Anticorpos (Ac) anti-oxLDL estão envolvidos no desenvolvimento da lesão aterosclerótica. Induzimos a produção desses Ac através da imunização com oxLDL. Avaliamos os perfis lipídico e de Ac anti-oxLDL e anti-pepD (pepD é um peptídeo derivado da apoB). Animais apoE-/- possuem maiores níveis de lípides sorológicos em comparação com animais C57Bl/6, exceção da HDL. A imunização oxLDL não alterou este perfil. A imunização aumentou os títulos dos Ac IgM, IgG e IgG1 anti-oxLDL. Animais C57Bl/6, mas não os apoE-/- tiveram aumento dos Ac IgM anti-pepD após a imunização. Acreditamos que o aumento de anticorpos IgG1 deve-se ao adjuvante usado. A aterosclerose pode ocorrer com maior freqüência, em indivíduos portadores de doenças renais crônicas. Para estudar essa interferência submetemos os animais a um modelo de isquemia e reperfusão renal (I/R). A I/R promoveu aumento da concentração de TG, podendo agravar a aterosclerose. O aumento de Ac IgG anti-oxLDL promovidos pela I/R sugere que o processo inflamatório desencadeado por este procedimento, aumenta a oxidação de LDL. Desta forma, concluímos que a I/R pode ser considerado um procedimento pró-aterosclerótico que não pode ser revertido pela imunização com oxLDL. / Oxidized LDL (oxLDL) antibodies (Ab) are involved in the development of the atherosclerotic lesion. We induced the formation of such Ab through immunization with oxLDL. We evaluate the lipids and antibody (anti-oxLDL and anti-pepD pep D is a peptide derived from apolipoprotein B) profile. The apoE-/- mice used have higher seric levels of most lipids than C57Bl/6 animals, the exception being HDL. The oxLDL immunization does not change this profile. The immunization increased anti-oxLDL Ab e IgM, IgG and IgG1 titers. Anti-pepD IgM Ab increased with immunization in C57Bl/6 animals only, and not in apoE-/-. We believe the increased IgG1 Ab due to the adjuvant used. Atherosclerosis has a higher incidence in individuals with chronic renal conditions. In order to study such interference, we submitted the animals to a renal ischemia and reperfusion model (I/R). The I/R procedure increased TG concentration, what can make atherosclerosis more severe. The increase in IgG anti-oxLDL antibody caused by I/R suggests that the inflammation process set by the procedure increases LDL oxidation. This way, we conclude that the I/R can be considered a pro-atherosclerotic procedure. The oxLDL immunization was not able to revert the atherogenic effect caused by I/R model.

Introdução São escassas as evidências do impacto dos padrões de atividade física (AF) e local de residência (rural e urbano) para a dislipidemia e ao perfil lipídio. Objetivos Avaliar a associação dos padrões de atividade física e local de residência (urbano e rural) com dislipidemia. Associar a correlação do gasto energético estimado em Mets e valores e frações de colesterol e triglicérides. Metodologia Estudo Transversal em coorte prospectiva, com 4551 indivíduos com idade entre 35 e 70 anos de áreas urbana e rural. A AF foi avaliada utilizando o Questionário Internacional de Atividade Física (IPAQ). Foi realizada regressão múltipla para a associação de AF e local de residência com o tipo de dislipidemia. A regressão linear foi realizada para avaliar o efeito da AF e local de residência nos valores e frações de colesterol e triglicérides. Resultados A prevalência de baixa AF no rural foi de 5,1 por cento (mulheres 4,3 por cento e homens 6,0 por cento ), urbano 11,2 por cento (7,9 por cento mulheres e 5,5 por cento homens), p<0,001. A dislipidemia ocorreu em 75,3 por cento do rural e 68 por cento do urbano (p<0,001). Em mulheres 80,6 por cento do rural e 65,7 por cento do urbano (p<0,001), homens 69,1 por cento do rural e 71 por cento do urbano (p=0,369). O perfil lipídico das mulheres em mediana foi: colesterol total (CT) rural 194,51 mg/dl, e urbano 201,5 mg/dl (p<0,001), HDL rural 44,5 mg/dl e urbano 50,1 mg/dl (p<0,001) LDL rural 120,3 mg/dl e urbano 122,6 mg/dl (p=0,35), triglicérides rural 127,1 mg/dl e urbano 117,8 (p<0,001). Em homens as medianas foram: CT rural 189,5 mg/dl e urbano 196,1 mg/dl (p=0,002), HDL rural 40,2 mg/dl e urbano 41,8 mg/dl (p<0,001), LDL rural 118,3 mg/dl e urbano 121,4 mg/dl (p=0,5), triglicérides rural 131,1 mg/dl l e urbano 147,0 mg/dl (p=0,001). A dislipidemia por HDL baixo foi a mais frequente. AF domiciliar, transporte e trabalho estão associados com a redução do risco de dislipidemia. Em mulheres, o efeito no HDL da AF domiciliar foi no rural 1,001 mg/dl (p=0,021) e no urbano 1,114 mg/dl (p<0,001). Em homens do grupo urbano, o efeito de AF de transporte foi -1,42 mg/dl (p<0,001) no LDL. O efeito da AF de trabalho no HDL foi no rural 1,002 mg/dl (p<0,001) e urbano 0,042 mg/dl (p=0,04) Conclusão Existem fortes indícios da interação da atividade física e local de residência com as dislipidemias e perfil lipídico. As associações encontradas entre as atividades físicas domiciliar, transporte e trabalho e dislipidemias sugerem caminhos complementares nas estratégias de prevenção de doenças cardiovasculares na população brasileira. / Introduction There is scarce evidence about the impact of physical activity (PA) and residence area (rural and urban) on dyslipidemia and lipid profile. Objectives To assess the association between patterns of physical activity, residence area (rural or urban) and dyslipidemia. To assess the correlation between energy expenditure in estimated Mets and total cholesterol, HDL, LDL and l triglycerides levels. Methodology A cross sectional evaluation in a prospective cohort with 4551 individuals aged between 35 and 70 years from rural and urban areas. The PA was assessed using the International Physical Activity Questionnaire (IPAQ). Multiple logistic regression was performed to determine the association between PA and residence area and the type of dyslipidemia. Linear regression was used to determine the effect of PA on the cholesterol, HDL, LDL and triglycerides levels. Results The prevalence of low PA in rural individuals was 5.1 per cent (4.3 per cent women and 6.0 per cent men), urban 11.2 per cent (7.9 per cent women and 5.5 per cent men), p<0,001. Dyslipidemia was present in 75.3 per cent of rural and 68 per cent of urban individuals (p<0.001). In rural women 80.6 per cent and urban women 65.7 per cent (p <0.001), rural men 69.1 per cent and urban men 71 per cent (p=0.369). The medians of lipid levels among women were: total cholesterol (CT) 164,5 mg/dl rural and 201,5 mg/dl urban (p<0,001), HDL 44,5 mg/dl rural and 50,1mg/dl (p<0,001), LDL 120,3 mg/dl rural e 122,6 mg/dl urban (p=0,35), triglycerides 127,1 mg/dl rural e 117,8 urban (p<0,001). Among men the medians of lipid levels were CT 189,5 mg/dl rural e 196,1 mg/dl urban (p=0,002), HDL 40,2 mg/dl rural e 41,8 mg/dl urban (p<0,001), LDL 118,3 mg/dl rural e 121,4 mg/dl urban (p=0,5), triglycerides 131,1 mg/dl rural e 147,0 mg/dl urban (p=0,001). Low HDL level was the most frequent. Household, transportation and occupational PA are associated with reduced risk of dyslipidemia. The effect of household PA on HDL level was 1,001 mg/dl among rural women (p=0.021) and 1,114 mg/dl (p<0.001) among urban women. In urban men, the effect of transportation PA on LDL level was -1.42 mg/dl (p<0.001). The effects of occupational PA on HDL level was 1.002 mg/dl among rural men (p<0.001) and 0.042 mg/dl among urban men (p=0.04). Conclusion There are strong indications about the interaction of physical activity and residence area with dyslipidemia and lipid profile. The observed associations between household, transportation, occupational physical activities and dyslipidemia suggest complementary ways in strategies for cardiovascular disease prevention in the Brazilian population.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-09-04T15:40:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_ivania_deliberalli.pdf: 709384 bytes, checksum: faef8c8ebde38dfcf18e902b5a0410ad (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-09-13T17:55:10Z (GMT) No. of bitstreams: 2 dissertacao_ivania_deliberalli.pdf: 709384 bytes, checksum: faef8c8ebde38dfcf18e902b5a0410ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-09-13T17:55:50Z (GMT) No. of bitstreams: 2 dissertacao_ivania_deliberalli.pdf: 709384 bytes, checksum: faef8c8ebde38dfcf18e902b5a0410ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-13T17:56:13Z (GMT). No. of bitstreams: 2 dissertacao_ivania_deliberalli.pdf: 709384 bytes, checksum: faef8c8ebde38dfcf18e902b5a0410ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A leptospirose é uma doença causada por espiroquetas do gênero Leptospira. É uma zoonose de ampla distribuição geográfica, sendo um problema de saúde pública e veterinária, principalmente em países subdesenvolvidos e em desenvolvimento de clima tropical e subtropical. Em medicina veterinária, a leptospirose é uma doença importante tanto para a clínica quanto para a produção, devido ao risco à saúde pública, perdas reprodutivas e óbitos. A vacinação animal é realizada como medida de prevenção da enfermidade, entretanto, a proteção conferida pelas vacinas comerciais é limitada e não evita a condição de portador. Os antígenos protéicos da membrana externa parecem ser a melhor alternativa para substituir as vacinas atualmente disponíveis, porém, após diversos estudos, nenhum apresentou resultados satisfatórios. O objetivo deste trabalho foi avaliar antígenos recombinantes e preparações vacinais, capazes de conferir proteção de amplo espectro, em hamsters (Mesocricetus auratus). Neste estudo, quatro novas proteínas recombinantes de Leptospira spp foram obtidas e testadas no modelo de leptospirose aguda em hamster para avaliar vacinas experimentais. A análise in silico do genoma de L. interrogans sorogrupo Icterohaemorrhagie sorovar Copenhageni cepa FIOCRUZ L1-130 foi realizada para identificar genes alvo potenciais para o desenvolvimento de vacinas de subunidade. Quatro genes foram selecionados para expressão de proteínas recombinantes em sistema de expressão heteróloga em Escherichia coli: lic10260, lic10365, lic11207 e lic11360, que codificam para prováveis lipoproteína de membrana externa. Os genes foram amplificados a partir do DNA genômico de L. borgpeterseni sorogrupo Ballum cepa 4E. Os procedimentos de clonagem resultaram nos vetores recombinantes pAE/lic10260, pAE/lic10365, pAE/lic11207 e pAE/lic11360. As proteínas recombinantes foram eficientemente expressas nesse sistema de expressão e posteriormente, utilizadas na imunização de hamsters que foram subsequentemente desafiados com dose letal de L. borgpeterseni sorovar Ballum 4E. A resposta imune humoral induzida pelas proteínas foi avaliada por ensaio imunoenzimático (ELISA) e verificou-se que, pelo menos uma das formulações vacinais para cada proteína produziu resposta humoral significativa estatisticamente (p < 0,05). As proteínas rLIC10260, rLIC10365, rLIC11207 e rLIC11360 mostraram-se imunogênicas, tornando promissora sua utilização como vacinas contra leptospirose. / Leptospirosis is a widespread zoonosis caused by spirochetes from genus Leptospira. The disease is considered a problem in both public health and veterinary areas, affecting mainly tropical and subtropical developing countries. In veterinarian medicine, leptospirosis is an important disease for both clinic and production sectors due to the public health risks, reproductive failures and death, culminating in considerable economic losses for the area. Animal vaccination is taken as a prevention measure, however the acquired protection by the vaccines is, in most cases, weak and allows the spreading of the disease. The outer membrane antigens seems to be the best alternative to substitute the currently available vaccines, however, after several studies, none of them presented satisfactory results. The aim of the present work was the production and evaluation of four recombinant antigens in order to elicit broad spectrum protection in hamster, the susceptible experimental model (Mesocricetus auratus). An in silico analysis in L. interrogans serogroup icterohaemorrhagie serovar Copenhageni strain FIOCRUZ L1-130 was run in order to identify potential genes for subunit vaccine development. Four genes were selected, amplified, cloned into vectors and expressed in Escherichia coli: lic10260, lic10365, lic11207, lic11360. These gene products are potential outer membrane lipoproteins and were amplified from L. borgpeterseni serugroup Ballum strain 4E. The cloning procedures resulted in the recombinant vectors pAE/LIC10260, pAE/LIC10365, pAE/LIC11207 and pAE/LIC11360 and its purified products were inoculated in hamsters and their humoral response was evaluated per Enzyme-linked Immunossorbent Assay (ELISA). It was possible to verify that at least one of the vaccinal preparations for each protein was able to induce statistically significant humoral response (p < 0.05). The four obtained proteins showed immunogenic response, making them promising recombinant antigens for use against leptospirosis.

A disfunção endotelial é um dos componentes básicos tanto da origem como das complicações de algumas doenças cardiovasculares, principalmente aquelas devidas a aterosclerose. Fatores de risco que compõe a Síndrome Metabólica (SMet) interferem na integridade endotelial por causarem marcante estresse oxidativo e conseqüente disfunção endotelial. Os benefícios de um treinamento físico de longa duração sobre o endotélio e sobre a concentração das lipoproteínas já são conhecidos. Entretanto, ainda restam lacunas de conhecimento dos efeitos que um treinamento de curta duração produziria em pacientes portadores de Síndrome Metabólica. Para elucidarmos este assunto, estudamos 40 indivíduos sedentários sendo 30 portadores de SMet e 10 normais para controle. Vinte dos pacientes com SMet (10 mulheres e 10 homens) realizaram treinamento físico (TF) aeróbio de moderada intensidade, em bicicleta ergométrica, por um período de 3 meses. A reatividade vascular e testes funcionais in vitro das lipoproteínas HDL e LDL foram realizados antes e depois do TF. Os resultados indicam melhora na unção endotelial, porém sem mudanças do índice de massa corpórea e dos níveis lipídicos. Houve redução da circunferência abdominal e dos níveis de Triglicérides. Os testes funcionais revelaram que, apesar de não terem ocorrido alterações na concentração, houve melhora funcional das lipoproteínas. Portanto, exercício de curta duração melhora a funcionabilidade endotelial e das lipoproteínas. / Endothelial dysfunction is one of the basic components of origin and complications of some cardiovascular diseases, especially those consequent to atherosclerosis. Risk factors that compose the Metabolic Syndrome (MetS) modify the endothelial integrity causing significant oxidative stress and consequent endothelial dysfunction. The long-term exercise training benefits in lipoproteins concentration and endothelium are already known. However, the effects of short-term training in endothelial function and in LDL, HDL quantitative and functional profile are still doubtful, especially in patients with MetS. To address this issue, we studied 40 sedentary persons, 30 with MetS and 10 controls. Twenty of those with MetS (10 women and 10 men) were subjected to a 3 times/week moderate intensity controlled training load for 3 months on a bicycle ergometer. Vascular reactivity and in vitro HDL, LDL functional laboratorial tests were analyzed before and after the training. The results revealed that exercise training improved the endothelial function. There was no significant change in body mass index, but some reduction in the abdominal circumference was observed. Total cholesterol and lipoprotein concentrations were not affected by exercise, but triglyceride levels were reduced and lipoprotein subfractions functional tests significantly improved. Therefore, short-term exercise improves endothelium and lipoprotein functionability.

Entre 5,3 e 12% das mulheres com endometriose apresentam a forma mais severa da doença, com comprometimento profundo do intestino, cujo principal tratamento é cirúrgico, porém com alta taxa de complicações: recorrência das lesões, fístulas, hemorragias, infecção, suboclusão intestinal e disfunção vesical e/ou intestinal. O tratamento medicamentoso visa inibir o crescimento dos focos por meio do bloqueio ovariano, com boa redução da dor, mas sem diminuição dos nódulos endometrióticos, e maiores efeitos colaterais pela longa duração do tratamento. Atualmente, drogas podem ser acopladas a nanopartículas que tenham afinidade com tecidos doentes, de forma a serem entregues diretamente aos mesmos, sem efeitos sistêmicos significativos e com eficácia superior no controle das lesões. Há evidências de que células em alta divisão apresentam maior captação de LDL que células normais, para síntese de membrana. Em diversos tipos de câncer, doenças reumatológicas, aterosclerose, mieloma múltiplo e na cardiomiopatia diabética, a necessidade significativamente maior de LDL permitiu bons resultados terapêuticos acoplando-se um quimioterápico à LDE (nanoemulsão artificial semelhante à LDL e que se liga ao mesmo receptor). Na endometriose, assim como no câncer, há fatores de crescimento e citocinas associados à multiplicação celular, e também há superexpressão dos receptores de LDL nas lesões de endometriose. Além disso, mulheres com endometriose intestinal apresentam níveis plasmáticos menores de LDL que o grupo sadio. Este estudo avaliou a captação da emulsão de LDE marcada radioativamente pelos receptores na superfície do foco endometriótico, para posteriormente testar a capacidade da LDE de interiorização celular. Tratou-se de um estudo-piloto comparativo entre três grupos: endometriose intestinal e não-intestinal, e sem endometriose. Antes do tratamento cirúrgico, foi determinado o perfil lipídico das pacientes e foi injetada a LDE marcada. Foi dosada a radioatividade das amostras de tecidos coletados (endometriose intestinal e não-intestinal, peritôneosadio e endométrio tópico). Na comparação entre os grupos, não houve diferença significativa nos parâmetros (p > 0,01), possivelmente devido à amostra pequena. Mas o grupo com endometriose intestinal tendeu a apresentar um IMC mais baixo que os demais, com maior sintomatologia álgica e níveis mais altos de LDL. A captação de LDE no peritôneosadio foi maior no grupo com endometriose não intestinal. O endométrio tópico e os focos de endometriose intestinal captaram mais LDE que os de não-intestinal, sugerindo maior divisão celular e menos fibrose. A comparação da captação da LDE por local mostrou uma tendência a captação decrescente entre endométrio tópico, peritôneo sadio e lesão de endometriose. Como os focos de endometriose (intestinal ou não) captaram de maneira significativa a LDE, conclui-se que há consumo aumentado de LDL nas lesões de endometriose, assim como no câncer e nas doenças inflamatórias. Não houve diferença dos níveis de colesterol plasmático na captação da LDE, comprovando a entrega direta da nanoemulsão aos tecidos em alta divisão celular, independente das lipoproteínas séricas. Estes resultados abrem caminho para novos estudos de avaliação da nanotecnologia como opção terapêutica às cirurgias radicais, com menores complicações e sem efeitos colaterais sistêmicos / Between 5.3% and 12% of women with endometriosis present the most severe form of disease, with deep intestinal involvement, mainly treated by surgery, which has a high rate of complications: recurrence of lesions, fistulas, hemorrhage, infection, intestinal subocclusion and bladder and / or bowel dysfunction. Drug treatment aims to inhibit foci growth by means of ovarian blockage, with good reduction of pain, but with no effect on endometriotic nodules, and frequent side effects due to the long duration of treatment. Currently, drugs can be coupled to nanoparticles that have affinity with compromised tissues, in order to be delivered directly to them, without significant systemic effects and with superior efficacy in the control of lesions. There are evidences that cells in high division have higher uptake of LDL than normal cells, for membrane synthesis. In several types of cancer, rheumatic diseases, atherosclerosis, multiple myeloma and in diabetic cardiomyopathy, the significantly greater need for LDL allowed good therapeutic results by coupling a chemotherapeutic to LDE (an artificial nanoemulsion similar to LDL that binds to the same receptor). In endometriosis, as well as in cancer, there are growth factors and cytokines associated with cell multiplication, and there is also overexpression of LDL receptors in endometriosis lesions. In addition, women with intestinal endometriosis have higher plasma LDL levels than healthy ones. This study evaluated the uptake of radioactively labeled LDE emulsion by receptors on the surface of endometriotic foci, to later test the LDE capacity of cellular interiorization. It was a comparative pilot study between three groups: patients with intestinal and non-intestinal endometriosis, and without endometriosis. Before surgical treatment, the lipid profile of patients was determined and labeled LDE nanoemulsion was injected. The radioactivity of the collected tissue samples (intestinal and non-intestinal endometriosis, healthy peritoneum and topical endometrium) was measured. In the comparison between groups, there was no significant difference in parameters (p > 0.01), possibly due to the small sample. But the group with intestinal endometriosis tended to present a lower BMI than others, with greater pain symptomatology and higher levels of LDL. LDE uptake in healthy peritoneum was higher in the non-intestinal endometriosis group. The topical endometrium and foci of intestinal endometriosis uptaked more LDE than non-intestinal ones, suggesting greater cell division and less fibrosis. Comparison of LDE uptake per site showed a tendency toward decreasing uptake between topical endometrium, healthy peritoneum and endometriosis lesion. As endometriosis foci (intestinal or not) have uptaked LDE on a significant level, the conclusion might be that there is an increased consumption of LDL by endometriosis lesions, as well as in cancer and inflammatory diseases. There was no difference between plasma cholesterol levels and LDE uptake, demonstrating the direct delivery of the nanoemulsion to tissues in high cell division, independent of serum lipoproteins. These results open the way for new studies evaluating nanotechnology as a therapeutic option for radical surgeries, with lower complications and no systemic side effects

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.

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in nitric oxide (NO) production by macrophages. A major goal of this study was to identify the lipid components in oxLDL modulating NO production. The effect of a water soluble antioxidants (N-acetylcysteine) and lipid soluble antioxidant (alpha-tocopherol) on NO production in macrophages was also determined. A second goal was to determine if the effects of oxLDL occurred at the transcriptional level. Human LDL was oxidized using an azo-initiator 2,2$\sp\prime$-azobis (2-amidinopropane) HCI (ABAP). OxLDL markedly decreased the production of NO in LPS stimulated RAW264.7 macrophages. This inhibition depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. In contrast, acetylated LDL (AcLDL) and native LDL showed only minor inhibition. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the primary products formed during LDL oxidation. 13-Hydroperoxyl octadecadienoic acid (13-HPODE) markedly inhibited NO production, whereas lysoPC showed only slight inhibition. Furthermore, the effects of 13-HPODE and lysoPC did not require their uptake in an AcLDL carrier. Pre-treatment of macrophages with alpha-tocopherol attenuated the inhibition due to oxLDL. Similarly, pre-treatment with N-acetylcysteine attenuated the inhibition caused by oxLDL or 13-HPODE. OxLDL was found to decrease iNOS protein and mRNA levels in RAW264.7 macrophages induced by LPS. The activation of NF-$\kappa$B was slightly suppressed after 45 minutes of treatment. 13-HPODE showed much stronger reduction of iNOS protein levels than lysoPC. These results suggest that oxLDL may inhibit NO production in macrophages at transcriptional level. 13-HPODE is likely to be the most important lipid component in oxLDL for the inhibitory effect. Antioxidants were found to preserve NO production in macrophages treated with either oxLDL or 13-HPODE. The physiological consequences of decreased NO production in macrophages caused by oxLDL are discussed with respect to atherosclerosis.

<p>Primary hyperparathyroidism (pHPT), characterized by elevated serum levels of calcium and parathyroid hormone (PTH), is associated with a number of metabolic derangements causing secondary manifestations. These include osteoporosis and increased risk of fractures, but also risk factors for cardiovascular morbidity and mortality. These risk factors include impaired glucose tolerance (IGT), dyslipidemia, increased body mass index and hypertension. While the skeletal abnormalities are mainly due to elevated PTH, the latter disturbances are still unexplained. Non-insulin dependent diabetes mellitus (NIDDM), IGT, dyslipidemia and hypertension are all included in the metabolic syndrome, also associated with morbidity and mortality in cardiovascular diseases.</p><p>In this thesis, decreased bone mineral density (BMD) and variables of the metabolic syndrome are explored in patients with mild and normocalcemic pHPT before and after parathyroidectomy. To further investigate the relationship between insulin sensitivity and calcium, a community-based cohort was investigated.</p><p>In two different patient cohorts of pHPT, lipoprotein alterations with decreased levels of HDL-cholesterol and elevated triglycerides were found in association with a high frequency of IGT, NIDDM and decreased insulin sensitivity. Parathyroidectomy had effects on the dyslipidemia and in part on the glucose metabolism. The disturbed glucose metabolism in pHPT was substantiated by results from the general population by a negative association between insulin sensitivity, measured by hyperinsulinemic clamp, and serum calcium.</p><p>In conclusion, normocalcemic, mild and overt pHPT are associated with a range of risk factors for cardiovascular diseases, development of NIDDM and decreased BMD in cortical as well as trabecular bone. These findings explain, at least in part, the elevated morbidity and mortality from cardiovascular disease as well as fractures, reported in pHPT patients. Moreover, in the general population, serum calcium is associated with decreased insulin sensitivity. Parathyroidectomy has positive effects on several, but not all, of the investigated metabolic parameters.</p>

Primary hyperparathyroidism (pHPT), characterized by elevated serum levels of calcium and parathyroid hormone (PTH), is associated with a number of metabolic derangements causing secondary manifestations. These include osteoporosis and increased risk of fractures, but also risk factors for cardiovascular morbidity and mortality. These risk factors include impaired glucose tolerance (IGT), dyslipidemia, increased body mass index and hypertension. While the skeletal abnormalities are mainly due to elevated PTH, the latter disturbances are still unexplained. Non-insulin dependent diabetes mellitus (NIDDM), IGT, dyslipidemia and hypertension are all included in the metabolic syndrome, also associated with morbidity and mortality in cardiovascular diseases. In this thesis, decreased bone mineral density (BMD) and variables of the metabolic syndrome are explored in patients with mild and normocalcemic pHPT before and after parathyroidectomy. To further investigate the relationship between insulin sensitivity and calcium, a community-based cohort was investigated. In two different patient cohorts of pHPT, lipoprotein alterations with decreased levels of HDL-cholesterol and elevated triglycerides were found in association with a high frequency of IGT, NIDDM and decreased insulin sensitivity. Parathyroidectomy had effects on the dyslipidemia and in part on the glucose metabolism. The disturbed glucose metabolism in pHPT was substantiated by results from the general population by a negative association between insulin sensitivity, measured by hyperinsulinemic clamp, and serum calcium. In conclusion, normocalcemic, mild and overt pHPT are associated with a range of risk factors for cardiovascular diseases, development of NIDDM and decreased BMD in cortical as well as trabecular bone. These findings explain, at least in part, the elevated morbidity and mortality from cardiovascular disease as well as fractures, reported in pHPT patients. Moreover, in the general population, serum calcium is associated with decreased insulin sensitivity. Parathyroidectomy has positive effects on several, but not all, of the investigated metabolic parameters.

Alexandra Flocke Untersuchung von Stoffwechselparametern und Lipoproteinen im Blutserum von einlings- und zwillingsträchtigen Merino- und Schwarzköpfigen Fleischschafen im peripartalen Zeitraum Medizinische Tierklinik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im Februar 2012 (94 Seiten, 15 Abbildungen, 8 Tabellen, 199 Literaturangaben, 12 Tabellen im Anhang) Schlüsselwörter: Lipoproteine, Schaf, Stoffwechsel, Endotoxin, Ketose Problemstellung: Der Lipoproteinstatus in der kritischen Phase der Hochträchtigkeit wurde beim Schaf bisher nur wenig untersucht. Die Fähigkeit der Lipoproteine, sowohl Lipide zu transportieren und im Körper umzuverteilen, als auch ihre Rolle in der Endotoxinneutralisation machen sie zu einem wichtigen Parameter in stoffwechselbelasteten Situationen. Das Ziel dieser Arbeit ist es, den Lipoproteinstatus zweier verschiedener Leistungsrassen zu analysieren und zu prüfen, ob und inwiefern peripartal Unterschiede bei Ein- und Zwillingsträchtigkeiten bestehen. Zusätzlich werden die Endotoxine der Tiere bestimmt und mögliche Zusammenhänge mit der Stoffwechselsituation und dem Lipoproteinstatus evaluiert. Tiere, Material und Methoden: Untersucht wurden gesunde einlingsträchtige Merinofleischschafe (MFS1), zwillingsträchtige Merinofleischschafe (MFS2) sowie zwillingsträchtige Schwarzköpfige Fleischschafe (SKF2). Im Abstand von je sieben Tagen wurden den Tieren im Zeitraum von der 5. Woche a.p. bis eine Woche p.p. Blutproben aus der V. jugularis externa entnommen. Aus dem Serum wurden die Konzentrationen von β-Hydroxybutyrat (BHB), Glucose, Freie Fettsäuren (FFS), Triacylglycerol (TG), Cholesterol, Bilirubin, Gesamtprotein (alle Hitachi 912), Insulin (RIA-Kit, Firma IBL, Hamburg), α- und β-Lipoproteine (LIPIDOPHOR All In12®, - ohne prä-β-Lipoproteine), freies Endotoxin (LAL-Test) und Anti-Lipid A Antikörper (ALAAK; ELISA) bestimmt. Ergebnisse: Die α-Lipoproteinkonzentration stieg bei den MFS1 vor dem Ablammen signifikant an und erreichte eine Konzentration von = 208,0 ± 40,1 mg/dl (1. Woche a.p.). Die MFS2 wiesen vor dem Lammen einen konstanten Verlauf zwischen = 180,4 ± 31,1 mg/dl (5. Woche a.p.) und = 196,1 ± 42,1 mg/dl (3. Woche a.p.) auf. Die SKF2 zeigten in der 5. Woche a.p. mit = 206,5 ± 39,0 mg/dl eine größtenteils signifikant höhere Konzentration gegenüber den folgenden Untersuchungszeitpunkten. Bei beiden Rassen ließ sich bei den Zwillingsmuttern nach dem Lammen ein signifikanter Abfall der α-Lipoproteinkonzentration auf = 144,3 ± 46,1 mg/dl (MFS2) bzw. = 170,2 ± 48,8 mg/dl (SKF2) feststellen. Es bestand eine gesicherte Korrelation zu Cholesterol. Die β-Lipoproteinkonzentration lag in der 1. Woche p.p. bei den Zwillingsmuttern (MFS2 und SKF2) signifikant niedriger als vor dem Lammen. Die SKF2 wiesen in der 5. und 4. Woche a.p. signifikant niedrigere Konzentrationen als die MFS1 auf. Gesicherte Korrelationen bestanden zu TG, Cholesterol und ALAAK (MFS1). Für die α-Lipoproteinkonzentration wurde ein Referenzbereich von 144,3 – 208,0 mg/dl und für die β-Lipoproteinkonzentration von 13,6 – 28,0 mg/dl bei Muttertieren der beschriebenen Rassen ermittelt. Ausgenommen die Insulinkonzentration in der 1. Woche a.p. bestanden keine signifikanten Differenzen zwischen MFS1 und MFS2. Im Vergleich der Rassen zeigten sich signifikante Differenzen in der Glucosekonzentration in der 2. Woche a.p. und in der Konzentration von Bilirubin in der 3. und 1. Woche a.p.. Die BHB-Konzentration zeigte einen konstanten Verlauf und stieg bei den MFS1 bis auf = 0,96 ± 0,36 mmol/l eine Woche p.p., bei den MFS2 auf = 0,73 ± 0,35 mmol/l und bei den SKF2 auf = 0,63 ± 0,14 mmol/l eine Woche a.p. signifikant an. Gesicherte Korrelationen bestanden zu FFS (MFS2) und Insulin (MFS2; SKF2). Die Glucosekonzentration zeigte sich bei den Zwillingsmuttern nach zunächst abfallenden Konzentrationen in der Hochträchtigkeit eine Woche p.p. signifikant höher ( = 3,63 ± 0,59 mmol/l; MFS2 und = 3,28 ± 0,42 mmol/l; SKF2) als vor dem Lammen. Gesicherte Korrelationen ließen sich zu FFS (MFS1; MFS2), BHB (MFS2) und dem Zeitpunkt der Untersuchung (MFS2; SKF2) feststellen. Die FFS-Konzentration stieg bei den SKF2 zur 1. Woche a.p. auf = 265 ± 131 μmol/l an, fiel eine Woche p.p. signifikant auf = 128 ± 95 μmol/l ab und korrelierte gesichert mit TG. Die TG-Konzentration fiel von der 1. Woche a.p. bis zur 1. Woche p.p. signifikant auf = 0,19 mmol/l (MFS1; SKF2) bzw. = 0,18 ± 0,05 mmol/l (MFS2) ab und korrelierte mit Gesamtprotein (MFS1), Cholesterol (MFS1; MFS2), β-Lipoproteinen (MFS1; MFS2) und freiem Endotoxin (MFS1). Die Cholesterolkonzentration fiel eine Woche p.p. bei den Zwillingsmuttern signifikant ab, bei den MFS1 war der Verlauf konstant. Die Bilirubinkonzentration stieg eine Woche p.p. signifikant an (MFS1; MFS2) und korrelierte mit der Konzentration von freiem Endotoxin. Das Gesamtprotein sank zum Zeitpunkt des Ablammens hin signifikant ab, erreichte jedoch eine Woche p.p. wieder seine Ausgangskonzentration. Eine gesicherte Korrelation konnte mit ALAAK festgestellt werden (MFS2; SKF2). Freies Endotoxin konnte zu jedem Zeitpunkt der Untersuchungsreihe nachgewiesen werden. Dabei lagen die festgestellten Konzentrationen bei x̃ = 0,06 – 1,30 EU/ml. Eine gesicherte Korrelation fand sich mit der Konzentration der ALAAK (MFS2). ALAAK konnten ebenfalls zu jedem Zeitpunkt in Konzentrationen von x̃ = 22,2 - 56,8 (OD-Wert) nachgewiesen werden. Fazit: Die Lipoproteinkonzentrationen in den einzelnen Gruppen zeigten sich a.p. zunächst regulativ den erhöhten peripartalen Belastungen des Lipidstoffwechsels angepasst, p.p. demonstrierte auch die Abnahme der Lipoproteinkonzentrationen das Ende der lipolytischen Aktivität bei den Schafen. Die vorliegenden Ergebnisse der Stoffwechselparameter lassen auf eine stärkere Belastung der Zwillingsmuttern schließen, welche auch die Lipoproteine mit einbezieht. Freies Endotoxin konnte auch bei gesunden Schafen zwar zu jedem Zeitpunkt nachgewiesen, eine gesicherte Korrelation mit der Lipoproteinkonzentration jedoch nicht festgestellt werden. / Alexandra Flocke Evaluation of metabolic parameters and lipoproteins in the serum of merino and blackhead sheep for meat production with one or two lambs in the peripartal period Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig Submitted in February 2012 (94 pages, 15 figures, 8 tables, 199 references, 12 tablets in the appendix) Keywords: lipoproteins, sheep, metabolism, endotoxin, ketosis Objective: The lipoprotein status in the critical phase of the peripartal period of sheep has been few reviewed. The ability of lipoproteins to transport and redistribute lipids in the organism and their capacity for endotoxinneutralisation makes them a considerable parameter in metabolic bonded situations. The objective of this research is to show the lipoprotein status of two competitive breeds and to proof if and to what extend differences between ewes with one and two lambs exist. Additionally, the endotoxic situation is assigned and possible correlations with the metabolic system and the lipoprotein status are evaluated. Animals, materials and methods: The research contains healthy merino sheep with one lamb (MFS1), merino sheep with two lambs (MFS2) and blackhead sheep with two lambs (SKF2). At intervals of seven days, blood samples out of the V. jugularis externa were taken from the animals in a period from the 5th week a.p. to one week p.p.. Concentrations in the serum of the following parameters were defined: β-hydroxybutyrat (BHB), glucose, free fatty acids (FFS), triacylglycerol (TG), cholesterol, bilirubin, total protein (all Hitachi 912), insulin (RIA-kit, IBL, Hamburg), α- and β-lipoprotein (LIPIDOPHOR All In12® - without pre-β-lipoprotein), free endotoxin (LAL-test) and anti-lipid A antibodies (ALAAK). Results: The concentration of α-lipoproteins of the MFS1 increased significantly before parturition and finally reached 208,0 ± 40,1 mg/dl (1st Week a.p.). The group of MFS2 showed a constant deviation between = 180,4 ± 31,1 mg/dl (5th Week a.p.) and = 196,1 ± 42,1 mg/dl (3rd week a.p.) before parturition. The SKF2 evidenced with a concentration of = 206,5 ± 39,0 mg/dl in the 5th week a.p. a predominantly significant higher result as during following points in time. Both groups carrying twins showed a significant decrease of the α-lipoprotein concentration down to = 144,3 ± 46,1 mg/dl (MFS2) or = 170,2 ± 48,8 mg/dl (SKF2) after parturition. There were statistically reliable correlations between the α-lipoprotein concentration and cholesterol. The β-lipoprotein concentration from both twin pregnant groups had been significantly lower one week after parturition than before parturition. The results of the SKF2 in week 5 and 4 a.p. were significantly lower than those of the MFS1. Reliable correlations could be determined with TG, cholesterol and ALAAK (MFS1). An α-lipoprotein reference range for ewes of the introduced breeds could be determined from 144,3 - 208,0 mg/dl and for β-lipoproteins from 13,6 - 28,0 mg/dl. There were no significant differences between MFS1 and MFS2 except the concentration of insulin in the 1st week a.p.. In the comparison of the different breeds, significant differences in the concentration of glucose (2nd week a.p.) and bilirubin (3rd week a.p. and 1st week a.p.) could be shown. The concentration of BHB showed a constant deviation and increased significantly to = 0,96 ± 0,36 mmol/l (MFS1; 1st week p.p.), = 0,73 ± 0,35 mmol/l (MFS2, 1st week a.p.) or = 0,63 ± 0,14 mmol/l (SKF2, 1st week a.p.), respectively. Reliable correlations existed with FFS (MFS2) and insulin (MFS2; SKF2). The concentration of glucose of the twin pregnant ewes decreased initially before parturition and increased significantly again in the 1st week p.p. ( = 3,63 ± 0,59 mmol/l; MFS2 and = 3,28 ± 0,42 mmol/l; SKF2). Reliable correlations had been determined with FFS (MFS1; MFS2), BHB (MFS2) and the time of sampling (MFS2; SKF2). Concentrations of FFS increased in the group of SKF2 to = 265 ± 131 μmol/l in the 1st week a.p., decreased again significantly to = 128 ± 95 μmol/l one week p.p. and showed a correlation with TG. The TG concentration decreased significantly from one week a.p. to one week p.p. down to = 0,19 mmol/l (MFS1; SKF2) and = 0,18 ± 0,05 mmol/l (MFS2), respectively. A reliable correlation existed between TG and total protein (MFS1), cholesterol (MFS1; MFS2), β-lipoproteins (MFS1; MFS2) and free endotoxin (MFS1). Both twin pregnant groups had a significant decrease in the cholesterol concentration one week after parturition, in contrast the group of MFS1 showed a constant deviation. The concentration of bilirubin increased significantly one week p.p. (MFS1; MFS2) and showed a reliable correlation with the concentration of free endotoxin. Total protein decreased significantly towards parturition, but reached its initial concentration again one week p.p.. A statistically reliable correlation could be determined with ALAAK (MFS2; SKF2). Free endotoxin could be detected at any point of the exploration. The observed concentrations ranged from x̃ = 0,06 – 1,30 EU/ml. A reliable correlation could be shown with the concentration of ALAAK (MFS2). ALAAK was detected as well in concentrations from x̃ = 22,2 - 56,8 (OD-value) in any taken sample. Conclusion: The lipoprotein concentrations in each group appeared a.p. initially adapted to the peripartal bonds of the increased lipid metabolism. After parturition, the decrease in lipoprotein concentrations, too, demonstrated the end of the lipolytic activity of the ewes. The results of the metabolic parameters lead to the conclusion of a stronger liability of the ewes with twins. This is also valid for the lipoproteins. Free endotoxin could be detected in healthy sheep at any time, but a reliable correlation with the concentration of lipoproteins was not visible.