2.71%, 4.8%, and 3.7% for the different samples analyzed. The mean recovery of the butter sample was 106.5%. The samples under investigation were common food items such as butter, lard, and two different types of cheese.

Cardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the pathogenesis of atherosclerosis and associated CVDs is strongly linked to elevated secretion of liver-specific lipoproteins called very-low-density-lipoprotein (VLDL). VLDLs are crucial lipoproteins responsible for transportation of triacylglycerides (TAGs), chemically inert particles that are physiologically significant for their energy storing capacity, from the liver to peripheral tissues. These VLDL particles are synthesized in the lumen of the endoplasmic reticulum (ER) of hepatocytes, transported from the ER to the cis-Golgi in special transport vesicles called VLDL-transport-vesicles (VTVs) and secreted into plasma through a highly regulated secretory pathway. Previous studies from our laboratory have shown that VTV-mediated ER-to-Golgi VLDL trafficking is the rate-limiting step in overall VLDL secretion from hepatocytes into plasma. In this project, we investigated intracellular VLDL trafficking and VLDL secretion in Ames dwarf (Prop1df, df/df) mice, a mutant mouse model homozygous for a recessive mutation at Prop1 gene locus (Prop1df) having deficiency of growth hormone (GH), thyroid stimulating hormone (TSH) and prolactin (PRL). This model is characteristic of prolonged longevity (~50% longer) and improved insulin sensitivity in comparison to their wild-type (N) counterparts. Ames dwarf (df/df) mice have recently been shown to have highly reduced plasma TAG levels, associating them with reduced susceptibility to atherosclerosis and associated CVDs. The underlying mechanism responsible for reduced VLDL secretion in Ames dwarf mice is yet to be characterized. We hypothesize that VTV-mediated trafficking of VLDL is reduced in Ames dwarf mice because of reduced expression of proteins regulating VLDL and VTV formation. To test our hypothesis, we first performed VTV-budding assay using cellular fractions isolated separately from Ames dwarf (df/df) and wild-type (N) mice livers. Our results show a significant (45%) reduction in VTV-budding process in Ames dwarf (df/df) mice compared to wild-type (N). Next we performed 2-dimensional differential gel electrophoresis (2-DIGE) on VTV and whole cell lysate (WCL) samples in order to examine the differences in protein expression and to have highly specific protein separation. ExPASy database was used to analyze protein spots that allowed us in identifying proteins specifically expressed in each of the mouse groups. Employing western blotting, samples (ER, cytosol, VTV and WCL) from both sets of mice were tested for expression levels of VLDL and VTV associated proteins (ApoB100, Sec22b, CideB, MTP, Apo-A1 and Apo-AIV) with ?-actin as the loading control. Significant differences in expression level of these proteins were observed which strongly suggest that the formation of VTV from ER in male Ames dwarf (df/df) mice is reduced compared to wild-type (N). Overall, we conclude that the differential expression of proteins required for VLDL transport causes reduced VLDL secretion in male Ames dwarf (df/df) mice.