Left Ventricular Diastolic Dysfunction Among Youth with Obesity and History of Elevated Blood Pressure.

OBJECTIVE: To assess prevalence of and factors associated with left ventricular diastolic dysfunction (LVDD) in youth with obesity and elevated blood pressure (BP). STUDY DESIGN: This was a cross-sectional analysis of baseline and follow-up visits of 83 youth, 5-21 years, evaluated for overweight/obesity and elevated BP in a multidisciplinary clinic. LVDD was defined according to established adult criteria (LVDDadult; E/A < 1, E/e' > 14, or e'/a' < 0.8) and pediatric criteria (LVDDpeds; E/A <10th percentile, E/e' >99th percentile, or e'/a' <1st percentile) based on data from 103 age-sex matched healthy controls. Baseline factors associated with LVDDpeds were examined using Wilcoxon rank sum and χ2 tests. Multiple logistic regression analyses using generalized estimating equations to account for repeated measures evaluated the associations of adiposity and BP with LVDDpeds. RESULTS: The prevalence of LVDD ranged from 1.2% to 2.7% when we used adult criteria and 19% to 28% when we used pediatric criteria. Those with LVDDpeds were older, predominantly male, and non-African American and had greater weight, BP, BP medication use, and non-high-density lipoprotein cholesterol than those without LVDDpeds. Diastolic BP z score was associated with LVDDpeds by E/A (OR 1.95, 95% CI 1.15-3.32, P = .014) after we adjusted for age, sex, race, BP medications, and body mass index z score. CONCLUSIONS: LVDD was present in a substantial proportion of youth with overweight/obesity and elevated BP using pediatric criteria. Those with LVDDpeds had significantly greater measures of adiposity and BP compared with those without LVDDpeds, and diastolic BP z score was an independent predictor of LVDDpeds by E/A. These data emphasize the importance of prevention and treatment of cardiovascular disease risk factors in childhood.

Urea-Assisted Reconstitution of Discoidal High-Density Lipoprotein.

High-density lipoprotein (HDL) is a naturally occurring composite of lipids and lipid-binding proteins. The cholate dialysis method, first reported by Jonas in 1969, is the most widely used approach for reconstituting discoidal HDL (dHDL) in test tubes with phospholipids and the most dominant protein, apolipoprotein A-1 (apoA-I). Here, we show that a dHDL-relevant complex can also be prepared by gently mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and apoA-I or its mutants in ethanol/H2O solutions containing urea at a concentration of a few molar and then incubating the mixture at the gel-liquid crystalline phase transition temperature in test tubes. Subsequent purification steps involve quick dialysis following size exclusion chromatography. The yields (73 ± 3% and 70 ± 1% protein and DMPC, respectively) of the resulting HDL-like nanoparticles, designated as uHDL, were comparable to the values of 68 ± 9% and 71 ± 12% obtained in the cholate dialysis method. Using apoA-I and two mutants, the key factor in this method was found to be urea at the folded and unfolded transition midpoint concentration. By using this urea-assisted method in the presence of a hydrophobic drug, all-trans-retinoic acid (ATRA), one-step preparation of ATRA-loaded uHDL was also possible. The loading efficiency was comparable to that in the mixing of ATRA and uHDL or dHDL reconstituted by the cholate dialysis method. Atomic force microscopy analysis revealed that uHDL and ATRA-loaded uHDL were discoidal. Our urea-assisted method is an easy and efficient method for reconstituting dHDL and can be utilized to prepare various drug-dHDL complexes.

Probing the Assembly of HDL Mimetic, Drug Carrying Nanoparticles Using Intrinsic Fluorescence.

Reconstituted high-density lipoprotein (HDL) containing apolipoprotein A-I (Apo A-I) mimics the structure and function of endogenous (human plasma) HDL due to its function and potential therapeutic utility in atherosclerosis, cancer, neurodegenerative diseases, and inflammatory diseases. Recently, a new class of HDL mimetics has emerged, involving peptides with amino acid sequences that simulate the the primary structure of the amphipathic alpha helices within the Apo A-I protein. The findings reported in this communication were obtained using a similar amphiphilic peptide (modified via conjugation of a myristic acid residue at the amino terminal aspartic acid) that self-assembles (by itself) into nanoparticles while retaining the key features of endogenous HDL. The studies presented here involve the macromolecular assembly of the myristic acid conjugated peptide (MYR-5A) into nanomicellar structures and its characterization via steady-state and time-resolved fluorescence spectroscopy. The structural differences between the free peptide (5A) and MYR-5A conjugate were also probed, using tryptophan fluorescence, FÓ§rster resonance energy transfer (FRET), dynamic light scattering, and gel exclusion chromatography. To our knowledge, this is the first report of a lipoprotein assembly generated from a single ingredient and without a separate lipid component. The therapeutic utility of these nanoparticles (due to their capablity to incorporate a wide range of drugs into their core region for targeted delivery) was also investigated by probing the role of the scavenger receptor type B1 in this process. SIGNIFICANCE STATEMENT: Although lipoproteins have been considered as effective drug delivery agents, none of these nanoformulations has entered clinical trials to date. A major challenge to advancing lipoprotein-based formulations to the clinic has been the availability of a cost-effective protein or peptide constituent, needed for the assembly of the drug/lipoprotein nanocomplexes. This report of a robust, spontaneously assembling drug transport system from a single component could provide the template for a superior, targeted drug delivery strategy for therapeutics of cancer and other diseases (Counsell and Pohland, 1982).

The HDL lipidome is widely remodeled by fast food versus Mediterranean diet in 4 days.

INTRODUCTION: HDL is associated with increased longevity and protection from multiple chronic diseases. The major HDL protein ApoA-I has a half-life of about 4 days, however, the effects of diet on the composition of HDL particles at this time scale have not been studied. OBJECTIVES: The objective of this study is to investigate the short term dietary effect on HDL lipidomic composition. METHODS: In this randomized order cross-over study, ten healthy subjects consumed a Mediterranean (Med) and a fast food (FF) diet for 4 days, with a 4-day wash-out between treatments. Lipidomic composition was analyzed in isolated HDL fractions by an untargeted LC-MS method with 15 internal standards. RESULTS: HDL phosphatidylethanolamine (PE) content was increased by FF diet, and 41 out of 170 lipid species were differentially affected by diet. Saturated fatty acids (FAs) and odd chain FA were enriched after FF diet, while very-long chain FA and unsaturated FA were enriched after Med diet. The composition of phosphatidylcholine (PC), triacylglycerol (TG) and cholesteryl ester (CE) were significantly altered to reflect the FA composition of the diet whereas the composition of sphingomyelin (SM) and ceramides were generally unaffected. CONCLUSION: Results from this study indicate that the HDL lipidome is widely remodeled within 4 days of diet change and that certain lipid classes are more sensitive markers of diet whereas other lipid classes are better indicators of non-dietary factors.

Triglyceride-to-High-Density Lipoprotein Cholesterol Ratio and Systemic Inflammation in Patients with Idiopathic Pulmonary Arterial Hypertension.

BACKGROUND Idiopathic pulmonary arterial hypertension (IPAH) patients are characterized by elevated triglyceride (TG)-to-HDL cholesterol (HDL-C) ratio, which has been proposed to be an important prognostic factor in this population. The mechanism of this phenomenon remains unknown. We therefore investigated the potential determinants of increased TG/HDL-C ratio in IPAH patients. MATERIAL AND METHODS We prospectively recruited consecutive clinically stable IPAH patients between January 2016 and February 2017. Patients with diabetes or using statins were excluded. Anthropometric measurements included body mass index (BMI) and skinfold thickness; body fat mass was calculated using age and sex-specific equations. We assessed lipid profile, homeostatic model assessment of insulin resistance (HOMA-IR), serum adipokine levels (adiponectin, resistin, leptin, and visfatin), and circulating cytokines (IL-1ß, IL-6, MCP-1, and TNF-α). RESULTS We assessed 47 IPAH patients: 9 of them had been diagnosed with diabetes and 10 were treated with statins; therefore, were excluded them from further analysis. Age, sex distribution, and BMI were similar irrespectively of TG/HDL-C ratio. Patients with increased TG/HDL-C ratio (>3) as compared to patients with TG/HDL-C ≤3 were characterized by higher levels of IL-1ß, MCP-1, and IL-6. TG level was correlated with IL-1ß (R=0.76, p<0.001), IL-6 (R=0.52, p=0.005), TNF-α (R=0.62, p<0.001), and MCP-1 (R=0.63, p<0.001). IL-1ß was also inversely correlated with HDL-C (R=-0.44, p=0.02). We found no differences in concentration of fasting glucose, insulin, HOMA-IR, body fat content, or adipokine levels between patients with higher and lower TG/HDL-C ratios. CONCLUSIONS In IPAH patients, elevated TG/HDL-C ratio is a marker of systemic inflammation.

Resolution of Lipoprotein Subclasses by Charge Detection Mass Spectrometry.

Lipoproteins are micelle-like assemblies that are key players in the pathogenesis of atherosclerosis. High-density lipoprotein (HDL), low-density lipoproteins (LDL), and very low density lipoprotein (VLDL) are the three major classes present in fasting plasma. Within each class, there is a broad size distribution with wide variations in protein and lipid content. The development of better metrics for cardiovascular risk is thought to depend on better characterization of lipoprotein subclasses. Using charge detection mass spectrometry (CDMS), the mass distributions of HDL, LDL, and VLDL have been directly measured for the first time. In the case of HDL, seven distinct subpopulations were resolved using a two-dimensional correlation of charge and mass. The resolved components are assigned to HDL particles containing different numbers of the key structural proteins apolipoprotein A-I and apolipoprotein A-II.

LDL and HDL lipoprotein subfractions in multiple sclerosis patients with decreased insulin sensitivity.

OBJECTIVES: Increased metabolic and cardiovascular morbidity has been reported in multiple sclerosis (MS) patients. Previously, we have found decreased insulin sensitivity and hyperinsulinemia in a group of newly diagnosed MS patients. We hypothesize that these features may be associated with an altered lipid profile and low, intermediate, or high density lipoprotein (LDL, IDL, HDL) subclasses accelerating atherosclerosis and thus contributing to the cardiovascular risk increase in these patients. SUBJECTS AND METHODS: In a group of 19 newly diagnosed untreated MS patients with previously found hyperinsulinemia and insulin resistance and a matched group of 19 healthy controls, the lipoprotein subclasses profile was determined. Polyacrylamide gel electrophoresis was used to separate and measure the LDL (large LDL and small dense LDL), HDL (large, intermediate and small), and IDL (A, B and C) subclasses with the Lipoprint© System (Quantimetrix Corporation, Redondo Beach, CA, USA). RESULTS: No difference was found either in the conventional lipid or lipoprotein subclasses profile between the MS patients and healthy controls. We found an inverse association between the level of IDL-B with fasting insulin (r=-0.504, p=0.032), the insulin resistance estimated by homeo-static model assessment - insulin resistance (HOMA-IR) (r=-0.498, p=0.035), insulin response expressed as area under the curve (AUC; r=-0.519, p=0.027), and area above the baseline (AAB; r=-0.476, p=0.045) and positive association with insulin sensitivity estimated by insulin sensitivity index (ISI) Matsuda (r=0.470, 0.048) in MS patients, but not in healthy controls suggesting the first signs in lipoprotein subclasses profile change. CONCLUSIONS: Our data indicate that changes in lipoprotein profile and subclasses are preceded by insulin resistance and hyperinsulinemia in patients with newly diagnosed MS.

A Novel Method for Estimating Low-Density Lipoprotein (LDL) Levels: Total Cholesterol and Non-High-Density Lipoprotein (HDL) Can Be Used to Predict Abnormal LDL Level in an Apparently Healthy Population.

BACKGROUND We aimed to predict the abnormal LDL level by using TG, TC, HDL, and non-HDL in this study. MATERIAL AND METHODS Triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) data were obtained from the Laboratory Information System (LIS) for 4 years (Oct 1, 2013 to Sept 30, 2017) from among 34 270 healthy Chinese patients at Shuyang People's Hospital. TG, TC, HDL, and LDL (direct clearance method) were measured using a TBA2000FR biochemical analyzer. The non-HDL was calculated as TC minus HDL. Correlations between TG, TC, non-HDL, and LDL were analyzed using Spearman's rank correlation. Receiver operating characteristics (ROC) curve analysis was used to evaluate the predictive utility of TG, TC, and non-HDL for the abnormal LDL level (<130 mg/dL). RESULTS Both TC (r=0.870) and non-HDL (r=0.893) were significantly positively correlated with LDL. The area under curve of TC and non-HDL can be used to predict abnormal LDL levels. Optimal thresholds were 182.5 mg/Dl (4.72 mmol/L) for TC and 135.3 mg/Dl (3.50 mmol/L) for non-HDL. Based on these optimal thresholds, less than 0.5% and 0.4% of tests with elevated LDL were missed using TC and non-HDL, respectively, but the value of these missed LDL levels was not very high (<147.3 mg/dL). CONCLUSIONS If the value of non-HDL is less than 135.3 mg/Dl (3.50 mmol/L) and/or TC is less than 182.5 mg/Dl (4.72 mmol/L) for the apparently healthy populations, the LDL level will be less than 130 mg/Dl (3.36 mmol/L). TC and non-HDL can be used to predict the abnormal LDL level in apparently healthy populations.

Intracellular APOL1 Risk Variants Cause Cytotoxicity Accompanied by Energy Depletion.

Population genetic approaches have uncovered a strong association between kidney diseases and two sequence variants of the APOL1 gene, called APOL1 risk variant G1 and variant G2, compared with the nonrisk G0 allele. However, the mechanism whereby these variants lead to disease manifestation and, in particular, whether this involves an intracellular or extracellular pool of APOL1 remains unclear. Herein, we show a predominantly intracellular localization of APOL1 G0 and the renal risk variants, which localized to membranes of the endoplasmic reticulum in podocyte cell lines. This localization did not depend on the N-terminal signal peptide that mediates APOL1 secretion into the circulation. Additionally, a fraction of these proteins localized to structures surrounding mitochondria. In vitro overexpression of G1 or G2 lacking the signal peptide inhibited cell viability, triggered phosphorylation of stress-induced kinases, increased the phosphorylation of AMP-activated protein kinase, reduced intracellular potassium levels, and reduced mitochondrial respiration rates. These findings indicate that functions at intracellular membranes, specifically those of the endoplasmic reticulum and mitochondria, are crucial factors in APOL1 renal risk variant-mediated cell injury.

Automation of PRM-dependent D3-Leu tracer enrichment in HDL to study the metabolism of apoA-I, LCAT and other apolipoproteins.

We developed an automated quantification workflow for PRM-enabled detection of D3-Leu labeled apoA-I in high-density lipoprotein (HDL) isolated from humans. Subjects received a bolus injection of D3-Leu and blood was drawn at eight time points over three days. HDL was isolated and separated into six size fractions for subsequent proteolysis and PRM analysis for the detection of D3-Leu signal from ∼0.03 to 0.6% enrichment. We implemented an intensity-based quantification approach that takes advantage of high-resolution/accurate mass PRM scans to identify the D3-Leu 2HM3 ion from non-specific peaks. Our workflow includes five modules for extracting the targeted PRM peak intensities (XPIs): Peak centroiding, noise removal, fragment ion matching using Δm/z windows, nine intensity quantification options, and validation and visualization outputs. We optimized the XPI workflow using in vitro synthesized and clinical samples of D0/D3-Leu labeled apoA-I. Three subjects' apoA-I enrichment curves in six HDL size fractions, and LCAT, apoA-II and apoE from two size fractions were generated within a few hours. Our PRM strategy and automated quantification workflow will expedite the turnaround of HDL apoA-I metabolism data in clinical studies that aim to understand and treat the mechanisms behind dyslipidemia.

The Changing Face of HDL and the Best Way to Measure It.

BACKGROUND: HDL cholesterol (HDL-C) is a commonly used lipid biomarker for assessing cardiovascular health. While a central focus has been placed on the role of HDL in the reverse cholesterol transport (RCT) process, our appreciation for the other cardioprotective properties of HDL continues to expand with further investigation into the structure and function of HDL and its specific subfractions. The development of novel assays is empowering the research community to assess different aspects of HDL function, which at some point may evolve into new diagnostic tests. CONTENT: This review discusses our current understanding of the formation and maturation of HDL particles via RCT, as well as the newly recognized roles of HDL outside RCT. The antioxidative, antiinflammatory, antiapoptotic, antithrombotic, antiinfective, and vasoprotective effects of HDL are all discussed, as are the related methodologies for assessing these different aspects of HDL function. We elaborate on the importance of protein and lipid composition of HDL in health and disease and highlight potential new diagnostic assays based on these parameters. SUMMARY: Although multiple epidemiologic studies have confirmed that HDL-C is a strong negative risk marker for cardiovascular disease, several clinical and experimental studies have yielded inconsistent results on the direct role of HDL-C as an antiatherogenic factor. As of yet, our increased understanding of HDL biology has not been translated into successful new therapies, but will undoubtedly depend on the development of alternative ways for measuring HDL besides its cholesterol content.

Effects of curcumin on HDL functionality.

Curcumin, a bioactive polyphenol, is a yellow pigment of the Curcuma longa (turmeric) plant. Curcumin has many pharmacologic effects including antioxidant, anti-carcinogenic, anti-obesity, anti-angiogenic and anti-inflammatory properties. Recently, it has been found that curcumin affects lipid metabolism, and subsequently, may alleviate hyperlipidemia and atherosclerosis. Plasma HDL cholesterol (HDL-C) is an independent negative risk predictor of cardiovascular disease (CVD). However, numerous clinical and genetic studies have yielded disappointing results about the therapeutic benefit of raising plasma HDL-C levels. Therefore, research efforts are now focused on improving HDL functionality, independent of HDL-C levels. The quality of HDL particles can vary considerably due to heterogeneity in composition. Consistent with its complexity in composition and metabolism, a wide range of biological activities is reported for HDL, including antioxidant, anti-glycation, anti-inflammatory, anti-thrombotic, anti-apoptotic and immune modulatory activities. Protective properties of curcumin may influence HDL functionality; therefore, we reviewed the literature to determine whether curcumin can augment HDL function. In this review, we concluded that curcumin may modulate markers of HDL function, such as apo-AI, CETP, LCAT, PON1, MPO activities and levels. Curcumin may subsequently improve conditions in which HDL is dysfunctional and may have potential as a therapeutic drug in future. Further clinical trials with bioavailability-improved formulations of curcumin are warranted to examine its effects on lipid metabolism and HDL function.

A Single In-Vial Dual Extraction Strategy for the Simultaneous Lipidomics and Proteomics Analysis of HDL and LDL Fractions.

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount.

Variability in follicular fluid high density lipoprotein particle components measured in ipsilateral follicles.

PURPOSE: The purpose of this study was to examine the biological variability of follicular fluid (FF) high density lipoprotein (HDL) particle components measured in ipsilateral ovarian follicles. METHODS: We collected FF from two ipsilateral follicles among six women undergoing in vitro fertilization (IVF). We measured concentrations of 19 FF HDL particle components, including HDL cholesterol, free cholesterol, four cholesteryl esters, phospholipids, triglycerides, paraoxonase and arylesterase activities, apolipoproteins A-1 and A-2 (ApoA-1 and ApoA-2), and seven lipophilic micronutrients, by automated analysis and with high-performance liquid chromatography. We assessed biological variability using two-stage nested analysis of variance and compared values with those previously published for contralateral follicles. RESULTS: For most FF HDL analytes, there was little variability between follicles relative to the variability between women (i.e., %σ(2) F:%σ(2) B <0.5). Intraclass correlation coefficients were >0.80 for HDL cholesterol (0.82), phospholipids (0.89), paraoxonase (0.96), and arylesterase (0.91) activities, ApoA-1 (0.89), and ApoA-2 (0.90), and single specimen collections were required to estimate the subject-specific mean, demonstrating sufficient reliability for use as biomarkers of the follicular microenvironment in epidemiologic and clinical studies. CONCLUSIONS: These preliminary results raise the possibility for tighter regulation of HDL in follicles within the same ovary vs. between ovaries. Thus, collection of a single FF specimen may be sufficient to estimate HDL particle components concentrations within a single ovary. However, our results should be interpreted with caution as the analysis was based on a small sample.

Combined High-Density Lipoprotein Proteomic and Glycomic Profiles in Patients at Risk for Coronary Artery Disease.

OBJECTIVES: To test whether recently developed methods for comprehensive profiling of the high-density lipoprotein (HDL) glycome combined with the HDL proteome can distinguish individuals with coronary artery disease (CAD) from those without. METHODS: Twenty subjects at risk for CAD, who underwent diagnostic coronary arteriography, were analyzed. Ten subjects had CAD, and ten did not. HDL was extracted from fasting plasma samples by ultracentrifugation, followed by shotgun proteomic, glycomic, and ganglioside analyses using LC-MS. CAD vs non-CAD subjects' data were compared using univariate and multivariate statistics. RESULTS: Principal components analysis showed a clear separation of CAD and non-CAD subjects, confirming that combined HDL proteomic and glycomic profiles distinguished at-risk subjects with atherosclerosis from those without. CAD patients had lower HDL apolipoprotein content (specifically ApoA-I, A-II, and E, p < 0.05), and lower serum amyloid A2 (SAA2, p = 0.020) and SAA4 (p = 0.007) but higher sialylated glycans (p < 0.05). CONCLUSION: Combined proteomic and glycomic profiling of isolated HDL was tested as a novel analytical approach for developing biomarkers of disease. In this pilot study we found that HDL proteome and glycome distinguished between individuals who had CAD from those who did not within a group of individuals equally at risk for heart disease.

Native protein mapping and visualization of protein interactions in the area of human plasma high-density lipoprotein by combining nondenaturing micro 2DE and quantitative LC-MS/MS.

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high-density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions.

A piezoelectric-based immunosensor for high density lipoprotein particle measurement.

A piezoelectric-based immunosensor was developed for high density lipoprotein particle (HDL-P) measurement. Monoclonal anti-human apolipoprotein A1 antibody was used as a specific binding molecule for the major apolipoprotein of HDL-P. This sensing element was fabricated by immobilizing the anti-human apolipoprotein A1 on a 12 MHz AT-cut quartz crystal via a 3-mercaptopropionic acid (MPA) self-assembled monolayer. The frequency shift from the mass change of the antigen-antibody binding refers to the amount of HDL-P. The optimal antibody immobilization was performed to achieve the maximum potential of the antibody. The appropriate quantity and immobilization time of the antibody were 0.1 mg ml(-1) and 90 minutes, respectively. The immobilized antibody in the HDL-P immunosensor accomplished perfect binding with HDL-P within 60 minutes. The dose-response curve for HDL-P showed a linear response from 0.21 to 2.50 mg protein per ml equivalent to 0.40 × 10(10) to 3.65 × 10(10) particles per µl without significant interference from other lipoproteins. The intra- and inter-assay imprecision (CV) were 7.8 and 18.5%, respectively. The analytical accuracy of this measurement was 96.29-96.31%. The HDL-P concentration obtained from the sensor revealed a 2.05 mg protein per ml with 0.26 mg protein per ml of expanded uncertainty at the 95% confidence level. This immunosensor gave an assay result which correlated with the homogeneous enzymatic colorimetric assay (R(2) = 0.902).

Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that apolipoprotein (apo) AI and apoAII were present in all 5 subfractions; apoCI was present only in H1, and apoCIII and apoE were most abundantly present in H4 and H5. HDL-associated antioxidant enzymes such as lecithin-cholesterol acyltransferase, lipoprotein-associated phospholipase A2, and paraoxonase 1 were most abundant in H4 and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time-of-flight mass spectrometry. To quantify other proteins in the HDL subfractions, we used the isobaric tags for the relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity, with the exception of H5, which promoted the least efflux activity. In conclusion, anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions, it may be possible to uncover HDL-specific proteins that play a role in disease.

HDL cholesterol subfractions and the effect of testosterone replacement in hypogonadism.

Metabolic disorders and cardiovascular events are increased in hypogonadism. Serum HDL composition is a better cardiovascular predictor than the HDL counts. However, there is no information about the HDL subfractions in patients with hypogonadism. We designed a prospective study to investigate the HDL subfractions in treatment naïve subjects with hypogonadism and the effects of 2 different testosterone replacement regimens on the HDL subfractions. Seventy young male patients with congenital hypogonadotropic hypogonadism (CHH) and 70 age and BMI-matched healthy males were enrolled in the present study. The patients were assigned to receive intramuscular injections of testosterone esters 250 mg every 3 weeks and transdermal testosterone applications 50 mg daily. Biochemical investigations including HDL subfractions and insulin resistance were done. Patients with CHH had higher levels of insulin, HOMA-IR, WC, triglyceride, and diastolic blood pressure. Although, the HDL cholesterol concentrations were similar in both groups, hypogonadal patients had lower HDL2 and higher HDL3 levels. The total testosterone levels were independent determinants of the HDL2 subfractions. During the follow-up, a significant increase in the BMI and WC values and a significant decrease in the levels of total cholesterol, HDL cholesterol, and HDL3 were observed. No difference was present between the 2 treatment arms. These results show that patients with hypogonadism have unfavorable HDL compositions in addition to the other dysmetabolic features. However, testosterone replacement for about six months neither improves the metabolic problems nor the HDL composition. Mechanistic studies are warranted to better understand the cardiovascular effects of unfavorable HDL compositions in hypogonadism.

Nitric oxide production, systemic inflammation and lipid metabolism in periodontitis patients: possible gender aspect.

AIM: Nitric oxide (NO) plays a crucial role in vascular tone regulation and is involved in pathogenesis of periodontitis. In this cross-sectional study, we investigated the serum and saliva levels of NO metabolites in periodontal disease and their relationship with serum C-reactive protein (CRP) levels, lipids metabolism and periodontal disease severity. MATERIAL AND METHODS: Serum and saliva were collected from non-smoking patients with generalized severe periodontitis (n = 89) and healthy controls (n = 56). Serum and salivary levels of NO metabolites, serum levels of high density lipoproteins (HDL), low density lipoproteins (LDL), triglycerides, cholesterol and CRP were measured. Data were analysed in whole population and in different gender groups. RESULTS: Periodontitis patients exhibited significantly lower serum and saliva levels of NO metabolites and significantly higher LDL, cholesterol and CRP levels than control group. Similar findings were observed within male but not within female population. Serum NO metabolites levels exhibited significant negative correlation with CRP in whole population and in male population. Significant positive correlation of serum NO metabolite levels with HDL levels was observed in whole population. CONCLUSION: NO production is reduced in periodontitis, especially in male population. Gender might be an important factor in assessing risk of cardiovascular disease in periodontitis.