Every year, millions of people in the world die from cardiovascular diseases. Most of thesevictims show no symptoms of disease for decades as the disease silently progresses, but maysuddenly become afflicted with a myocardial infarction or stroke. At present there is aregrettable lack of important disease markers that could help identify patients at risk withregard to secondary cardiovascular events. Biomarkers for this would of course be extremelyvaluable. If such markers could distinguish between patients with different responses totreatment and overall outcome, that would be of major added value as this potentially couldpave the way for differentiated preventive measures. In this study we show that specificproteins isolated from plasma-derived exosomes are indeed associated with an increased riskfor secondary cardiac events.

Density gradient ultracentrifugation (DGU) is a powerful method for analyzing lipoprotein particles in great detail. It yields considerable amounts of information regarding the density distribution of these particles when coupled with fluorometric analysis and is an invaluable tool in determining their relative abundance. This union allows relationships between subclasses of lipoproteins to be established that gives researchers a more focused path to aid them in developing methods to predict the early onset of coronary artery disease (CAD). The research presented here focuses on the pairing of DGU with post-separatory techniques including matrix-assisted laser desorption mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis (CE), isoelectric focusing (IEF) and apoptosis studies involving cell cultures. It is becoming clearer that cholesterol concentrations themselves do not provide sufficient data to assess the quality of cardiovascular health. As a result, research is becoming more focused on identifying better markers that may be indicative of development of CAD in a patient. Of specific interest is group of particles known as high density lipoproteins (HDL). Classically, this molecule is considered the “good cholesterol”, but literature from the last decade suggests that there may be atherogenic variants to this group. By utilizing DGU as a preparatory method for secondary analyses, new dimensions can be added to the density distribution analysis to allow a better determination of markers of cardiovascular health. The aim of this work is to utilize the principles involved with these various techniques to develop a comprehensive set of methods to aid in the detection of potential risk markers. In this study, the properties of metal ion complexes of EDTA as solute systems for analysis of lipoproteins by DGU are analyzed. We show that by varying the complexing ion and counter-ion of these metal-ion complexes, we gain the ability to control the separation of lipoprotein subclasses for subsequent analyses. Qualitative and quantitative data is presented that describes the analysis of different density regions of HDL for apolipoprotein content. Trends between control and atherogenic samples are also described and a clinical link between the biological activity of these regions and the chemical analysis is discussed.

A hemoglobina de Glossoscolex paulistus (HbGp) é caracterizada por uma massa molecular de 3,6 MDa, alta estabilidade oligomérica, resistência a auto-oxidação, e alta afinidade em ligar oxigênio. A estrutura quaternária desta macromolécula apresenta 144 cadeias com grupo heme (globinas) e 36 cadeias sem grupo heme (linkers), dispostos em duas camadas hexagonais. No presente trabalho estudos de caracterização das subunidades da HbGp, bem como da estabilidade da HbGp em diferentes formas, em função do pH, e em diferentes concentrações de ureia, por diferentes técnicas biofísicas, foram realizados. Os estudos de caracterização por eletroforese SDS-PAGE, MALDI-TOF-MS e ultracentrifugação analítica (AUC) das subunidades isoladas mostraram que apenas o monômero d obtido da cromatografia de exclusão por tamanho (SEC) tem alto grau de pureza. Para as demais frações mais de uma contribuição foi observada em solução. Assim, para a fração trimérica, duas espécies estão presentes em solução, a espécie predominante (87 %) é atribuída ao trímero abc e a outra espécie (13 %) pode ser associada ao complexo (abc + L). Os dados espectroscópicos e de AUC mostraram que a estabilidade da HbGp depende fortemente do estado de oxidação do heme, do ligante coordenado no centro metálico e da concentração de proteína. Assim, a forma oxidada, a meta-HbGp, mostrou-se menos estável em meio alcalino e na presença de ureia, seguida pelas formas oxi- e cianometa-HbGp. Desta forma, no pH 8,0, a meta-HbGp está totalmente dissociada em trímero abc e monômero d, enquanto a oxi-HbGp está apenas parcialmente dissociada com uma contribuição de 88 % de proteína íntegra em solução e a cianometa-HbGp não sofre dissociação oligomérica. Os valores de coeficiente de sedimentação s20,w e massa molecular (MM) determinados para as espécies em solução são similares aos observados para as correspondentes espécies isoladas por SEC. Na presença de ureia a mesma tendência foi observada para as três formas da HbGp. Porém, para uma caracterização melhor de processo de desnaturação, os dados espectroscópicos foram analisados usando modelos de dois e três estados para obter informações sobre os parâmetros termodinâmicos do sistema. Assim, bons ajustes foram obtidos usando ambos os modelos, no entanto, o modelo de três estados foi mais adequado para descrever o processo. Por este modelo o processo de desnaturação da HbGp pode ser descrito por duas etapas. A primeira etapa, na faixa de 1,0 - 3,0 mol/L de ureia, está associada a transição do estado nativo para o estado intermediário (N → I), e é caracterizada pela dissociação do oligômero nas diferentes subunidades da HbGp. O estado intermediário apresenta propriedades físico-químicas similares ao estado nativo, sugerindo que o processo de dissociação oligomérica não induz mudanças significativas na estrutura secundária e na região do grupo heme da proteína. Os parâmetros termodinâmicos associados à primeira transição apresentaram erros consideráveis, que podem ser atribuídos à complexidade do estado intermediário com diferentes espécies em solução bem como à semelhança ao estado nativo. A segunda etapa (I → U) com transição bem definida entre 4,5 - 5,0 mol/L de desnaturante é caracterizada pela desnaturação das subunidades dissociadas. Os dados de AUC e SAXS são consistentes com os dados obtidos por espectroscopia, onde a primeira etapa do processo foi caracterizada pela dissociação oligomérica do oligômero em dodecâmero (abcd)3, tetrâmero abcd, trímero abc e monômero d. Para concentrações acima de 4,0 e 5,0 mol/L de ureia, para oxi-HbGp e cianometa-HbGp, respectivamente, aumentos significativos nos valores de I(0), Dmax e Rg sugerem que as subunidades da HbGp estão desnaturadas em solução. As massas moleculares (MM) obtidas por espectrometria de massas e AUC, e os coeficientes de sedimentação s20,w são consistentes com outros resultados reportados para hemoglobinas ortólogas. Além disso, os resultados aqui apresentados representam um avanço importante na caracterização do processo de desnaturação de proteínas oligoméricas complexas. / Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3.6 MDa, a high oligomeric stability, a high resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this current work the characterization of the HbGp subunits and the effect of pH and urea upon the oligomeric stability were studied by several biophysical techniques. Our results obtained by electrophoresis SDS-PAGE, MALDI-TOF-MS and analytical ultracentrifugation (AUC) showed that only the monomer d isolated by size exclusion chromatography (SEC) presented high purity. For the other fractions various species were observed in the solution. Thus, for the trimeric fraction, two species are present in the equilibrium, the main species with percentage contribution of 87 % is assigned to the trimer abc and the species with 13 % in the solution is associated to the complex (abc + L). Additionally, the data obtained by several spectroscopic techniques and AUC show clearly that the oligomeric stability of HbGp depends on the iron oxidation state, the specific ligand coordinated to the iron and the protein concentration. Therefore, our results show that the met-HbGp form is the less stable one in the alkaline medium and in the presence of urea, followed by the oxy- and cyanomet- forms. In this way, at pH 8.0, the met- form is fully dissociated into smaller subunits, such as, trimer abc and monomer d, while the oxy-HbGp is partially dissociated with a significant percentage contribution (88 %) of undissociated protein, and the cyanomet-HbGp does not undergo oligomeric dissociation. The sedimentation coefficients (s20,w) and molecular masses (MM) values for species present in the solution, at different pH, are very close to the values obtained for isolated species. In the presence of urea the same behavior was observed for the three HbGp forms as compared to the alkaline medium. However, for a full characterization of the unfolding process the thermodynamic parameters were obtained by spectroscopic data analysis using models of two and three states. Adequate fits were obtained for both models, but the three states model was very appropriate to describe the HbGp denaturation process. Thus, the denaturation process of HbGp is defined by two phases. The first phase between 1.0 and 3.0 mol/L, of urea is assigned to the transition of native state to an intermediate state (N → I), and is characterized by dissociation of the oligomer in several subunits. The strong similarity of the intermediate state to the native one suggests that oligomeric dissociation induces little changes in the secondary structure and the region of heme group of the protein. As a consequence, the thermodynamic parameters associated to the first transition have large errors due to the complexity of the intermediate state with different species in the solution, as well as its great similarity to the native state. The second phase (I → U), associated with a cooperative transition at 4,5 - 5,0 mol/L of denaturant agent, is attributed to the unfolding of the dissociated subunits. Our AUC and SAXS data are very consistent with spectroscopic data. Thus, in the first phase the oligomeric dissociation of whole protein in dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d was observed. For urea concentrations above 4.0 - 5,0 mol/L, for oxy-HbGp and cyanomet-HbGp, respectively, the significant increase in I(0), Dmax and Rg values suggests that the HbGp subunits are denatured in the solution. The molecular masses values (MM) obtained by mass spectrometry and AUC, and the sedimentation coefficients (s20,w) are consistent with others results reported in the literature for orthologous hemoglobins. In addition, the results of this work correspond to an important advance in the characterization of the denaturation process of this complex oligomeric protein.

The relationship between hyperlipidemia and pancreatitis remains obscure in dogs. The aim of the present study was to investigate any possible association between hyperlipidemia and pancreatitis in dogs. In the first part of the study, Miniature Schnauzers with hypertriglyceridemia were found to have significantly higher serum cPLI concentrations than Miniature Schnauzers with normal serum triglyceride concentrations (P=0.0001). Also, Miniature Schnauzers with severe hypertriglyceridemia (>862 mg/dL) had 4.5 times higher odds (P=0.0343) for having a serum cPLI concentration consistent with pancreatitis. In the second part of the study, 17 Miniature Schnauzers prospectively enrolled with a history of pancreatitis were significantly more likely to have hypertriglyceridemia (71 percent) after resolution of pancreatitis than 34 age-matched Miniature Schnauzers without a history of pancreatitis (33 percent; odds ratio=5.02; P=0.0163). For the third part of the study, assessment of the feasibility and usefulness of a novel density gradient ultracentrifugation method using NaBiEDTA for lipoprotein profiling in dogs was attempted. Density gradient ultracentrifugation using NaBiEDTA was found to be useful for the study of lipoprotein profiles in dogs. Significant differences were detected in the lipoprotein profiles (mainly involving TRL and specific LDL fractions) among healthy Miniature Schnauzers, dogs of various other breeds, and hypertriglyceridemic Miniature Schnauzers. In the fourth part of the study, the effect of a commercially available low-fat diet on serum lipid and pancreas-specific lipase (Spec cPL) concentrations and lipoprotein profiles in Miniature Schnauzers with primary hypertriglyceridemia was evaluated. The study diet was found to be effective in significantly reducing serum triglyceride and cholesterol concentrations and changing the lipoprotein profiles of the dogs studied within 2 months. However, there was no significant effect of the study diet on serum Spec cPL concentrations. In the last part of the study, serum triglyceride and cholesterol concentrations and lipoprotein profiles were compared between dogs with naturally occurring pancreatitis and healthy dogs. The majority of dogs with naturally occurring pancreatitis had normal serum triglyceride and cholesterol concentrations. Important differences were identified in lipoprotein profiles between dogs with pancreatitis (higher LDL2, LDL3, and LDL4 fractions and lower TRL, HDL2a, and HDL3c fractions) and healthy control dogs.

A method for the oxidation of lipoproteins in vitro was developed using the free radical initiator, 2,2?-azobis-(2-amidinopropane) dihydrochloride (AAPH). Following in vitro oxidation, the susceptibility to oxidation of the serum samples was studied using density gradient ultracentifugation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Shifts in mean buoyant density of the lipoprotein particles, specifically low density lipoprotein (LDL) and high density lipoprotein (HDL), were observed in the density profile following centrifugation. The degree of shift in the density proved to be proportional to the extent of oxidation. Changes in apolipoproteins were studied with MALDI-TOF-MS. Observed variations in the mass spectra include m/z shifts due to chemical modifications and change in isoform distributions. The oxidation procedure and analysis techniques were applied to a clinical application to study the effects of table grape consumption on lipoprotein susceptibility to oxidation. The main objective of the research, to show feasibility that these methods could be used in a clinical setting, was achieved.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

With the severity of cardiovascular disease (CVD) and the related mortality rate to this disease, new methods are necessary for risk assessment and treatment prior to the onset of the disease. The current paradigm in CVD risk assessment has shifted towards the multivariate approach over the individual use of traditional risk factors or lipid measurements. Through a combination of analytical techniques and multivariate statistical analysis, a novel method of cardiovascular risk assessment was developed. The analytical techniques employed include density gradient ultracentrifugation (DGU) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) applied to human serum. These techniques provided detailed information about the characterization of the lipoproteins and their structural components, specifically the apolipoproteins belonging to high density lipoproteins (HDL). This information when combined with multivariate statistical analysis provided a method that accurately identified the presence of CVD in clinical studies between cohorts of subjects that had been previously diagnosed with CVD and cohorts of subjects that had been identified as healthy controls (CTRL) based on a clear angiography. The lipoprotein density profiles were divided into subclasses based on their density and measured using a fluorescent probe to tag the lipoprotein particles. Use of multiple ethylenediaminetetraacetic acid (EDTA) based solutes allowed for the manipulation of the density gradient formation in order to separate the lipoproteins by specific density ranges in order to achieve better baseline separation of the profiles. Application of the integrated fluorescence intensities for each subclass of lipoprotein to linear discriminant analysis/sliced inverse regression (LDA/SIR) and quadratic discriminant analysis (QDA) yielded an advanced and accurate form of risk assessment for CVD. This method was found to be highly accurate as well as identify potential atherogenic lipoprotein subclasses through studying the LDA/SIR prediction equation generated. It was also shown that the LDA/SIR equation could be used to monitor medical treatment and lifestyle change for their effects on the risk assessment model. Further study into the atherogenicity of HDL through analysis of the apolipoproteins using MALDI-MS led to identification of potential risk factors that could be added to the statistical analyses. These risk factors included mass differences in the Apolipoprotein A-I (Apo A-I) and Apolipoprotein C-I (Apo C-I) between CVD and CTRL samples as well as the presence of specific mass peaks related to Apolipoprotein A-II (Apo A-II) that were primarily found in the CVD samples. These differences, in addition to the lipoprotein density profile data, were found to increase the potential accuracy of CVD risk assessment. The combination of these methods has shown great potential in the assessment of CVD risk as well as the ability to increase researchers' understanding of the nature of VD and how to treat it.

Th. univ.--Phys. des liquides--Paris 6, 1998. / Bibliogr. p. 110-119. Résumé en français et en anglais. 1999 d'après la déclaration de dépôt légal.

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