Objective To assess potential effects of variants in six lipid modulating genes (SORT1, HMGCR, MLXIPL, FADS2, APOE and MAFB) on early development of dyslipidemia independent of the degree of obesity in children, we investigated their association with total (TC), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) cholesterol and triglyceride (TG) levels in 594 children. Furthermore, we evaluated the expression profile of the candidate genes during human adipocyte differentiation. Results Expression of selected genes increased 101 to >104 fold during human adipocyte differentiation, suggesting a potential link with adipogenesis. In genetic association studies adjusted for age, BMI SDS and sex, we identified significant associations for rs599839 near SORT1 with TC and LDL-C and for rs4420638 near APOE with TC and LDL-C. We performed Bayesian modelling of the combined lipid phenotype of HDL-C, LDL-C and TG to identify potentially causal polygenic effects on this multi-dimensional phenotype and considering obesity, age and sex as a-priori modulating factors. This analysis confirmed that rs599839 and rs4420638 affect LDL-C. Conclusion We show that lipid modulating genes are dynamically regulated during adipogenesis and that variants near SORT1 and APOE influence lipid levels independent of obesity in children. Bayesian modelling suggests causal effects of these variants.

Kolesterol transporteras i blodet med hjälp av lipoproteinpartiklar. Höga nivåer av low-density lipoprotein (LDL)-kolesterol i blodet är en riskfaktor för kardiovaskulär sjukdom. Koncentrationen av LDL-kolesterol kan beräknas med hjälp av Friedewalds formel men det finns även metoder där LDL-kolesterol kan analyseras direkt. Syftet med arbetet var att verifiera metoden direkt LDL-kolesterol på analysinstrumentet Beckman Coulter AU680. Metodens inomserie- och totalimprecision analyserades. Två korrelationsstudier utfördes mellan direkt LDL-kolesterol och beräknat LDL-kolesterol, en med 43 patientprover med triglycerider < 4,5 mmol/L och en med 11 patientprover med triglycerider > 4,5 mmol/L. Friedewalds formel ska egentligen inte användas vid triglycerider > 4,5 mmol/L, men i detta fall användes formeln ändå för att utvärdera eventuella skillnader mellan metodernas resultat vid höga triglyceridkoncentrationer. Vid analys av metodens inomserieimprecision blev variationskoefficienten (CV) omkring 0,5 % vid analys av både den låga kontrollen (A1) och den höga kontrollen (A2). CV för totalimprecisionen blev 1,21 % vid analys av A1 och 1,11 % vid analys av A2. Korrelationsstudierna visade ett linjärt samband mellan metoderna men den direkta metoden gav något högre resultat vid lägre koncentrationer och något lägre resultat vid högre koncentrationer jämfört med beräknat LDL-kolesterol. Vid triglycerider > 4,5 mmol/L gav den direkta metoden betydligt högre resultat än beräknat LDL-kolesterol. Slutsatsen blev att metoden hade god precision. Överensstämmelsen mellan metodernas resultat var relativt bra för proverna med triglycerider < 4,5 mmol/L. Vid triglycerider > 4,5 mmol/L var differensen mellan metoderna stor, troligtvis på grund av falskt för låga resultat från beräknat LDL-kolesterol. / Cholesterol is transported in the blood by lipoproteins. High levels of low-density lipoprotein (LDL)-cholesterol in the blood is a risk factor for cardiovascular disease. The concentration of LDL-cholesterol can be calculated using the Friedewald formula but there are also methods that measure LDL-cholesterol directly. The aim of this study was to verify the method P-LDL-cholesterol on a Beckman Coulter AU680 analyzer. Within-run imprecision and total imprecision were analyzed. The correlation between direct LDL-cholesterol and calculated LDL-cholesterol was examined using 43 patient samples with triglyceride levels < 4,5 mmol/L and 11 patient samples with triglyceride levels > 4,5 mmol/L. The Friedewald formula is not supposed to be used on triglyceride levels > 4,5 mmol/L, but in this case the formula was used anyway to evaluate differences between the methods at high triglyceride concentrations. The coefficient of variation (CV) for the within-run imprecision was about 0,5 %, both for the low control (A1) and the high control (A2). Total imprecision had a CV of 1,21 % for A1 and 1,11 % for A2. There was a linear relationship between the methods, but the direct method gave slightly higher results at low concentrations and slightly lower results at high concentrations compared to calculated LDL-cholesterol. At triglyceride levels > 4,5 mmol/L the results from the direct method was considerably higher than calculated LDL-cholesterol. The conclusion is that the precision of the method was good. The correlation between the results from direct LDL-cholesterol and calculated LDL-cholesterol was relatively high for samples with triglyceride levels < 4,5 mmol/L. At triglyceride levels > 4,5 mmol/L there was a big difference between the methods, probably because of falsely low results from calculated LDL-cholesterol.

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No. of bitstreams: 1 Dissertação- Bianca L Vargas.pdf: 978654 bytes, checksum: 5e8b0997366945157f9b6106f7221c08 (MD5) Previous issue date: 2012-08-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Currently, chronic diseases such as diabetes, cardiovascular disease (CVD) and cancer account for 60% of all deaths worldwide. Oxidative stress caused by the action of free radicals is involved in the pathogenesis of many chronic diseases such as CVD. Flavonoids and vitamin C are antioxidant nutrients found in fruits and vegetables, and its regular consumption is associated with decreased risk of developing chronic diseases. Camu-camu (Myrciaria dubia [Kunth] McVaugh) is a fruit from the Amazon region that has significant levels of antioxidants, especially vitamin C and flavonoids. This study aimed to evaluate the effect of dust from the pulp of camu-camu on glucose and lipid profile in young adults. The pulp of camu-camu was lyophilized and its nutritional composition was analyzed using the methods indicated by Instituto Adolpho Lutz for the centesimal composition and HPLC method for vitamin C. For the intervention, was conducted a not randomized, double-blind, controlled clinical trial with 18 volunteers of both genders, aged between 21 and 35 years. The volunteers were divided into two groups: 1) intervention group that received capsules of lyophilized camu-camu containing 320 mg of vitamin C; 2) control group that received capsules containing 320 mg of synthetic vitamin C. The ingestion of capsules was daily for 15 days and blood samples were collected and analyzed before and after the intervention. Statistical differences in the levels of vitamin C in plasma, fasting glucose and lipid profile were verified by means of Student's “t” test. The lyophilized camu-camu presented 20,310 mg of vitamin C/100g and 12.89 mg of flavonoids/100g. At the end of the intervention was reported a significant decrease in fasting glucose, total cholesterol and HDL-cholesterol (p <0.05) levels in group that received capsules of camu-camu and a significant reduction only in fasting glucose (p <0.05) levels in group that received capsules of synthetic vitamin C. When considering only those individuals who correctly followed the protocol, there was also a significant decrease in LDL-cholesterol (p <0.05) levels in intervention group. In both groups there was a tendency of reduction in triglyceride levels, although it has not been significant (p> 0.05). It was concluded that the capsules of camu-camu were more efficient, presenting hypolipidemic and hypoglycemic action in volunteers studied. These results demonstrate the potential benefit of vitamin C and camu-camu health, presenting the fruit as an excellent source of this vitamin. / As doenças crônicas, como diabetes, doenças cardiovasculares (DCV) e câncer, representam atualmente 60% de todas as mortes no mundo. O estresse oxidativo, desencadeado pela ação de radicais livres, está envolvido na patogênese de inúmeras doenças crônicas, a exemplo as DCV. Alguns nutrientes presentes em frutas e verduras possuem ação antioxidante, como é o caso dos flavonóides e da vitamina C, e seu consumo regular está associado à diminuição do risco de desenvolvimento de doenças crônicas. O camu-camu [Myrciaria dubia (Kunth) McVaugh] é um fruto exclusivo da região amazônica que possui teores significativos de antioxidantes, principalmente vitamina C e flavonóides. O presente trabalho teve como objetivo avaliar o efeito do pó da polpa de camu-camu sobre a glicemia e o perfil lipídico de adultos jovens. A polpa do camu-camu foi liofilizada e sua composição nutricional foi analisada utilizando-se os métodos indicados pelo Instituto Adolpho Lutz para a composição centesimal e o método de CLAE para vitamina C. Para a intervenção, foi conduzido um ensaio clínico controlado, duplo-cego, em 18 voluntários de ambos os gêneros, com idades entre 21 e 35 anos. Os participantes foram divididos em dois grupos: 1) grupo intervenção, que recebeu cápsulas de camu-camu liofilizado contendo 320 mg de vitamina C; 2) grupo controle, que recebeu cápsulas contendo 320 mg vitamina C sintética. A ingestão das cápsulas foi diária durante 15 dias e duas coletas de sangue foram procedidas, uma antes do início do estudo e outra ao final. Foram verificadas as diferenças estatísticas nos níveis de vitamina C no plasma, glicemia de jejum e perfil lipídico por meio do Teste t de Student. O camu-camu liofilizado apresentou 20.310 mg de vitamina C/100g e 12,89 mg de flavonóides/100g. Ao final da intervenção registrou-se queda significativa nos níveis de glicemia de jejum, colesterol total e HDL (p<0,05) no grupo que recebeu cápsulas de camu-camu e redução significativa apenas da glicemia de jejum (p<0,05) no grupo que recebeu cápsulas de vitamina C sintética. Quando avaliados somente aqueles indivíduos que seguiram corretamente o protocolo, observou-se diminuição significativa também nos valores de LDL (p<0,05) no grupo intervenção. Em ambos os grupos houve tendência de redução nos níveis de triglicerídeos, porém não significativa (p>0,05). Conclui-se que as cápsulas de camu-camu foram mais eficientes, apresentando ação hipolipidêmica e hipoglicemiante nos voluntários estudados. Tais resultados demonstram o potencial benéfico da vitamina C e do camu-camu à saúde, apresentando o fruto como uma excelente fonte desta vitamina.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

La proprotéine convertase subtilisine/kexine-9 (PCSK9) a été identifiée comme le troisième locus impliqué dans l’hypercholestérolémie autosome dominante (ADH). Les deux autres gènes impliqués dans l’ADH encodent le récepteur des lipoprotéines de faible densité (LDLR) et l’apolipoprotéine B. La PCSK9 est une convertase qui favorise la dégradation du LDLR dans les hépatocytes et augmente le niveau plasmatique de cholestérol des LDL (LDL-C). Les mutations « gain de fonction » de la PCSK9 sont associées à un phénotype d’hypercholestérolémie familiale, tandis que les variantes « perte de fonction » sont associées à un LDL-C réduit et à un risque coronarien plus faible. Pour élucider le rôle physiologique de la PCSK9, nous avons étudié sa régulation génique. En utilisant le RT-PCR quantitatif dans des hépatocytes humains, nous avons analysé la régulation de PCSK9 sous différentes conditions modulant l’expression des gènes impliqués dans le métabolisme du cholestérol. Nous avons démontré que l’expression de la PCSK9 était induite par les statines de manière dose-dépendante et que cette induction était abolie par le mévalonate. De plus, le promoteur de PCSK9 contenait deux motifs conservés pour la régulation par le cholestérol : le sterol regulatory element (SRE) et un site Sp1. La PCSK9 circule dans le plasma sous des formes mature et clivée par la furine. Grâce à notre anticorps polyclonal, nous avons mis au point un test ELISA mesurant la PCSK9 plasmatique totale. Une étude transversale a évalué les concentrations plasmatiques de PCSK9 chez des sujets sains et hypercholestérolémiques, traités ou non par des statines ou une combinaison statine/ezetimibe. Chez 254 sujets sains, la valeur moyenne de PCSK9 (écart-type) était de 89,5 (31,9) µg/L. La concentration plasmatique de la PCSK9 corrélait avec celle de cholestérol total, du LDL-C, des triglycérides (TG), de la glycémie à jeun, l’âge et l’indice de masse corporelle. Le séquençage de PCSK9 chez des sujets aux extrêmes de la distribution des concentrations de PCSK9 de notre cohorte a révélé la présence d’une nouvelle variation « perte de fonction » : R434W. Chez 200 patients hypercholestérolémiques, la concentration de PCSK9 était plus élevée que chez les sujets sains (P<0,04). Elle a augmenté avec une dose croissante de statine (P<0,001), et a augmenté encore plus suite à l’ajout d’ezetimibe (P<0,001). Chez les patients traités, ceux présentant une hypercholestérolémie familiale (HF; due à une mutation du LDLR) avaient des concentrations plus élevées de PCSK9 que les non-HF (P<0,005), et la réduction de LDL-C corrélait positivement avec la concentration de PCSK9 atteinte de la même manière dans les deux sous-catégories (P<0,02 et P<0,005, respectivement). Par ailleurs, une incubation des cellules HepG2 (hépatocytes) et Caco-2 (entérocytes) avec de l’ezetimibe a provoqué une augmentation de l’ARNm de PCSK9 et de NPC1L1 de 1,5 à 2 fois (P<0,05), mais aucune variation significative de PCSK9 sécrétée n’a été observée, suggérant que ces lignées cellulaires ne sont pas un modèle idéal. Nous avons également mesuré le niveau de PCSK9 chez 1 739 Canadiens-français âgés de 9, 13 et 16 ans. La valeur moyenne (écart-type) de PCSK9 dans cette cohorte était de 84,7 (24,7) µg/L, légèrement plus basse que dans la cohorte d’adultes (89,5 (31,9) µg/L). Chez les garçons, la PCSK9 circulante diminuait avec l’âge, tandis que c’était l’inverse chez les filles. Il y avait des associations positives et significatives entre la PCSK9 et la glycémie à jeun, l’insulinémie, le HOMA-IR, et les paramètres lipidiques (TC, LDL-C, TG, HDL-C, apoAI et apoB). Dans l’analyse multivariée, une hausse de 10% de l’insulinémie à jeun était associée à une augmentation de 1 à 2% de PCSK9. La régulation de PCSK9 est typique de celle d’un gène impliqué dans le métabolisme des lipoprotéines et est probablement la cible du facteur de transcription «sterol regulatory element-binding protein » (SREBP-2). La concentration plasmatique de la PCSK9 est associée avec l’âge, le sexe, et de multiples marqueurs métaboliques chez les enfants et les adultes. La détection de la PCSK9 circulante chez les sujets HF et non-HF signifie que ce test ELISA spécifique à PCSK9 pourrait servir à suivre la réponse à la thérapie chez un grand éventail de sujets. PCSK9 semble être une cible thérapeutique prometteuse dans le traitement de l’hypercholestérolémie et de la maladie cardiovasculaire. / Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The two other known genes implicated in ADH encode the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 is a protein convertase that post-translationally promotes the degradation of the LDLR in hepatocytes and increases plasma LDL cholesterol concentration (LDL-C). Heterozygote “gain-of-function” mutations of PCSK9 are associated with the familial hypercholesterolemia phenotype, whereas “loss-of-function” variants are associated with reduced LDL-C concentrations and lower coronary risk. As an approach toward the elucidation of the physiological role(s) of PCSK9, we studied its transcriptional regulation. Using quantitative RT-PCR, we assessed PCSK9 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that PCSK9 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. The PCSK9 promoter contains two typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. PCSK9 circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal and hypercholesterolemic subjects treated or untreated with statins or statin plus ezetimibe. In 254 healthy subjects, the mean plasma PCSK9 (SD) concentration was 89 (32) µg/L. PCSK9 levels correlated positively with plasma cholesterol, LDL-C, triglycerides, fasting glucose, age and body mass index. Sequencing PCSK9 from subjects at the extremes of PCSK9 plasma distribution revealed a new loss-of-function R434W variant. In 200 hypercholesterolemic patients, circulating PCSK9 was higher than in controls (P<0.04), increased with increasing statin dose (P<0.001), and further increased when ezetimibe was added (P<0.001). In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDLR gene mutations) had higher PCSK9 values than non-FH (P<0,005), and LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (P<0.02 and P<0.005, respectively). However, incubation with ezetimibe of HepG2 (hepatocytes) and Caco-2 (enterocytes) cells caused an increase in PCSK9 and NPC1L1 mRNA of 1.5 to 2-fold (P<0.05), but no significant rise in PCSK9 protein secretion, suggesting that these transformed cells are not an ideal model. We also studied PCSK9 levels in 1,739 French Canadian youth ages 9, 13, and 16 years old. The mean (SD) plasma PCSK9 concentration, measured by ELISA, was 84.7 (24.7) µg/L in the cohort, slightly lower than in the adult cohort (89.5 (31.9) µg/L. In boys, plasma PCSK9 decreased with age, whereas the inverse was true for girls. There were significant positive associations between PCSK9 and fasting glucose, insulin, and HOMA-IR (homeostasis model assessment of insulin resistance). In multivariable analysis, a 10% higher fasting insulin was associated with a 1%-2% higher PCSK9 in both sexes. There were also positive associations between PCSK9 and total cholesterol, LDL-C, and triglycerides, as well as with HDL-C and apolipoproteins A1 and B. PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of the transcription factor “sterol response element-binding protein” (SREBP)-2. The PCSK9 plasmatic concentration is associated with age, sex, and multiple metabolic markers in youth and adult samples. The detection of circulating PCSK9 in both FH and non-FH subjects means that this PCSK9 ELISA test could be used to monitor response to therapy in a wide range of patients. PCSK9 seems to be a promising drug target in the treatment of hypercholesterolemia and coronary heart disease.

INTRODUÇÃO: portadores de diabetes mellitus tipo 1 (DM1) apresentam, progressivamente, complicações vásculo-neurais. Os fatores que aumentam o risco de coronariopatia - hipertensão, dislipidemia e idade avançada - explicam, em parte, a alta mortalidade cardiovascular, entretanto diabéticos tipo 1 podem morrer de coronariopatia precoce e não apresentar os fatores de risco clássicos para aterosclerose. Modificações estruturais e funcionais nas lipoproteínas, alterando a sua composição e trocas lipídicas poderiam justificar o aumento de eventos vasculares, entretanto estas alterações podem não ser detectadas através das dosagens rotineiras de lípides plasmáticos. OBJETIVOS: através de nanoemulsão lipídica artificial (LDE) que simula a estrutura lipídica da LDL avaliamos, em portadores de DM1, normolipidêmicos, intensivamente tratados e sem complicações significativas da doença, a taxa de esterificação do colesterol, a remoção da nanoemulsão da circulação, o tamanho da partícula HDL e as transferências de lípides entre a nanoemulsão e as partículas HDL. Secundariamente, determinamos a influência do controle glicêmico, resistência à insulina (RI) e insulinização no metabolismo lipídico. MÉTODOS: estudamos 36 indivíduos diabéticos e 37 controles não-diabéticos pareados para idade, sexo e índice de massa corpórea. Nanoemulsão lipídica artificial com marcação radioativa nos lípides éster de colesterol (CE), colesterol livre (CL), triglicérides (TG) e fosfolípides (PL) foi utilizada para os estudos. Nanoemulsão com marcação 14C-CE e 3H-CL foi injetada nos participantes e amostras de sangue foram coletadas durante 24 horas para mensuração da radioatividade. Remoção dos lípides da circulação foi calculada por análise compartimental. A taxa da esterificação do colesterol livre foi calculada após extração e separação de lípides do plasma por cromatografia em camada delgada. Para estudo da transferência de lípides, nanoemulsões com marcação 14C-CE e 3H-CL ou 14C-PL e 3H-TG foram incubadas com plasma e a radioatividade dos lípides transferidos para as HDL foi contada após a precipitação de lipoproteínas contendo apoB. O diâmetro da HDL foi mensurado por método de dispersão da luz. A RI nos diabéticos foi mensurada por fórmula que estima a taxa de captação da glicose. RESULTADOS: hemoglobina glicada foi de 8,8±1,3 mg/dl e concentrações de LDLc foram menores nos diabéticos (85±22 vs. 98±26 mg/dl), p=0, 035. Não houve diferenças em relação às taxas de esterificação, transferências de lípides da nanoemulsão para as HDL e tamanho da partícula HDL entre os grupos. Não encontramos relação entre as análises cinéticas e HbA1c, glicemia, índices de RI e dose de insulina. A taxa de remoção do 14C-CE foi mais rápida em diabéticos tipo 1 que nos controles (0, 059±0, 022 vs.0, 039±0, 022 h-1), p=0, 019. 16 CONCLUSÕES: apesar de controle glicêmico ruim nos DM1, as transferências de lípides da nanoemulsão para as HDL, a taxa de esterificação e a remoção da 3H-CL são semelhantes às dos controles. O controle glicêmico, perfil lipídico, índices de RI e dose de insulina não influenciaram nas transferências de lípides e na taxa de esterificação. A remoção do 14C-CE é mais rápida em indivíduos diabéticos, o que poderia justificar as concentrações de LDLc mais baixas encontradas nesta população. Acreditamos que a terapia insulínica intensiva pode justificar estes achados / INTRODUTION: people with type 1 diabetes mellitus (DM1) have progressively neuro-vascular complications. Factors that increase the risk of coronary artery disease hypertension, dislipidemia and advanced age explains part of increased cardiovascular mortality, however some DM1 died of early coronary artery disease and often do not have atherosclerosis classical risk factors. Structural and functional changes in lipoproteins, altering their composition and activities of lipid exchange could justify the increase in vascular events but these changes are generally not detected by routine clinical laboratory plasma lipid exams. OBJETIVES: in normolipidemic DM1, intensively treated and without significant complications of disease we evaluated, by an artificial lipid nanoemulsion that resembles the lipid structure of LDL, rates of cholesterol esterification, nanoemulsion removal of the circulation, HDL particle size and lipid transfer from nanoemulsion to HDL. Secondarily, we determine the influence of glycemic control, insulin resistance (IR) and insulinization on lipid metabolism. METHODS: we studied 36 diabetics and 37 non-diabetic controls paired by age, sex and body mass index. Artificial lipid nanoemulsion labeled with radioactive lipids cholesterol ester (CE), cholesterol (CL), phospholipids (PL) and triglycerides (TG) was used for studies. Intravenous infusion of nanoemulsion 14C-CE e 3H-CL was injected in participants and blood was sampled over 24 hours for radioactivity measurement. Circulation lipid removal was calculated through compartmental analysis. Rate of cholesterol esterification was calculated after lipid extraction and separation by thin-layer chromatography. Nanoemulsion was incubated with plasma and radioactivity of lipids 14C-EC, 3H-CL, 14C-PL and 3H-TG transferred to the HDL was quantified after the precipitation of other apoB lipoproteins. The HDL diameter was measured by laser light scattering. The insulin resistance in diabetic patients was measured by formula that estimates the rate of uptake of glucose. RESULTS: glycated hemoglobin was 8,8±1,3 mg/dl and LDL concentrations were lower in diabetic patients (85 ± 22 vs. 98 ± 26 mg / dl), p = 0035. There were no differences between groups regarding rates of cholesterol esterification, lipids transfer from nanoemulsion to HDL and HDL particle size. We found no relationship between the kinetic analyses and HbA1c, blood glucose, measures of IR and dose of insulin. The rate of removal of 14C-EC was faster in diabetics type 1 than controls (0.059 ± 0.022 vs.0.039 ± 0.022 h- 1), p = 0.019. CONCLUSIONS: despite suboptimal glycemic control in diabetics, lipids transfer from nanoemulsion to HDL, rate of cholesterol esterification and removal of 3H-CL are similar to those of non-diabetic individuals. Glycemic control, lipid profile, measures of IR and dose of insulin did not influence lipids transfer and rate of cholesterol esterification. Removal of 14C-EC from diabetic circulation is faster than controls which could justify the 18 lower LDL concentration found in this population. We believe that intensive insulin therapy could explain these findings

A determinação de Intervalos de Referência (IR) é uma árdua tarefa para os laboratórios clínicos, porém indispensável e de fundamental importância para a tomada de decisão médica. Para parâmetros como os lípides séricos, os IR tem sido estabelecidos consensos nacionais ou internacionais definindo limites de decisão (CLSI, 2008). O objetivo deste estudo foi determinar os IR para colesterol total, HDL-colesterol, colesterol não-HDL, LDL- colesterol e triglicérides séricos em crianças e adolescentes saudáveis do município de Cuiabá, capital do estado de Mato Grosso. Trata-se de um estudo transversal, descritivo, realizado em 1.866 crianças e adolescentes saudáveis de creches e escolas municipais desta capital, obtidas por amostragem aleatória. Foi aplicado um questionário avaliando antecedentes do indivíduo e de seus familiares além de dados demográficos e antropométricos. Foram definidos como critérios de inclusão do estudo, crianças e adolescentes nas faixas etárias de 1 a 12 anos 11 meses e 29 dias, sem nenhuma doença de base diagnosticada ou queixas clínicas no momento da coleta. Além disto, os participantes não deveriam fazer uso regular de medicamento. As amostras foram coletadas em jejum. As amostras foram processadas no equipamento cobas® 6000 (analyser series - Roche Diagnostics), em laboratório da rede DASA na cidade de Cuiabá. Em relação à metodologia estatística, foi analisada a homogeneidade das variâncias através do teste de Bartlett para cada analito por idade e, posteriormente, testados pelo teste ANOVA ou Kruskal-Wallis para verificar a existência de diferença entre as faixas etárias. O teste \"post hoc\" de Bonferroni foi aplicado quando se constatou a diferença para reagrupar as faixas etárias similares, constituindo assim novos grupos etários. Aplicou-se então, o teste de Bartlett e, conforme seu resultado, realizou-se ANOVA ou Kruskal-Wallis para verificar se os agrupamentos nas faixas se mantinham. Em seguida, procedeu-se a exclusão de valores extremos (outiliers) tomados como sendo aqueles valores acima ou abaixo da média ± 3 desvios-padrão. Depois de excluídos os outiliers, obteve-se o IR como sendo a média ± 2 desvios-padrão dos valores remanescentes. Adicionalmente, foi calculada a distribuição em percentis, sendo adotado o critério do NHLBI 2012 como proposta de limite de decisão para a população estudada. Em todos os testes, o nível de significância adotado foi de 5%. As análises estatísticas foram realizadas pelos programas MINITAB (versão 15) e SPSS (versão 16). Este projeto foi aprovado pelas Comitês de Ética em Pesquisa das instituições envolvidas e das Secretarias Municipais de Educação e Saúde de Cuiabá. Os valores encontrados para o colesterol total, nos percentis 75 e 95 foram: 1 a 2 anos de 160 mg/dL e 189 mg/dL; 3 a 8 anos de 170 mg/dL e 199 mg/dL; 9 a 12 anos de 176 mg/dL e 205 mg/dL, respectivamente. Para o colesterol não-HDL, na única faixa etária de 1 a 12 anos, os valores nestes percentis foram de 122 mg/dL e 150 mg/dL, respectivamente. Para o LDL-colesterol, os valores correspondentes aos percentis acima, na faixa etária de 1 a 8 anos e de 9 a 12 anos, foram de 104 mg/dL e 132 mg/dL; 106 mg/dL e 139 mg/dL, respectivamente. Para os triglicérides, os valores correspondentes aos referidos percentis foram: 1 ano de 127 mg/dL e 189 mg/dL; 2 a 5 anos de 98 a 139 mg/dL; 6 a 12 anos de 92 mg/dL e 139 mg/dL. Para as faixas etárias propostas para o HDL-colesterol os valores correspondentes ao percentil 10 foram: 1 ano de 24 mg/dL; 2 anos de 28 mg/dL; 3 anos de 32 mg/dL e de 4 a 12 anos foi de 36 mg/dL. Os valores dos parâmetros aqui avaliados, definidos em diferentes faixas etárias em crianças e adolescentes brasileiros da cidade de Cuiabá, podem representar limites de decisão para a população pediátrica brasileira contribuindo para aprimorar o diagnóstico neste grupo específico em nosso país / Establishment of Reference Intervals (RI) is an arduous task for clinical laboratories however vital and fundamental importance to medical decision making. For some parameters, such as serum lipids, RI have been established by national and international consensus defining decision limits (CLSI 2008). The aim of this study was to determine pediatric RI of total cholesterol, HDL-cholesterol, cholesterol non- HDL, LDL-cholesterol and triglycerides in healthy children and adolescents in Cuiabá, capital of Mato Grosso. This is a descriptive study, conducted in 1,866 healthy children and adolescents from kindergartens and schools of the capital city, obtained by random sampling. A questionnaire assessing the individual background and their relatives besides demographic and anthropometric data had beeb also carried out. Were defined as inclusion criteria of the study, children and adolescents in the group 1-12 years 11 months and 29 days without any underlying disease or diagnosed clinical complaints at the time of collection. In addition, participants should not take any regular medication. The samples were collected during fasting period and were determined using cobas® 6000 (analyser series - Roche Diagnostics). Regarding statistic methodology, we did analyse the homogeneity of variances by Bartlett´s test for each parameter by age and subsequently by ANOVA or Kruskal-Wallis test to check the differences between age groups. The test \"pos hoc\" Bonferroni was applied when it was found the difference to regroup similar age groups, thus constituting a new age bracket. After this procedure the Bartllet test was applied, as it result, we did conduct ANOVA or Kruskal-Wallis to check if the groups remained. Then proceeded to the exclusion of extreme values (outliers) taken as those values above or below the mean ± 3 standard deviations. After excluding outliers, obtained the RI as the mean ± 2 standard deviations of the remaining values. Additionally, we calculated the percentile distribution, and adopted the criteria of NHLBI 2012 as proposed decision limit for the population studied. In all tests, the significance level was 5%. Statistical analyzes were performed by Minitab software (version 15) and SPSS (version 16). The project was approved by the Research Ethics Committees of the institutions involved and Municipal Departments of Education and Health of Cuiabá city The values obtained for total cholesterol, 75 and 95 percentiles, were: 1 to 2 years, 160 mg/dL and 189 mg/dL; 3 to 8 years, 170 mg/dL and 199 mg/dL; 9 to 12 years, 176 mg/dL and 205 mg/dL, respectively. For the non-HDL cholesterol, the only age group 1 to 12 years, this percentiles values were 122 mg/dL and 150 mg/dL, respectively. For the LDLcholesterol, the values corresponding to the percentiles above, aged 1 to 8 years and 9 to 12 years, were 104 mg/dL and 132 mg/dL; 106 mg/dL and 139 mg/dL, respectively. For the triglycerides, the values corresponding to these percentiles were: 1 year, 127 mg/dL and 189 mg/dL; 2 to 5 years, 98 to 139 mg/dL; 6 to 12 years, 92 mg/dL and 139 mg/dL. For ages proposed for HDL-cholesterol the corresponding values to 10th percentile were: 1 year, 24 mg/dL; 2 years, 28 mg/dL; 3 years, 32 mg/dL and 4 to 12 years were 36 mg/dL. The values of the parameters evaluated here, defined in different age groups in Brazilian children and adolescents in the city of Cuiabá, can represent decision limits for the Brazilian pediatric population contributing to improve the diagnosis in this particular group in our country

OBJETIVO: A via metabólica da lipoproteína de baixa densidade (LDL) em pacientes com lúpus eritematoso sistêmico (LES) em uso de difosfato de cloroquina (DFC) foi avaliada através do comportamento cinético de uma nanoemulsão radioativa rica em colesterol (LDE) que se assemelha à estrutura lipídica da LDL. MÉTODOS: LDE foi marcada com 14C-colesterol éster (14C-CE), sendo a seguir injetada endovenosamente em pacientes do sexo feminino com LES inativo: 10 tomando DFC (grupo DFC), 10 sem tratamento (grupo SEM TRATAMENTO); e 10 mulheres normais (grupo CONTROLE). Os grupos foram pareados pela idade e seguiram rigorosos critérios de seleção de condições que pudessem interferir no perfil lipídico. Amostras de sangue foram coletadas em intervalos pré-estabelecidos após a infusão para mensuração da radioatividade. Níveis séricos de jejum de lipoproteínas foram determinados no início dos estudos cinéticos. RESULTADOS: Idade e índice de massa corpórea (IMC) foram similares nos grupos estudados. A taxa fracional de remoção (TFR) de 14C-CE foi significativamente maior no grupo DFC comparada ao grupo SEM TRATAMENTO (0,076 ± 0,037 vs. 0,046 ± 0,021 h-1; p < 0,05) e CONTROLE (0,0516 ± 0,0125 h-1; p < 0,05). Em concordância, níveis significativamente menores de colesterol total e LDL foram observados no grupo DFC (156 ± 16 e 88 ± 16 mg/dl) comparando-se com SEM TRATAMENTO (174 ± 15 e 108 ± 17 mg/dl; p < 0,05) e CONTROLE (200 ± 24 e 118 ± 23 mg/dl; p < 0,05). Além disso, o incremento em 50% na TFR de 14C-CE no grupo DFC foi acompanhado por uma redução em 20% no LDL-C comparando-se a SEM TRATAMENTO. CONCLUSÃO: Esta é a primeira demonstração in vivo que a remoção de LDE do plasma encontra-se aumentada em pacientes com LES em uso de DFC. Estes dados suportam o benefício desta droga no tratamento do LES e identificam o receptor de LDL como um mecanismo promissor de DFC na redução de lípides em pacientes tomando corticosteróides. / OBJECTIVE: Low-density lipoprotein (LDL) pathway in systhemic lupus erythematosus (SLE) patients taking chloroquine diphosphate (CDP) was evaluated through the kinetic behavior of a radioactive cholesterol-rich nanoemulsion (LDE) that resembles the LDL lipidic structure. METHODS: LDE was labeled with 14C-cholesteryl ester (14C-CE), then IV injected in inactive female SLE patients: 10 taking CDP (CDP), 10 without therapy (NO THERAPY); and 10 normal subjects (CONTROL). Groups were age-matched and followed rigorous selection criteria of conditions that interfere in the lipid profile. Blood samples were collected in pre-established intervals after infusion for radioactivity measurement. Fasting lipoproteins were determined in the beginning of kinetic studies. RESULTS: Age and body mass index (BMI) were similar in the studied groups. Fractional clearance rate (FCR) of 14C-CE was significantly greater in CDP compared to NO THERAPY (0.076 ± 0.037 vs. 0.046 ± 0.021 h-1; p < 0.05) and CONTROL (0.0516 ± 0.0125 h-1; p < 0.05). Accordingly, a significant lower total and LDL cholesterol were observed in CDP (156 ± 16 and 88 ± 16 mg/dl) compared to NO THERAPY (174 ± 15 and 108 ± 17 mg/dl; p<0.05) and CONTROL (200 ± 24 and 118 ± 23 mg/dl; p < 0.05). Moreover, the 50% increase in 14C-CE FCR in CDP was paralleled by 20% decrease in LDL-c compared to NO THERAPY. CONCLUSION: This is the first in vivo demonstration that removal of LDE from plasma was increased in SLE patients taking CDP. These data support its beneficial use in SLE and identify the LDL receptor as a promising CDP mechanism for lowering lipids in patients taking corticosteroids.

OBJETIVO: A via metabólica da lipoproteína de baixa densidade (LDL) em pacientes com lúpus eritematoso sistêmico (LES) em uso de difosfato de cloroquina (DFC) foi avaliada através do comportamento cinético de uma nanoemulsão radioativa rica em colesterol (LDE) que se assemelha à estrutura lipídica da LDL. MÉTODOS: LDE foi marcada com 14C-colesterol éster (14C-CE), sendo a seguir injetada endovenosamente em pacientes do sexo feminino com LES inativo: 10 tomando DFC (grupo DFC), 10 sem tratamento (grupo SEM TRATAMENTO); e 10 mulheres normais (grupo CONTROLE). Os grupos foram pareados pela idade e seguiram rigorosos critérios de seleção de condições que pudessem interferir no perfil lipídico. Amostras de sangue foram coletadas em intervalos pré-estabelecidos após a infusão para mensuração da radioatividade. Níveis séricos de jejum de lipoproteínas foram determinados no início dos estudos cinéticos. RESULTADOS: Idade e índice de massa corpórea (IMC) foram similares nos grupos estudados. A taxa fracional de remoção (TFR) de 14C-CE foi significativamente maior no grupo DFC comparada ao grupo SEM TRATAMENTO (0,076 ± 0,037 vs. 0,046 ± 0,021 h-1; p < 0,05) e CONTROLE (0,0516 ± 0,0125 h-1; p < 0,05). Em concordância, níveis significativamente menores de colesterol total e LDL foram observados no grupo DFC (156 ± 16 e 88 ± 16 mg/dl) comparando-se com SEM TRATAMENTO (174 ± 15 e 108 ± 17 mg/dl; p < 0,05) e CONTROLE (200 ± 24 e 118 ± 23 mg/dl; p < 0,05). Além disso, o incremento em 50% na TFR de 14C-CE no grupo DFC foi acompanhado por uma redução em 20% no LDL-C comparando-se a SEM TRATAMENTO. CONCLUSÃO: Esta é a primeira demonstração in vivo que a remoção de LDE do plasma encontra-se aumentada em pacientes com LES em uso de DFC. Estes dados suportam o benefício desta droga no tratamento do LES e identificam o receptor de LDL como um mecanismo promissor de DFC na redução de lípides em pacientes tomando corticosteróides. / OBJECTIVE: Low-density lipoprotein (LDL) pathway in systhemic lupus erythematosus (SLE) patients taking chloroquine diphosphate (CDP) was evaluated through the kinetic behavior of a radioactive cholesterol-rich nanoemulsion (LDE) that resembles the LDL lipidic structure. METHODS: LDE was labeled with 14C-cholesteryl ester (14C-CE), then IV injected in inactive female SLE patients: 10 taking CDP (CDP), 10 without therapy (NO THERAPY); and 10 normal subjects (CONTROL). Groups were age-matched and followed rigorous selection criteria of conditions that interfere in the lipid profile. Blood samples were collected in pre-established intervals after infusion for radioactivity measurement. Fasting lipoproteins were determined in the beginning of kinetic studies. RESULTS: Age and body mass index (BMI) were similar in the studied groups. Fractional clearance rate (FCR) of 14C-CE was significantly greater in CDP compared to NO THERAPY (0.076 ± 0.037 vs. 0.046 ± 0.021 h-1; p < 0.05) and CONTROL (0.0516 ± 0.0125 h-1; p < 0.05). Accordingly, a significant lower total and LDL cholesterol were observed in CDP (156 ± 16 and 88 ± 16 mg/dl) compared to NO THERAPY (174 ± 15 and 108 ± 17 mg/dl; p<0.05) and CONTROL (200 ± 24 and 118 ± 23 mg/dl; p < 0.05). Moreover, the 50% increase in 14C-CE FCR in CDP was paralleled by 20% decrease in LDL-c compared to NO THERAPY. CONCLUSION: This is the first in vivo demonstration that removal of LDE from plasma was increased in SLE patients taking CDP. These data support its beneficial use in SLE and identify the LDL receptor as a promising CDP mechanism for lowering lipids in patients taking corticosteroids.

A determinação de Intervalos de Referência (IR) é uma árdua tarefa para os laboratórios clínicos, porém indispensável e de fundamental importância para a tomada de decisão médica. Para parâmetros como os lípides séricos, os IR tem sido estabelecidos consensos nacionais ou internacionais definindo limites de decisão (CLSI, 2008). O objetivo deste estudo foi determinar os IR para colesterol total, HDL-colesterol, colesterol não-HDL, LDL- colesterol e triglicérides séricos em crianças e adolescentes saudáveis do município de Cuiabá, capital do estado de Mato Grosso. Trata-se de um estudo transversal, descritivo, realizado em 1.866 crianças e adolescentes saudáveis de creches e escolas municipais desta capital, obtidas por amostragem aleatória. Foi aplicado um questionário avaliando antecedentes do indivíduo e de seus familiares além de dados demográficos e antropométricos. Foram definidos como critérios de inclusão do estudo, crianças e adolescentes nas faixas etárias de 1 a 12 anos 11 meses e 29 dias, sem nenhuma doença de base diagnosticada ou queixas clínicas no momento da coleta. Além disto, os participantes não deveriam fazer uso regular de medicamento. As amostras foram coletadas em jejum. As amostras foram processadas no equipamento cobas® 6000 (analyser series - Roche Diagnostics), em laboratório da rede DASA na cidade de Cuiabá. Em relação à metodologia estatística, foi analisada a homogeneidade das variâncias através do teste de Bartlett para cada analito por idade e, posteriormente, testados pelo teste ANOVA ou Kruskal-Wallis para verificar a existência de diferença entre as faixas etárias. O teste \"post hoc\" de Bonferroni foi aplicado quando se constatou a diferença para reagrupar as faixas etárias similares, constituindo assim novos grupos etários. Aplicou-se então, o teste de Bartlett e, conforme seu resultado, realizou-se ANOVA ou Kruskal-Wallis para verificar se os agrupamentos nas faixas se mantinham. Em seguida, procedeu-se a exclusão de valores extremos (outiliers) tomados como sendo aqueles valores acima ou abaixo da média ± 3 desvios-padrão. Depois de excluídos os outiliers, obteve-se o IR como sendo a média ± 2 desvios-padrão dos valores remanescentes. Adicionalmente, foi calculada a distribuição em percentis, sendo adotado o critério do NHLBI 2012 como proposta de limite de decisão para a população estudada. Em todos os testes, o nível de significância adotado foi de 5%. As análises estatísticas foram realizadas pelos programas MINITAB (versão 15) e SPSS (versão 16). Este projeto foi aprovado pelas Comitês de Ética em Pesquisa das instituições envolvidas e das Secretarias Municipais de Educação e Saúde de Cuiabá. Os valores encontrados para o colesterol total, nos percentis 75 e 95 foram: 1 a 2 anos de 160 mg/dL e 189 mg/dL; 3 a 8 anos de 170 mg/dL e 199 mg/dL; 9 a 12 anos de 176 mg/dL e 205 mg/dL, respectivamente. Para o colesterol não-HDL, na única faixa etária de 1 a 12 anos, os valores nestes percentis foram de 122 mg/dL e 150 mg/dL, respectivamente. Para o LDL-colesterol, os valores correspondentes aos percentis acima, na faixa etária de 1 a 8 anos e de 9 a 12 anos, foram de 104 mg/dL e 132 mg/dL; 106 mg/dL e 139 mg/dL, respectivamente. Para os triglicérides, os valores correspondentes aos referidos percentis foram: 1 ano de 127 mg/dL e 189 mg/dL; 2 a 5 anos de 98 a 139 mg/dL; 6 a 12 anos de 92 mg/dL e 139 mg/dL. Para as faixas etárias propostas para o HDL-colesterol os valores correspondentes ao percentil 10 foram: 1 ano de 24 mg/dL; 2 anos de 28 mg/dL; 3 anos de 32 mg/dL e de 4 a 12 anos foi de 36 mg/dL. Os valores dos parâmetros aqui avaliados, definidos em diferentes faixas etárias em crianças e adolescentes brasileiros da cidade de Cuiabá, podem representar limites de decisão para a população pediátrica brasileira contribuindo para aprimorar o diagnóstico neste grupo específico em nosso país / Establishment of Reference Intervals (RI) is an arduous task for clinical laboratories however vital and fundamental importance to medical decision making. For some parameters, such as serum lipids, RI have been established by national and international consensus defining decision limits (CLSI 2008). The aim of this study was to determine pediatric RI of total cholesterol, HDL-cholesterol, cholesterol non- HDL, LDL-cholesterol and triglycerides in healthy children and adolescents in Cuiabá, capital of Mato Grosso. This is a descriptive study, conducted in 1,866 healthy children and adolescents from kindergartens and schools of the capital city, obtained by random sampling. A questionnaire assessing the individual background and their relatives besides demographic and anthropometric data had beeb also carried out. Were defined as inclusion criteria of the study, children and adolescents in the group 1-12 years 11 months and 29 days without any underlying disease or diagnosed clinical complaints at the time of collection. In addition, participants should not take any regular medication. The samples were collected during fasting period and were determined using cobas® 6000 (analyser series - Roche Diagnostics). Regarding statistic methodology, we did analyse the homogeneity of variances by Bartlett´s test for each parameter by age and subsequently by ANOVA or Kruskal-Wallis test to check the differences between age groups. The test \"pos hoc\" Bonferroni was applied when it was found the difference to regroup similar age groups, thus constituting a new age bracket. After this procedure the Bartllet test was applied, as it result, we did conduct ANOVA or Kruskal-Wallis to check if the groups remained. Then proceeded to the exclusion of extreme values (outliers) taken as those values above or below the mean ± 3 standard deviations. After excluding outliers, obtained the RI as the mean ± 2 standard deviations of the remaining values. Additionally, we calculated the percentile distribution, and adopted the criteria of NHLBI 2012 as proposed decision limit for the population studied. In all tests, the significance level was 5%. Statistical analyzes were performed by Minitab software (version 15) and SPSS (version 16). The project was approved by the Research Ethics Committees of the institutions involved and Municipal Departments of Education and Health of Cuiabá city The values obtained for total cholesterol, 75 and 95 percentiles, were: 1 to 2 years, 160 mg/dL and 189 mg/dL; 3 to 8 years, 170 mg/dL and 199 mg/dL; 9 to 12 years, 176 mg/dL and 205 mg/dL, respectively. For the non-HDL cholesterol, the only age group 1 to 12 years, this percentiles values were 122 mg/dL and 150 mg/dL, respectively. For the LDLcholesterol, the values corresponding to the percentiles above, aged 1 to 8 years and 9 to 12 years, were 104 mg/dL and 132 mg/dL; 106 mg/dL and 139 mg/dL, respectively. For the triglycerides, the values corresponding to these percentiles were: 1 year, 127 mg/dL and 189 mg/dL; 2 to 5 years, 98 to 139 mg/dL; 6 to 12 years, 92 mg/dL and 139 mg/dL. For ages proposed for HDL-cholesterol the corresponding values to 10th percentile were: 1 year, 24 mg/dL; 2 years, 28 mg/dL; 3 years, 32 mg/dL and 4 to 12 years were 36 mg/dL. The values of the parameters evaluated here, defined in different age groups in Brazilian children and adolescents in the city of Cuiabá, can represent decision limits for the Brazilian pediatric population contributing to improve the diagnosis in this particular group in our country