HDL Fraction Research Articles

Metabolomic signature identifies HDL and apolipoproteins as potential biomarker for systemic sclerosis with interstitial lung disease

BackgroundHigh-density lipoproteins (HDL) affect endothelial functions such as the expression of endothelial cell adhesion molecules and exert anti-apoptotic/-thrombotic functionalities. Therefore, profound analysis of lipoproteins may unveil biomarkers for (micro-)vasculopathy in systemic sclerosis (SSc) and mortality determining disease manifestations like interstitial lung disease (SSc-ILD). Because nuclear magnetic resonance (NMR) spectroscopy provides a wide range of lipoprotein parameters beyond the capabilities of classical analyses it has been used herein to examine lipoprotein profiles in SSc. MethodsTo detect the metabolic and lipidomic profile serum samples from clinically well-characterized SSc patients (n = 100) and age-and sex-matched healthy controls (n = 40) were analyzed by 1H NMR spectroscopy using Bruker's in-vitro diagnostic research (IVDr) protocol. Statistical analyses were performed to validate significant findings and to search for associations between lipoproteins and clinical phenotypes. ResultsPatients with SSc-ILD and lung fibrosis displayed reduced HDL levels. Furthermore, a reduction in apolipoprotein A1 + A2 and its HDL fractions reflected a distinct lipoprotein profile for SSc-ILD patients. This association was independent of potential clinical confounders for dyslipidemia. Notably, in SSc-ILD HDL levels correlate with FVC (forced vital capacity), DLCO (diffusion capacity of the lungs for carbon monoxide), and the modified Rodnan-Skin-Score. ConclusionThese results suggest HDL and its lipoproteins may be considered as potential new biomarkers for SSc-ILD. Immune-mediated HDL effects on the endothelium facilitate microvasculopathy - one of the pathophysiological hallmarks in SSc. Therefore, a closer prospective evaluation of the capability of HDL-determination and its lipoproteins regarding a more individualized evaluation of SSc-ILD is warranted.

Triglyceride-rich lipoprotein, remnant cholesterol, and apolipoproteins CII, CIII, and E in patients with schizophrenia

Patients with schizophrenia show a disproportionally increased risk of cardiovascular disease. Hypertriglyceridemia is prevalent in this population; however, how this relates to levels of remnant cholesterol, triglyceride (TG)-rich lipoprotein (TRL) particle size and composition, TG turnover, and apolipoprotein (apo) and angiopoietin-like protein (ANGPTL) concentrations is unknown. Fasting levels of cholesterol (total [TC], LDL-C, HDL-C, non-HDL-C and remnant cholesterol) and TG were determined in 110 patients diagnosed with schizophrenia, and 46 healthy controls. TRL particle size, concentration and composition, and β-hydroxybutyrate (TG turnover marker) were assessed by NMR. Levels of apoCII, apoCIII, apoE, ANGPTL3, ANGPTL4, and ANGPTL8 were measured by ELISA, and apoCII, apoCIII and apoE were further evaluated in HDL and non-HDL fractions. Patients with schizophrenia had significantly elevated TG, TG:apoB ratio, non-HDL-C, remnant cholesterol, non-HDL-apoCII and non-HDL-apoCIII, and HDL-apoE (all P < 0.05), lower HDL-C and apoA-I (all P < 0.001), and comparable apoB, TC, TC:apoB ratio, LDL-C, β-hydroxybutyrate, ANGPTL3, ANGPTL4 and ANGPTL8 to healthy controls. Patients had a 12.0- and 2.5-fold increase in the concentration of large and medium TRL particles respectively, but similar cholesterol:TG ratio within each particle. Plasma TG, remnant cholesterol, and large and medium TRL particle concentrations correlated strongly with apoCII, apoCIII, and apoE in the non-HDL fraction, and with apoCIII and apoE in the HDL fraction in patients with schizophrenia. Differences in TG, HDL-C, TRL particle concentrations, apoCIII, and apoE persisted after adjustment for conventional risk factors. These results are consistent with impaired TRL lipolysis and clearance in patients with schizophrenia which may be responsive to targeting apoCIII.

Apo Lipoproteins A1 and B in Multidrug-Resistant Tuberculosis (MDR-TB)

Aims: The medical management of multidrug-resistant tuberculosis (MDR-TB) has become a major public health issue. Apolipoproteins play a key role in lipoprotein metabolism such as the recognition of receptors involved in lipoprotein metabolism. Thus, the study of the inter- relationships between apolipoproteins (A1 and B) and MDR-TB could represent an important approach to the biological management of MDR-TB patients. Methodology: This is an experimental study carried out on eighty-two (82) patients including thirty-eight (38) MDR-TB patients which age ranged from 18 to 60 years old recruited from three tuberculosis centers (CAT) in the city of Abidjan and forty-four (44) non-tuberculosis patients used as control aged 18 to 60 years old recruited at the National Blood Transfusion Center (CNTS) in Treichville (Abidjan, Côte d’Ivoire). Total cholesterol, HDL-cholesterol and triglycerides were measured by the colorimetric-enzymatic method. Apolipoproteins A1 and B were measured using the immunoturbidimetric method. Results and Conclusion: Showed a dyslipidemia concerning cholesterol and its HDL fraction, triglycerides and apolipoproteins A1 and B suggest an atherogenic profile in multidrug-resistant TB patients.

Are thyroid hormone levels and sarcopenia in elderly patients associated with type 2 diabetes mellitus and euthyroidism?

Aim to study the relationship between thyroid function parameters (free T3 (FT3), free T4 (FT4), FT3/FT4 ratio), thyroid-stimulating hormone (TSH) and parameters characterizing sarcopenia (muscle mass and strength, physical performance) in patients with type 2 diabetes mellitus (T2DM) and euthyroidism.&#x0D; Material and methods. The study included 312 elderly patients with T2DM in a state of euthyroidism (mean age 65.39 2.28 years). In all participants the following parameters were measured: anthropometric parameters, thyroid hormones (FT3, FT4), TSH, fasting blood glucose, glycated hemoglobin (HbA1c), fasting C-peptide and insulin, lipid profile, body composition, handgrip strength, walking speed.&#x0D; Results. Sarcopenia was prevalent in 26.9% of T2DM patients with euthyroidism. A binary logistic regression analysis adjusted for age, gender, smoking and alcohol consumption, T2DM duration, hypertension, HbA1c level, eGFR, TG, HDL and fat mass fraction showed that the high-normal FT3 level (OR = 0.522, 95% CI: 0.3040.895, p = 0.018), a low-normal FT4 level (OR = 1.126, 95% CI: 1.0091.258, p = 0.034) and an increased FT3/FT4 ratio (OR = 0.923, 95% CI: 0.8790.969, p = 0.001) were associated with a low prevalence of sarcopenia. FT3 concentration was positively associated with muscle strength (OR = 0.525, 95% CI: 0.3050.902, p = 0.020) and walking speed (OR = 0.443, 95% CI: 0.2590.758, p = 0.003), while FT4 concentration was negatively associated with muscle mass (OR = 1.114, 95% CI: 1.0091.232, p = 0.036). The FT3/FT4 ratio was positively associated with muscle mass (OR = 0.943, 95% CI: 0.9050.981, p = 0.006), muscle strength (OR = 0.945, 95% CI: 0.9010.992, p = 0.021), and walking speed (OR = 0.934, 95% CI: 0.8940.975, p = 0.002). TSH levels were not associated with sarcopenia.&#x0D; Conclusion. The high FT3/FT4 ratio is significantly associated with a low risk of sarcopenia in T2DM patients with euthyroidism.

B-003 Decrease in Serum apoE-rich HDL-cholesterol May Be Associated With Cholesterol Accumulation in the Liver of Rats Fed A High-cholesterol Diet

Abstract Background Free cholesterol accumulation in the liver has been suggested to be one of risk factors for the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis. Therefore, a non-invasive approach to assess hepatic cholesterol accumulation in the early stage without elevation of liver enzymes (AST and ALT) may be useful in clinical setting. In this study, we show that serum apoE-rich HDL-cholesterol level is a possible indicator for predicting hepatic cholesterol accumulation in rats with diet-induced simple liver steatosis. Methods Male Sprague-Dawley rats (8 weeks) were purchased from Charles River Laboratories. Four different diets were obtained from Oriental Yeast; control diet (MF), high-fat diet (HF; 68% MF, 30% palm oil, and 2% cholic acid), high-cholesterol diet (HC; 95.5% MF, 2.5% cholesterol, and 2% cholic acid), and high-fat/high-cholesterol diet (HFC; 81.75% MF, 15% palm oil, 1.25% cholesterol, and 2% cholic acid). The rats were randomly divided into 4 groups and fed as follows. The control and HFC groups received the MF and HFC diets for 8 weeks, respectively. The HF/HC group received the HF diet for the first 4 weeks and the HC diet for the next 4 weeks. The HC/HF group received the HC diet first, then the HF diet. Blood and liver tissue specimens were collected under anesthesia. Total HDL fractions were isolated from whole sera by the polyethylene glycol precipitation method, and applied into a cation-exchange column (HiTrap SPHP, GE healthcare) for determining apoE-rich HDL-cholesterol levels. Correlations between variables were assessed with Spearman’s correlation coefficient (rs). Results Total cholesterol content of the liver was significantly higher in the HFC (11.1-fold, P &amp;lt; 0.001), HF/HC (9.1-fold, P &amp;lt; 0.001), and HC/HF (5.8-fold, P &amp;lt; 0.001) groups than the control group. Hepatic free cholesterol was also higher in the HFC (5.5-fold, P &amp;lt; 0.001), HF/HC (3.9-fold, P &amp;lt; 0.001), and HC/HF (3.3-fold, P &amp;lt; 0.001) groups. Hepatic triglycerides were slightly increased in the HC/HF (1.6-fold, P &amp;lt; 0.01) and HFC (1.5-fold, P &amp;lt; 0.05) groups. Serum apoE-rich HDL-cholesterol was significantly decreased in the HF/HC (0.21-fold, P &amp;lt; 0.001), HC/HF (0.25-fold, P &amp;lt; 0.001) and HFC (0.42-fold, P &amp;lt; 0.01) groups. The HFC group had significantly higher serum ALT (3.0-fold, P &amp;lt; 0.05) and AST (2.3-fold, P &amp;lt; 0.01) levels, compared with the control groups. There was a significant increase in hepatic mRNA expression of TNF-α (24.8-fold, P &amp;lt; 0.01) and TGF-β (3.8-fold, P &amp;lt; 0.001) only in the HFC group, indicating liver inflammation and stellate cell activation. To exclude the effects of hepatocyte injury on apoE-rich HDL metabolism, rats with ALT less than 100 U/mL (n = 16) were included in the following analysis. ApoE-rich HDL-cholesterol levels were significantly and inversely correlated with hepatic total cholesterol (rs = −0.535, P &amp;lt; 0.05) and free cholesterol (rs = −0.532, P &amp;lt; 0.05) levels but not with hepatic triglycerides. Furthermore, the ratio of ALT to apoE-rich HDL-cholesterol was more highly correlated with hepatic total cholesterol (rs = 0.626, P &amp;lt; 0.01) and free cholesterol (rs = 0.632, P &amp;lt; 0.01). There was no such correlation for apoE-poor HDL-cholesterol. Conclusion: The present observations suggest that serum apoE-rich HDL-cholesterol levels may be a useful indicator for cholesterol accumulation in the liver of cholesterol-fed rats.

Diet and physical activity and metabolic disorders in patients with schizophrenia and bipolar affective disorder in the Polish population.

There are numerous reports of a higher prevalence of metabolic disorders in patients with schizophrenia and bipolar disorder (BD), yet its connections to diet and physical activity remain not fully explained. This article aimed to evaluate diet, physical activity and selected biochemical and anthropometric parameters associated with metabolism in patients with schizophrenia and BD and to analyse the relationships between these variables in the subjects. A total of 126 adults participated in the study: 47 patients with schizophrenia, 54 patients with BD and 25 patients in mental illness remission (reference group). Data were collected on the underlying illness and concomitant illnesses, and the severity of symptoms of the current episode was assessed using the following scales: PANSS, MADRS and YMRS. An assessment of the subjects' diet (KomPAN questionnaire) and their physical activity (International Physical Activity Questionnaire) was carried out. Anthropometric and blood pressure measurements were taken and BMI and WHR were calculated. Serum concentrations of fasting glucose, TSH, total cholesterol, LDL and HDL fractions, triglycerides and leptin, ghrelin and resistin were determined. For statistical analysis, the significance level was set at 0.05. For multiple comparisons one way ANOVA or Kruskal Wallis were used with post hoc Tukey and Dunn tests, respectively. To determine correlation of variables, Pearson's linear correlation coefficient or Spearman's rank correlation coefficient were used. A total of 50.8% of the subjects had at least one metabolic disorder-most commonly excessive body weight (66.7%) and abdominal obesity (64.3%). Patients did not differ significantly in terms of physical activity, but they did differ in mean time spent sitting-with this being significantly longer for all groups than in the general population. The subjects differed in diet: patients with BD consumed less unhealthy foods than patients with schizophrenia. The highest correlations between physical activity, diet and variables defining metabolic disorders were found in patients with BD. Only in patients with schizophrenia were there significant correlations between the course of the disease and physical activity. The results suggest the existence of associations between diet, physical activity, and metabolic disorders in both BD and schizophrenia patients. They also suggest a tendency among those patients to spend long periods of time sitting.

Abnormal HDL lipid and protein composition following pediatric cancer treatment: an associative study

BackgroundLong-term childhood cancer survivors (CCS) are at high risk of having dyslipidemia including low high density lipoprotein cholesterol (HDL-C). However, little is known about the prevalence of low HDL-C and the impact of therapy exposure on HDL composition early after treatment is terminated.MethodsThis associative study included 50 children and adolescents who had completed their cancer treatments (< 4 years). Clinical characteristics (demographic, diagnosis, treatment, anthropometric parameters), fasting plasma lipids, apoliporoteins (Apo) A-I and composition of HDL fractions (HDL2 and HDL3) were assessed. Data were stratified according to the presence of dyslipidemia and median doses of therapeutic agents and compared using Fisher exact or Mann–Whitney tests. Univariate binary logistic regression analyses were carried out to evaluate the associations between the clinical and biochemical characteristics and having low HDL-C. Composition of HDL2 and HDL3 particles was assessed in a sub-group of 15 patients and compared to 15 age- and sex-matched healthy controls using Wilcoxon paired test.ResultsOf the 50 pediatric cancer patients included in this study (mean age: 11.30 ± 0.72 y; mean time since end of treatment: 1.47 ± 0.12 y; male: 38%), 8 had low HDL-C (16%), all of which were adolescent at diagnosis. Higher doses of doxorubicin were associated with lower HDL-C and Apo A-I levels. In hypertriglyceridemic patients and compared to normolipidemics, triglycerides (TG) content was greater in HDL2 and HDL3 fractions whereas esterified cholesterol (EC) content was lower in HDL2. Enrich TG content of HDL3 and lower EC of HDL2 was found in patients exposed to ≥ 90 mg/m2 doxorubicin. Factors positively associated with the risk of having low HDL-C were age, being overweight or obese and exposure to doxorubicin ≥ 90 mg/m2. Compared to healthy controls, a sub-group of 15 patients showed higher TG and free cholesterol (FC) content of HDL2 and HDL3 and lower EC content in HDL3.ConclusionsOverall, we found abnormalities in HDL-C and Apo A-I levels and in HDL composition early after pediatric cancer treatment that are influenced by age, overweight or obesity status and exposure to doxorubicin.

THE PLASMA HDL-SUBFRACTION PROFILE IN HYPERTENSIVE PATIENTS WITH HIGH HDL-CHOLESTEROL LEVELS

Objective: The evaluation of cardiovascular risk (CVR) in arterial hypertension includes the estimation of high density lipoprotein (HDL-C). Although HDL-C is considered as a favorite CVR factor, the exact role that HDL-C subfraction profile play in the pathogenesis of atherosclerosis, especially in higher than usual HDL-C levels (&gt;60 mg/dl) is currently under investigation. The aim of this study was to evaluate the plasma HDL subfraction profile of high HDL-C levels in hypertensive patients compared to normotensive subjects. Design and method: We studied 74 hypertensive patients (60 + 13 years, 62 females) and 24 normotensives, apparently healthy subjects (49 + 14 years, 22 females) with HDL-C equal or above 55 mg/dl. Cholesterol, HDL-C, and TG were determined enzymatically while LDL-C was calculated using the Friedewald formula. Plasma HDL particles were separated and determined electrophorically using Lipoprint System HDL Subfractions Kit. Aortic stiffness (c-f PWV) and central BP (cBP) were evaluated by carotid-femoral pulse wave velocity (Complior apparatus). Results: We found that hypertensive patients had increased age, BMI, office systolic and diastolic BP, PWV compared to normotensives. Regarding HDL, 10 HDL fractions were separated: fractions 1-3 were denoted as Large-HDL (L-HDL), fractions 4-7 constituted the Intermediate-HDL (I-HDL), and fractions 8-10 were denoted as Small-HDL (S-HDL) subfraction. Although HDL-C levels were similar between groups (76 + 13 vs. 81 + 17 mg/dl, p = 0.10), the percent distribution (%) of L-HDL was lower in hypertensives compared to normotensives (29.8±4.7% vs. 36.6±6.3%, p&lt;0.001), % I-HDL was similar while % S-HDL was increased (27.3±3.7% vs. 20.4±4.4%, p&lt;0.001). We found a significant negative correlation between L-HDL and SBP (rho = -0.48, p = 0.01) as well as cBP (rho = -0.52, p = 0.008) in younger hypertensive females before menopause (n = 26, 49 + 5 years). Conclusions: L-HDL particles, the most antiatherogenic HDL-C subfraction, were reduced in hypertensive patients with high HDL-C levels compared to normotensive subjects, suggesting that these patients are probably less protected against CV disease compared to normotensive subjects with similar HDL-C and LDL-C levels. Therefore, plasma HDL-subfraction profile might replace the HDL-C evaluation as a better biomarker of CVR in hypertensive patients, especially females before menopause.

Circulating lipoprotein-carried miRNome analysis reveals novel VLDL-enriched microRNAs that strongly correlate with the HDL-microRNA profile

Lipoproteins have been described as microRNAs (miRNAs) carriers. Unfortunately, the bibliography on this topic is scarce and shows a high variability between independent investigations. In addition, the miRNA profiles of the LDL and VLDL fractions have not been completely elucidated. Here, we profiled the human circulating lipoprotein-carried miRNome. Lipoprotein fractions (VLDL, LDL and HDL) were isolated from the serum of healthy subjects by ultracentrifugation and purified by size-exclusion chromatography. A panel of 179 miRNAs commonly expressed in circulation was evaluated in the lipoprotein fractions using quantitative real-time PCR (qPCR) assays. A total of 14, 4 and 24 miRNAs were stably detected in the VLDL, LDL and HDL fractions, respectively. VLDL- and HDL-miRNA signatures were highly correlated (rho 0.814), and miR-16–5p, miR-142–3p, miR-223–3p and miR-451a were among the top 5 expressed miRNAs in both fractions. miR-125a-5p, miR-335–3p and miR-1260a, were detected in all lipoprotein fractions. miR-107 and miR-221–3p were uniquely detected in the VLDL fraction. HDL showed the larger number of specifically detected miRNAs (n = 13). Enrichment in specific miRNA families and genomic clusters was observed for HDL-miRNAs. Two sequence motifs were also detected for this group of miRNAs. Functional enrichment analysis including the miRNA signatures from each lipoprotein fraction suggested a potential role in mechanistic pathways previously associated with cardiovascular disease: fibrosis, senescence, inflammation, immune response, angiogenesis, and cardiomyopathy. Collectively, our results not only support the role of lipoproteins as circulating miRNA carriers but also describe for the first time the role of VLDL as a miRNA transporter.

Postprandial Lipid Response Following a High Fat Meal in Rats Adapted to Dietary Fiber

Rats were adapted to diets containing 5 g/100 g cellulose (CL), 5 g/100 g oat bran fiber (OB) or 5 g/100 g psyllium husk (Psy) for 4 wk. Following a 12-h fast, animals were either killed at 0 h (baseline) or fed 4.5 g of a test meal that provided 50% energy from fat, then killed at 1, 4 or 6 h postprandially. Fasting plasma and HDL cholesterol concentrations were lower in Psy-fed animals than in rats fed either CL or OB. Plasma triglycerides increased significantly from baseline (0 h) in all groups but did not differ among diet treatments. Increases in triglyceride content of the chylomicron/VLDL fraction occurred in the CL- and OB-fed groups and in the HDL fraction of the Psy-fed group during the postprandial period. In unfed animals the hepatic and intestinal levels of apolipoprotein A-IV mRNA were higher in the CL-fed group than in the groups fed OB and Psy. Apolipoprotein B mRNA was higher in the intestine of the OB-fed group than in the groups fed CL and Psy and had a significant gradient along the small intestine, increasing in the distal third. The results suggest that chronic consumption of fiber is less likely to modify the acute plasma triglyceride response to a fat-containing test meal than if a fiber supplement is incorporated into the meal.

First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency

BackgroundFamilial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without effective treatment. Patients with FLD develop severe low HDL, corneal opacity, hemolytic anemia, and renal injury. ObjectiveWe developed genetically modified adipocytes (GMAC) secreting LCAT (LCAT-GMAC) for ex vivo gene therapy. GMACs were prepared from the patient’s adipocytes to express LCAT by retroviral gene transduction to secrete functional enzymes. This study aimed to evaluate the safety and efficacy of LCAT-GMAC implantation in an FLD patient. MethodsProliferative preadipocytes were obtained from a patient using a ceiling culture and retrovirally transduced with LCAT. After obtaining enough cells by expansion culture of the transduced cells, the resulting LCAT-GMACs were implanted into a patient with FLD. To evaluate the safety and efficacy, we analyzed the outcome of the autologous implantation for 24 weeks of observation and subsequent 240 weeks of the follow-up periods. ResultsThis first-in-human autologous implantation of LCAT-GMACs was shown to be safe by evaluating adverse events. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. Consistent with increased LCAT activity, intermediate-density lipoprotein (IDL) and free cholesterol levels of the small and very small HDL fractions decreased. We found the hemoglobin/haptoglobin complex in the hemolyzed pre-implantation sera of the patient. After one week of the implantation, the hemoglobin/haptoglobin complex almost disappeared. Immediately after the implantation, the patient's proteinuria decreased temporarily to mild levels and gradually increased to the baseline. At 48 weeks after implantation, the patient's proteinuria deteriorated with the development of mild hypertension. By the treatment with antihypertensives, the patient's blood pressure normalized. With the normalization of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. ConclusionsLCAT-GMAC implantation in a patient with FLD is shown to be safe and appears to be effective, in part, for treating anemia and proteinuria in FLD.

Molecular Characterization of Plasma HDL, LDL, and VLDL Lipids Cargos from Atherosclerotic Patients with Advanced Carotid Lesions: A Preliminary Report.

Carotid atherosclerosis represents a relevant healthcare problem, since unstable plaques are responsible for approximately 15% of neurologic events, namely transient ischemic attack and stroke. Although statins treatment has proven effective in reducing LDL-cholesterol and the onset of acute clinical events, a residual risk may persist suggesting the need for the detection of reliable molecular markers useful for the identification of patients at higher risk regardless of optimal medical therapy. In this regard, several lines of evidence show a relationship among specific biologically active plasma lipids, atherosclerosis, and acute clinical events. We performed a Selected Reaction Monitoring-based High Performance Liquid Chromatography-tandem Mass Spectrometry (SRM-based HPLC-MS/MS) analysis on plasma HDL, LDL, and VLDL fractions purified, by isopycnic salt gradient ultracentrifugation, from twenty-eight patients undergoing carotid endarterectomy, having either a “hard” or a “soft” plaque, with the aim of characterizing the specific lipidomic patterns associated with features of carotid plaque instability. One hundred and thirty lipid species encompassing different lipid (sub)classes were monitored. Supervised multivariate analysis showed that lipids belonging to phosphatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG) classes mostly contribute to discrimination within each lipoprotein fraction according to the plaque typology. Differential analysis evidenced a significant dysregulation of LDL PE (38:6), SM (32:1), and SM (32:2) between the two groups of patients (adj. p-value threshold = 0.05 and log2FC ≥ |0.58|). Using this approach, some LDL-associated markers of plaque vulnerability have been identified, in line with the current knowledge of the key roles of these phospholipids in lipoprotein metabolism and cardiovascular disease. This proof-of-concept study reports promising results, showing that lipoprotein lipidomics may present a valuable approach for identifying new biomarkers of potential clinical relevance.

Abstract 113: Statins, But Not Pcsk9 Inhibitors, Reduce The Adipokine Chemerin In Familial Hypercholesterolemia: Focus On Lipoprotein Subfractions.

Background: Familial hypercholesterolemia (FH) is characterized by severe elevations in circulating LDL-cholesterol, and an increase in the risk of dyslipidemia-related cardiovascular disease (CVD). Chemerin, as a newly identified adipokine, is considered as an additional risk factor for CVD. Here we investigated whether it can be modified by cholesterol-lowering therapy. Methods: Lipoprotein subfractions were isolated by density gradient ultracentrifugation. Lipids and chemerin concentrations were determined both before and after cholesterol lowering with either a statin (atorvastatin, simvastatin, rosuvastatin, or fluvastatin) or a PCSK9 inhibitor (PCSK9i; alirocumab or evolocumab). Results: At baseline chemerin levels were 113±46 (statin group) and 95±44 (PCSK9i group) ng/ml, while triglyceride (TG) levels were 1.9±1.6 and 2.6±1.7 mmol/ml, high-density lipoprotein cholesterol (HDL-c) levels were 1.3±0.4 and 1.2±0.4 mmol/ml, and low-density lipoprotein cholesterol (LDL-c) levels were 5.7±1.5 and 4.9±1.4 mmol/ml (P=ns for difference between 2 groups). Chemerin correlated positively with triglycerides (r=0.45, P&lt;0.005) and negatively with HDL-c (r=-0.33, P&lt;0.01). Both statins and PCSK9i reduced LDL-c (by 41 and 62%, P&lt;0.0001), triglycerides (by 13 and 19%, P&lt;0.01), and increased HDL-c (by 8 and 23%, P&lt;0.01), but only statins additionally reduced chemerin (by 35%, P&lt;0.005). The lipoprotein subfraction profile revealed that chemerin accumulated particularly in the HDL3 fraction (containing &gt;60% of all chemerin in lipoprotein subfractions), with approximately 30% being present in the HDL2 fraction, and approximately 3% in the LDL fraction. Statins reduced HDL3-c and HDL3-TG, and the level of chemerin bound to all subfractions. PCSK9i reduced HDL3-c but did not affect HDL3-TG or the level of chemerin bound to HDL3 and HDL2. Conclusions: Circulating chemerin occurs in different lipoprotein subfractions, accumulating particularly in the HDL3 fraction. Statins, but not PCSK9i, lowers chemerin, possibly by interfering with its levels across lipoprotein subfractions. This may represent a novel cardiovascular protective function of statins.

Profiling the serum proteome during Schistosoma mansoni infection in the BALB/c mice: A focus on the altered lipid metabolism as a key modulator of host-parasite interactions.

Schistosomiasis represents a condition in which every aspect of the disease, starting from skin invasion of the cercariae to egg laying by adult worms, incites a tissue response from the vertebrate host. This response, whether acute or chronic, leads to the appearance of reporter molecules of tissue injury in bodily fluids that could be surveyed as markers for disease diagnosis, status and prognosis. In this scenario, the serum proteome associated with a schistosome infection remains poorly explored; particularly by the use of high-throughput mass spectrometric instrumentation. In this study, we aimed to comparatively examine the serum proteome of control versus infected BALB/c mice, spanning the interval between the onset of egg laying and the peak of the acute phase of infection. Compositional analysis of the sera, using one dimensional reversed-phase fractionation of tryptic peptides coupled to mass spectrometry, allowed identification of 453 constituents. Among these, over 30% (143 molecules) were differentially present comparing sera from infected and non-infected mice, as revealed by quantitative label-free shotgun approach. The majority of proteins exhibiting altered levels was categorised as belonging to immune response (acute phase-related proteins) followed by those linked to lipid transport and metabolism. Inspection of the lipid profile from control and infected individuals demonstrated more pronounced and significant alterations in triglycerides, VLDL and HDL fractions (p<0,001), attesting for a disturbance in circulating lipid molecules, and suggesting a key role in host-parasite interactions. Our findings provide a global view of the serum proteome in the context of experimental schistosomiasis during the acute phase of infection. It contributes by listing key molecules that could be monitored to inform on the associated inflammatory disease status. We hope it will shed light into uncovered aspects of the Schistosoma mansoni parasitism in the vertebrate host, particularly those related to modulation of the lipid metabolism mediating immune responses.

HDL functionality in type 1 diabetes: enhancement of cholesterol efflux capacity in relationship with decreased HDL carbamylation after improvement of glycemic control

BackgroundReduced cholesterol efflux capacity (CEC) of HDLs is likely to increase cardiovascular risk in type 1 diabetes (T1D). We aimed to assess whether improvement of glycemic control in T1D patients is associated with changes in CEC in relation with changes in carbamylation of HDLs.MethodsIn this open-label trial, 27 uncontrolled T1D patients were given a three-month standard medical intervention to improve glycemic control. HDL fraction was isolated from plasma, and CEC was measured on THP-1 macrophages. Carbamylation of HDLs was evaluated by an immunoassay. Control HDLs from healthy subjects were carbamylated in vitro with potassium cyanate.ResultsHbA1c decreased from 11.4% [10.2–12.9] (median [1st–3rd quartiles]) at baseline to 8.1% [6.6–9.0] after the three-month intervention (P < 0.00001). The CEC of HDLs increased after intervention in 19 (70%) patients (P = 0.038). At the same time, the carbamylation of HDLs decreased in 22 (82%) patients after intervention (P = 0.014). The increase in CEC significantly correlated with the decrease in carbamylated HDLs (r = −0.411, P = 0.034), even after adjustment for the change in HbA1c (β = −0.527, P = 0.003). In vitro carbamylation of control HDLs decreased CEC by 13% (P = 0.041) and 23% (P = 0.021) using 1 and 10 mmol/L of potassium cyanate, respectively.ConclusionsThe improvement of CEC in relation to a decrease in the carbamylation of HDLs may likely contribute to the beneficial cardiovascular effect of glycemic control in T1D patients.Trial registration: NCT02816099 ClinicalTrials.gov.

STATINS, BUT NOT PCSK9 INHIBITORS, REDUCE THE ADIPOKINE CHEMERIN IN FAMILIAL HYPERCHOLESTEROLEMIA: FOCUS ON LIPOPROTEIN SUBFRACTIONS

Objective: Familial hypercholesterolemia (FH) is characterized by severe elevations in circulating LDL-cholesterol, and an increase in the risk of dyslipidemia-related cardiovascular disease (CVD). Chemerin, as a newly identified adipokine, is considered as an additional risk factor for CVD. Here we investigated whether it can be modified by cholesterol-lowering therapy. Design and method: Lipoprotein subfractions were isolated by density gradient ultracentrifugation. Lipids and chemerin concentrations were determined both before and after cholesterol lowering with either a statin (atorvastatin, simvastatin, rosuvastatin, or fluvastatin) or a PCSK9 inhibitor (PCSK9i; alirocumab or evolocumab). Results: At baseline chemerin levels were 113 ± 46 (statin group) and 95 ± 44 (PCSK9i group) ng/ml, while triglyceride (TG) levels were 1.9 ± 1.6 and 2.6 ± 1.7 mmol/ml, high-density lipoprotein cholesterol (HDL-c) levels were 1.3 ± 0.4 and 1.2 ± 0.4 mmol/ml, and low-density lipoprotein cholesterol (LDL-c) levels were 5.7 ± 1.5 and 4.9 ± 1.4 mmol/ml (P = ns for difference between 2 groups). Chemerin correlated positively with triglycerides (r = 0.45, P &lt; 0.005) and negatively with HDL-c (r = -0.33, P &lt; 0.01). Both statins and PCSK9i reduced LDL-c (by 41 and 62%, P &lt; 0.0001), triglycerides (by 13 and 19%, P &lt; 0.01), and increased HDL-c (by 8 and 23%, P &lt; 0.01), but only statins additionally reduced chemerin (by 35%, P &lt; 0.005). The lipoprotein subfraction profile revealed that chemerin accumulated particularly in the HDL3 fraction (containing &gt; 60% of all chemerin in lipoprotein subfractions), with approximately 30% being present in the HDL2 fraction, and approximately 3% in the LDL fraction. Statins reduced HDL3-c and HDL3-TG, and the level of chemerin bound to all subfractions. PCSK9i reduced HDL3-c but did not affect HDL3-TG or the level of chemerin bound to HDL3 and HDL2. Conclusions: Circulating chemerin occurs in different lipoprotein subfractions, accumulating particularly in the HDL3 fraction. Statins, but not PCSK9i, lowers chemerin, possibly by interfering with its levels across lipoprotein subfractions. This may represent a novel cardiovascular protective function of statins.

A method for lipoprotein (a) Isolation from a small volume of plasma with applications for clinical research

High levels of circulating Lipoprotein (a) [Lp(a)] are an independent risk factor for CVD. One of the major limitations to investigating Lp(a) biology is the need for large volumes of plasma (4–10 mL) for its isolation. We developed an isolation technique requiring only 0.4 mL of plasma yielding an enriched Lp(a) fraction suitable for compositional and functional studies. We collected plasma from patients (n = 9) in EDTA presenting to our Center for Preventive Cardiology for CVD risk management and with circulating Lp(a) > 66 mg/dL. 0.4 mL of plasma was added to 90 µL of potassium bromide (1.33 g/mL) and subjected to our two-step density-gradient ultracentrifugation method. The first step separates VLDL and LDL from the Lp(a) and HDL fractions and the second step further separates VLDL from LDL and Lp(a) from HDL. Lp(a) is then dialyzed for up to 24 h in potassium phosphate buffer. We performed cholesterol gel electrophoresis, immunoblotting and LC-MS/MS proteomics on isolated lipoprotein fractions to confirm fraction enrichment. Functional studies including Lp(a)-dependent induction of macrophage gene expression and cholesterol efflux inhibition were performed on isolated Lp(a) to confirm its preserved bioactivity. Lp(a) yields (264 ± 82.3 µg/mL on average) correlated with Lp(a) plasma concentrations (r2 = 0.75; p < 0.01) and represented the relative distribution of circulating apo(a) isoforms. Proteomic analyses confirm lipoprotein fraction separation. Functional integrity was confirmed by the findings that isolated Lp(a) inhibited plasminogen-dependent cholesterol efflux in HEK293T cells expressing ABCA1 and increased expressions of Il1b, Nos2 and Ccl2. We developed a small-volume isolation technique for Lp(a) suited for a range of applications used in biomedical research. The use of this technique circumvents volume-dependent limitations and expands our ability to investigate the mysteries of this deleterious lipoprotein.

Abstract 417: Elevation Of Serum Hdl And Apoe Level In A Novel Vps35l Deficient Mouse Model

Aim: Defect of the endosomal protein sorting factor VPS35L causes novel congenital anomalies syndrome, in which cholesterol dysmetabolism is one of the symptoms. To reveal the biochemical mechanisms, the a model mouse was developed and characterized. Methods: Loss-of-function mutation, N995del, VPS35L knock-in (KI) mice were generated. Serum lipoprotein profiles were assessed by LipoSEARCH™ (Immuno-Biological Laboratories, Japan). For biochemical assays, 70 micro-L of serum was sub-fractionated with Superose 6 column in PBS and total and free cholesterol detected using LabAssay™cholesterol (Fujifilm Wako Chemicals, Japan) and Pureauto™S F-CHO-N (SEKISUI MEDICAL, Japan) assay kits. Negative images of fractioned lipoproteins were observed by TEM (JEM-1400Plus, JEOL, Japan). ApoA-I and apoE in the fractions were determined by use of a mouse apoA1 ELISA PRO kit and a mouse apoE ELISA PRO kit (Mabtech AB, Sweden). Mouse liver total RNA was isolated by ISOGEN (Nippon Gene, Japan) and first-strand cDNA generated using the Invitrogen™ SuperScript™IV First-Strand Synthesis system with random hexamers. For quantitative real-time PCR, the StepOnePlus™ real-time PCR system (Thermo Fisher) was used, and the PCR end product of each well confirmed by the melt curve analysis. Results: Lipoprotein analysis indicated the KI mice showed 1.9-fold higher total cholesterol than WT mice. LDL-C and HDL-C were increased 2.9- and 1.9-fold compared to WT mice, respectively. ApoA-I levels in the large HDL and HDL fractions were all significantly increased 1.5-, and 1.6-fold, whereas apoE levels were elevated 25-, and 8.4-fold, respectively. Conclusions: Ablation of VPS35L in MEF(3T3) cells caused loss of endocytosis of the LDL receptor and LRP1, and perhaps even SR-BI, which may lead to increased serum LDL and HDL levels in the loss-of-function KI model mice. Surprisingly. a high level of apoE was detected in KI mice, especially in the HDL fraction. In this case, apoE catabolism became abnormal due to the lack of LRP1 at the cell surface. Abnormal metabolism of apoE may affect the central nervous system or neuron generation. Further examination of the phenotype of this this model mouse line are warranted.

Abstract 527: Loss Of Hepatic Insulin Signaling Promotes Apolipoprotein Ai Retention In Liver And Alters HDL In Plasma In Mice

Background: Insulin signaling regulates a variety of features of hepatic lipid and lipoprotein metabolism. Liver-specific deletion of insulin receptor (LIRKO) reduces plasma HDL, an effect attributed to a reduction in deiodinase 2 expression and apolipoprotein (Apo) AI mRNA expression. We report a concomitant accumulation of ApoAI protein in the liver of LIRKO mice in a yet to be fully characterized compartment within hepatocytes. Methods: Mice harboring loxP sites within the insulin receptor gene (IR fl/fl ) were administered a control adenoassociated viral vector (AAV-Empty) or an AAV encoding Cre recombinase under the control of a liver-specific promoter (AAV_TBG-Cre) at 8 weeks of age to generate Control and LIRKO mice. Male and female mice were evaluated for the abundance of ApoAI in liver and total and FPLC-fractionated plasma. ApoAI secretion rates were evaluated in primary hepatocytes from both genotypes. Subcellular localization of ApoAI in liver of Control and LIRKO mice was evaluated by immunofluorescence microscopy and biochemical fractionation. Results: The loss of hepatic insulin receptor and signaling was confirmed by rtPCR, immunoblot analysis and glucose tolerance test. Whereas ApoAI was unaffected in LIRKO mice compared to controls, protein levels were reduced in plasma and elevated in liver. In contrast, no changes were observed for ApoB or ApoE in either liver or plasma. FPLC fractionation of plasma indicates a shift in HDL size and an accumulation of ApoM in the HDL fractions. Secretion rates of newly synthesized ApoAI were modestly reduced in primary hepatocytes isolated from LIRKO mice. Immunolocalization of ApoAI demonstrated accumulation in large, membrane-bound organelles located peripherally in cells. These organelles stained positively for LampI and other markers of the endosomal-lysosomal system. However, markers of the endosomal-lysosomal compartment in perinuclear regions of the cells were negative for ApoAI. Conclusions: Insulin signaling is a regulator of ApoAI secretion that alters the intracellular itinerary of ApoAI in the secretory and endocytic pathways and alters the plasma HDL proteome in mice.

Brazilian Green Propolis Modulates Cholesterol Homeostasis in a Preclinical Guinea Pig Model

This study investigates the effect of the hydroalcoholic extract of Brazilian green propolis on lipid metabolism in hypercholesterolemic guinea pigs. This product has been considered an important source of bioactive compounds with potential health benefits. Thirty‐six guinea pigs, Cavia porcellus (male), 90 days old, weight of 400 g, were divided into six groups of six animals each and fed a standard AIN‐93 M diet (C), hypercholesterolemic diet (H), hypercholesterolemic diet + hydroalcoholic extract (75 mg/kg body weight [HEtOH1]; 150 mg/kg body weight [HEtOH2], and 300 mg/kg body weight [HEtOH3]), or hypercholesterolemic diet + simvastatin (1.5 mg/kg body weight) (HS) for 10 weeks. The group‐specific diet and water were provided ad libitum throughout the experimental period. Blood and liver tissue samples were collected for biochemical analysis and histopathology. Statistical analysis was performed using GraphPad Prism software version 5.0. The experimental protocol for the use of animals was approved by the Ethics Committee on Animal Use of the University of São Paulo (N°18.1.999.60.0). Animals in the H groups had a lower food intake than in the C group. Fecal excretion was regulated by the administration of extracts, being higher in these groups when compared to H and HS groups. Liver weights were influenced by the diet and administration of the hydroalcoholic extract of green propolis. Hypercholesterolemic diets determined an increase of about 2 times in the relative weight of this organ when compared to the C group, being more expressive in the groups of animals that received the hydroalcoholic extract. A significant increase in serum levels of total and non‐HDL cholesterol was observed in H group, concomitant with a reduction in the concentration of cholesterol‐HDL when compared to group C. However, these high levels of total cholesterol were reduced when simvastatin and extracts were administered with the hypercholesterolemic diet. The same was observed for the non‐HDL cholesterol fraction, where administration with simvastatin and extracts significantly reduced the concentration of these fractions, especially in HS and HEtOH3 groups when compared to group H. Analysis of the HDL fraction demonstrates that the animals submitted to the hypercholesterolemic diet had the lowest levels for this parameter when compared to the groups that received the control diet and/or extracts and simvastatin. There was a significant reduction in serum triacylglycerol levels in the HEtOH2 and HEtOH3 groups. However, a change in liver metabolism was observed, favoring the deposition of lipids in the liver, a different profile from what was observed when administering simvastatin. C group presented a histological aspect compatible with the normality of the organ. The H and HS groups presented moderate to severe fat degeneration, with a slight degeneration in the HS group when compared to the H group. A profile similar to that of the HS group was observed in the HEtOH2 group. Our results suggest that the Brazilian green propolis extract induces positive effects on dyslipidemia.