Deepening our understanding of HDL proteome.

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection. Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed. Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.

Serum amyloid A does not affect high-density lipoprotein cholesterol measurement by a homogeneous assay.

BACKGROUND: Serum amyloid A (SAA), which is one of the acute phase proteins, alters the structure of HDL by associating with it during circulation. We focused on whether SAA influences the values of HDL-cholesterol (HDL-C) measurements when using a homogeneous assay. METHODS: HDLs were isolated by ultracentrifugation from serum samples of 248 patients that were stratified into three groups based on their serum SAA concentrations (low: SAA ≤ 8 µg/mL; middle: 8 < SAA ≤ 100 µg/mL; and high: SAA > 100 µg/mL). HDL-C concentrations of the serum samples measured by the homogeneous assay were compared with the total cholesterol concentrations of HDL fractions isolated by ultracentrifugation. RESULTS: HDLs obtained from patients with low SAA concentrations were separated into their general particle sizes and classified as HDL2 and HDL3 by native-gel electrophoresis. On the other hand, HDLs obtained from patients with high SAA concentrations occasionally showed distributions different from the typical sizes of HDL2 and HDL3, such as extremely small or large particles. Nevertheless, HDL-C concentrations measured using the homogeneous assay were strongly correlated with those measured using the ultracentrifugation method, regardless of the SAA concentrations. However, the ratios of HDL-C concentrations obtained by the homogeneous assay to those obtained by the ultracentrifugation method for patients with high SAA concentrations were significantly lower than those of patients with low SAA concentrations. CONCLUSIONS: A large amount of SAA attached to HDL altered the HDL particle size but did not essentially affect HDL-C measurement by homogeneous assay.

A novel approach to measuring macrophage-specific reverse cholesterol transport in vivo in humans.

Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans.

Impaired Cholesterol Efflux Capacity of High-Density Lipoprotein Isolated From Interstitial Fluid in Type 2 Diabetes Mellitus-Brief Report.

OBJECTIVE: Patients with type 2 diabetes mellitus (T2D) have an increased risk of cardiovascular disease, the mechanism of which is incompletely understood. Their high-density lipoprotein (HDL) particles in plasma have been reported to have impaired cholesterol efflux capacity. However, the efflux capacity of HDL from interstitial fluid (IF), the starting point for reverse cholesterol transport, has not been studied. We here investigated the cholesterol efflux capacity of HDL from IF and plasma from T2D patients and healthy controls. APPROACH AND RESULTS: HDL was isolated from IF and peripheral plasma from 35 T2D patients and 35 age- and sex-matched healthy controls. Cholesterol efflux to HDL was determined in vitro, normalized for HDL cholesterol, using cholesterol-loaded macrophages. Efflux capacity of plasma HDL was 10% lower in T2D patients than in healthy controls, in line with previous observations. This difference was much more pronounced for HDL from IF, where efflux capacity was reduced by 28% in T2D. Somewhat surprisingly, the efflux capacity of HDL from IF was lower than that of plasma HDL, by 15% and 32% in controls and T2D patients, respectively. CONCLUSION: These data demonstrate that (1) HDL from IF has a lower cholesterol efflux capacity than plasma HDL and (2) the efflux capacity of HDL from IF is severely impaired in T2D when compared with controls. Because IF comprises the compartment where reverse cholesterol transport is initiated, the marked reduction in cholesterol efflux capacity of IF-HDL from T2D patients may play an important role for their increased risk to develop atherosclerosis.

HDL cholesterol subclasses, myocardial infarction, and mortality in secondary prevention: the Lipoprotein Investigators Collaborative.

AIMS: High-density lipoprotein (HDL) is highly heterogeneous and the link of its subclasses to prognosis remains controversial. We aimed to rigorously examine the associations of HDL subclasses with prognosis in secondary prevention. METHODS AND RESULTS: We collaboratively analysed data from two, complementary prospective cohorts: the TRIUMPH study of 2465 acute myocardial infarction patients, and the IHCS study of 2414 patients who underwent coronary angiography. All patients had baseline HDL subclassification by vertical-spin density gradient ultracentrifugation. Given non-linearity, we stratified by tertiles of HDL-C and its two major subclasses (HDL2-C, HDL3-C), then compared multivariable-adjusted hazard ratios for mortality and mortality/myocardial infarction. Patients were middle-aged to elderly (TRIUMPH: 58.2 ± 12.2 years; IHCS: 62.6 ± 12.6 years), and the majority were men (TRIUMPH: 68.0%; IHCS: 65.5%). IHCS had lower mean HDL-C levels (34.6 ± 10.1 mg/dL) compared with TRIUMPH (40 ± 10.6 mg/dL). HDL3-C accounted for >3/4 of HDL-C (mean HDL3-C/HDL-C 0.78 ± 0.05 in both cohorts). During 2 years of follow-up in TRIUMPH, 226 (9.2%) deaths occurred, while death/myocardial infarction occurred in 401 (16.6%) IHCS patients over 5 years. No independent associations with outcomes were observed for HDL-C or HDL2-C. In contrast, the lowest tertile of HDL3-C was independently associated with >50% higher risk in each cohort (TRIUMPH: with middle tertile as reference, fully adjusted HR for mortality of HDL3-C, 1.57; 95% CI, 1.13-2.18; IHCS: fully adjusted HR for mortality/myocardial infarction, 1.55; 95% CI, 1.20-2.00). CONCLUSION: In secondary prevention, increased risk for long-term hard clinical events is associated with low HDL3-C, but not HDL2-C or HDL-C, highlighting the potential value of subclassifying HDL-C.

Quantification of HDL particle concentration by calibrated ion mobility analysis.

BACKGROUND: It is critical to develop new metrics to determine whether HDL is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P), the size and concentration of HDL in plasma. However, the 2 methods currently used to determine HDL-P yield concentrations that differ >5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA). METHODS: HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. We used a calibration curve constructed with purified proteins to correct for the ionization efficiency of HDL particles. RESULTS: The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n = 40) and cerebrovascular disease (n = 40) participants, 3 subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4 (2.4) µmol/L. HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in participants with cerebrovascular disease (P = 0.002), and this difference remained significant after adjustment for HDL cholesterol concentrations (P = 0.02). CONCLUSIONS: Calibrated IMA accurately determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting that the method could accurately quantify HDL particle concentration. The estimated stoichiometry of apolipoprotein A-I determined by calibrated IMA was 3-4 per HDL particle, in agreement with current structural models. Furthermore, HDL-P was associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.

Evaluación durante 6 años de la dislipidemia en un centro de salud. Importancia de las acciones de mejora / A 6-year evaluation of dyslipidemia in a health center: Importance of improvement actions

Introducción. La dislipidemia, sobre todo el aumento del colesterol LDL, se ha demostrado como uno de los factores de riesgo más importantes en la génesis de la afectación coronaria. La prevalencia de las dislipidemias en España es alta. El objetivo del presente trabajo es valorar la evolución de los pacientes dislipidémicos de nuestro centro de salud durante 6 años y ver si se ha producido una mejora en el control de los mismos tras la presentación de la evaluación de los 3 primeros años y la actualización del protocolo de dislipidemias del centro de salud. Pacientes y método. Evaluación Periodo 1 (2006-2008): 267 pacientes dislipidémicos. Evaluación Periodo 2 (2009-2011): 222 pacientes, excluidos exitus y cambios de domicilio. Variables: edad, sexo, antecedentes personales de ECV, factores de riesgo vascular, lípidos, número de analíticas, tratamiento farmacológico, niveles de riesgo CV y porcentajes en objetivos de control. Resultados. Edad media 66,2 años (DE 13,4), mujeres 66,3%. Periodo 1-Periodo 2: colesterol total: 221,9-196,6 mg/dl (p = 0,000); colesterol LDL: 147,9-115,8 mg/dl (p = 0,000). En objetivos terapéuticos, pacientes riesgo alto: 14-50,5% (p = 0,024); riesgo medio: 35-68,1% (p = 0,038); riesgo bajo: 44-68,2% (p = NS). Tratamiento farmacológico 68-77% (p = 0,000). Modificación tratamiento: 30-43% (p = 0,001). Cumplimiento terapéutico: 75-86% (p = 0,003). Sin tratamiento riesgo alto: 15,4-16,3% (p = NS). Conclusiones. Se ha producido una mejoría significativa en el Periodo 2, sobre todo en los pacientes de riesgo alto, tras presentar los resultados de la evaluación del Periodo 1 y haber actualizado, en el centro de salud, el protocolo de dislipidemias. Hay pacientes con riesgo alto sin tratamiento hipolipidemiante que se deben detectar y revisar. (AU)

Introduction. Dyslipidemia, especially an increased LDL-cholesterol, has been shown to be one of the most important risk factors in the genesis of coronary involvement. The prevalence of dyslipidemias in Spain is high. The objective of this study is to assess the progress of dyslipidemic patients in our health center over a 6-year period, and see if there has been any improvement in its control after the presentation of the evaluation of the first 3 years, as well as an updated dyslipidemia protocol. Patients and methods. Assessment Period 1 (2006-2008): 267 patients with dyslipidemia. Assessment Period 2 (2009-2011): 222 patients, excluding exitus and address changes. Variables: age, sex, personal history of CVD, vascular risk factors, lipids, drug treatment, risk levels, and percentages of CV control objectives. Results. Mean age was 66.2 years (SD 13.4), 66.3% women. Period 1-Period 2: Total cholesterol: 221.9-196.6 mg/dl (P = .000); LDL-cholesterol: 147.9-115.8 mg/dl (P = .000). In high risk patients, therapeutic targets: 14-50.5% (P = .024); medium risk: 35-68.1% (P = .038); low risk: 44-68.2% (P = NS). Pharmacotherapy 68-77% (P = .000). Changing treatment: 30-43% (P = .001). Adherence: 75-86% (P = .003). Untreated high risk: 15.4-16.3% (P = NS). Conclusions. There was a significant improvement in Period 2, especially in high-risk patients, after presenting the results of the evaluation for Period 1 and with the updated dyslipidemia protocol. There are high risk patients without lipid-lowering treatment to be detected and reviewed (AU)

Measurement of LDL-C after treatment with the CETP inhibitor anacetrapib.

Estimation of low-density lipoprotein cholesterol (LDL-C) using the Friedewald (FR) formula is often inaccurate when triglycerides are elevated or VLDL particle composition is altered. We hypothesized that LDL-C estimation by the FR formula and other measurement methods might also be inaccurate in individuals treated with a cholesteryl ester transfer protein (CETP) inhibitor. An assay comparison study was conducted using pre and posttreatment serum samples from 280 of the 811 patients treated with the CETP inhibitor anacetrapib in the DEFINE study (determining the efficacy and tolerability of CETP inhibition with anacetrapib). After 24 weeks of treatment with anacetrapib, mean LDL-C values by FR formula, Roche direct method (RDM) and Genzyme direct method (GDM) deviated from that measured by the ß-quantification (BQ) reference method by -12.2 ± 7.5, -10.2 ± 6.6, -10.8 ± 8.8 mg/dl, respectively. After treatment with anacetrapib, the FR formula and detergent-based direct methods provided lower LDL-C values than those obtained by the BQ reference method. The bias by the FR formula appeared to be due to an overestimation of VLDL-C by the TG/5 component of the formula. Evaluation of the clinical significance of these findings awaits comprehensive lipid and cardiovascular outcome data from ongoing Phase III clinical studies of anacetrapib.

[Effect of water on silica gel adsorption of blood plasma components].

In this work, the study of properties of silica gel as an adsorbent for plasmasorption has been performed. Investigations have been realized of the effect of silica gel preliminary treatment conditions and a period of plasma with silica gel contact on plasmasorption characteristics of human blood plasma components, such as protein, triglycerides, cholesterol (high-density and low-density one). The results obtained can be used for variation of silica gel adsorption properties, in situ at the adsorbent preparation process. For explanation of the experimental concentration and kinetic (temporal) characteristics of plasmasorption, the model of silica gel grains charging at the hydration was used.

Adjustment of direct high-density lipoprotein cholesterol measurements according to intercurrent triglyceride corrects for interference by triglyceride-rich lipoproteins.

BACKGROUND: Low plasma levels of high-density-lipoprotein cholesterol (HDL-C) and high triglyceride (TG) are strongly associated with cardiovascular disease (CVD). Clinical recognition of this high-risk population demands accurate measurement of HDL-C, whereas cost and clinical demand dictate that optimal HDL-C measurement requires fully automated methods that avoid manual precipitation. Commercial techniques use specific reagents to selectively expose and "directly" measure cholesterol in HDL. However, these "direct" methods may experience interference from the cholesterol content of triglyceride-rich-lipoproteins (TRL), leading to analytical overestimation of HDL-C, with subsequent underestimation of low-density-lipoprotein cholesterol (LDL-C) and of CVD risk. OBJECTIVE: The aim of this study was to develop a method to overcome this interference. METHODS: Serum/Li+-heparin plasma samples from consecutive patients were analyzed for HDL-C by the comparison of three generations of the Roche Diagnostics, HDL-C assay on a Hitachi-917 or Modular-PPE analyzer. HDL-C measurement was performed before and after removal of TRL by ultracentrifugation ("direct" HDL-C and HDL-UC, respectively). We examined the effect of TG on the relationship between HDL-UC and "direct" HDL-C. Analysis of variance multiregression analysis was performed for each generation of the commercial assay. RESULTS: We observed progressive TG interference that increased "direct" HDL-C by 10% to 15% or more in moderately hypertriglyceridemic samples (<600 mg/dL). Predictive equations were derived for each generation of the assay to estimate HDL-C in the absence of TRL. CONCLUSIONS: This study casts doubt on the specificity of "direct" HDL-C assays in the presence of hypertriglyceridemia. The use of assay-specific correction formulae to adjust for interference from TRL reduces the overestimation of HDL-C that influences CVD risk calculation, treatment, and follow-up of patients.

[Recent advances in the research on HDL subfraction].

High-density lipoprotein (HDL) is the smallest lipoprotein, with a density range of 1.063-1.210. HDL plays a crucial role in reverse cholesterol transport. Epidemiological studies have consistently shown that HDL-cholesterol (HDL-C) is inversely correlated with the incidence of coronary heart disease (CHD). HDL is composed of heterogeneous particles with different compositions and functions. For more precise prediction of CHD, researchers measure HDL subfractions which are separated by various methods based on their physicochemical properties. In this article, we briefly review the methods for measuring HDL subfractions and their clinical significance. We also address future perspectives in HDL subfraction measurements in the "Era of HDL therapy."

In vitro characterization of a monoclonal IgG(kappa) from a patient with planar xanthomatosis.

OBJECTIVE: To characterize a monoclonal IgG(kappa) (MAb) from a patient with planar xanthoma that precipitated with serum lipids at reduced temperature. METHODS: The molecular weight (Mr), sensitivity to proteases, and glycosylation of the purified MAb were analyzed. The specificity of the MAb was tested by measuring cryoprecipitation with pure high- (HDL) and low-density (LDL) lipoproteins. The effect of choline, phosphocholine, and glycerol 3-phosphate on the precipitation temperature of LDL by the MAb was studied. RESULTS: The MAb was larger than normal IgG due to hyperglycosylation of the MAb light chain. It formed cryoprecipitates with pure HDL and LDL as well as the lipids extracted from these lipoproteins. Fab fragments of the MAb lowered the temperature of its precipitation with LDL. Choline, phosphocholine, and glycerol 3-phosphate also lower the precipitation temperature. CONCLUSION: This is the first human IgG reported with apparent specificity for phosphocholine antigens. Its precipitation with lipids at reduced temperature suggests that it recognizes conformations of phospholipid head groups that develop below core body temperature.

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay.

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2) and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory.

Increased methionine sulfoxide content of apoA-I in type 1 diabetes.

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

Nonenzymatically glycated lipoprotein ApoA-I in plasma of diabetic and nephropathic patients.

ApoA-I, which constitutes 70% of the apolipoprotein content of high-density lipoproteins, acts as an acceptor for the transfer of phospholipids and free cholesterol from peripheral tissues and transports cholesterol in the liver and other tissue for excretion and steroidogenesis. In order to verify its possible structural alteration in pathological states, plasma samples from healthy, diabetic, and nephropathic subjects have been analyzed by two-dimensional gel electrophoresis. By this approach, clear differences among the three classes of subjects become evident and, in the case of diabetic and nephropathic patients, intense spots are present. The matrix-assisted laser desorption/ionization mass spectrometry analysis of their digestion products shows that an overexpression of unglycated and glycated ApoA-I is present in both pathological states, reasonably affecting the efficiency in cholesterol transport.

Valores de colesterol HDL en la población infantil y riesgo trombótico / Values of high-density lipoprotein cholesterol in the pediatric population and risk of thrombosis

Introducción Existen evidencias del papel protector que el colesterol unido a lipoproteínas de alta densidad (c-HDL) puede ejercer frente a la formación de la placa de ateroma y de su implicación en el transporte reverso de colesterol, así como de sus propiedades antioxidantes y moduladoras de la respuesta inflamatoria. También se han relacionado concentraciones bajas con un estado protrombótico. Objetivo Determinar la relación existente entre el c-HDL y los parámetros lipídicos y hemostáticos. Pacientes y métodos Un total de 110 niños (50 niñas, 60 niños) de entre 6 y 7 años. Se determinó el perfil lipídico, dímero-D, inhibidor del activador del plasminógeno y fibrinógeno. Resultados Los valores medios de los parámetros estudiados fueron colesterol total (192,92 ± 26,01 mg/dl), c-HDL (72,87 ± 15,69 mg/dl), colesterol unido a lipoproteínas de baja densidad (c-LDL) (109,46 ± 23,30 mg/dl), triglicéridos (56,24 ± 20,35 mg/dl), apolipoproteína B (apo B) (91,96 ± 14,93 mg/dl), apo A1 (168,4 ± 24,55 mg/dl), logaritmo lipoproteína(a) (1,76 ± 1,36 mg/dl), logaritmo del inhibidor del activador del plasminógeno tipo 1 (PAI-1) (3,77 ± 3,93 U/ml), logaritmo del dímero-D (5,53 ± 0,49 ng/ml) y fibrinógeno (268,61 ± 48,59 mg/dl). Al dividir la muestra en dos grupos, atendiendo a las concentraciones de c-HDL, los niños con valores más bajos presentaron concentraciones más elevadas y estadísticamente significativas de colesterol total/c-HDL, fibrinógeno y PAI. Los valores de c-HDL se asociaron directa y significativamente con colesterol total y apo A1 e inversa y significativamente con el cociente colesterol total/c-HDL, fibrinógeno y el PAI. Conclusión La población infantil estudiada presentó valores elevados de c-HDL, y éstos pudieron ser los responsables del incremento de colesterol total. Aumentos en su concentración se asociaron de manera significativa con una disminución del riesgo trombótico

Introduction There is evidence of the protective effect of high-density lipoprotein (HDL)-cholesterol against atheroma plaque formation and of its role in cholesterol efflux from cells, as well as its anti-oxidative and inflammatory modulating response properties. Low HDL-cholesterol levels have been associated with a prothrombotic state. Objective To determine the relationship between HDL-cholesterol and lipidic and hemostatic parameters. Patients and methods We studied 110 children (50 girls, 60 boys) aged between 6 and 7 years old. Lipid profile, D-dimer, plasminogen activator inhibitor (PAI) and fibrinogen were determined. Results The mean values of the studied parameters were as follows: total cholesterol (192.92 ± 26.01 mg/dl), HDL-cholesterol (72.87 ± 15.69 mg/dl), low-density lipoprotein-cholesterol (109.46 ± 23.30 mg/dl), triglycerides (56.24 ± 20.35 mg/dl), apolipoprotein B (91.96 ± 14.93 mg/dl), apolipoprotein A1 (168.4 ± 24.55 mg/dl), lipoprotein(a) logarithm (1.76 ± 1.36 mg/dl), plasminogen activator inhibitor-1 logarithm (PAI-1) (3.77 ± 3.93 U/ml), D-dimer logarithm (5.53 ± 0.49 ng/ml) and fibrinogen (268.61 ± 48.59 mg/dl).When the sample was divided into two groups according to HDL-cholesterol levels, children with lower levels showed significantly higher values of total cholesterol/HDL-cholesterol, fibrinogen and PAI. HDL-cholesterol levels were directly and significantly associated with total cholesterol and apolipoprotein A1 and negatively and significantly associated with the total cholesterol/HDL-cholesterol ratio, fibrinogen and PAI. Conclusion The children studied had high HDL-cholesterol levels, which could be responsible for the high total cholesterol levels. High values of HDL-cholesterol are significantly associated with a reduction in thrombotic risk

Acception of cholesterol from cells in men of the Russian population correlates with concentration of pre-beta1 high-density lipoproteins.

We analyzed subfraction composition of HDL and cholesterol-acceptor properties of the plasma in Russian men with high and low HDL cholesterol. HDL were subfractionated by two-dimensional electrophoresis in agarose-polyacrylamide gel. The content of pre-beta1 HDL increased in individuals with high concentration of HDL cholesterol and strictly correlated with acception of cellular cholesterol in both groups.

LCAT facilitates transacylation of 17 beta-estradiol in the presence of HDL3 subfraction.

It has been shown that estrogens need to be metabolized to their hydrophobic estrogen ester derivatives to act as antioxidants in lipoproteins. Data suggest that 17beta-estradiol (E(2)) becomes esterified in LCAT-induced reactions and the esters are transported from HDL particles to LDL and VLDL particles by a CETP-dependent mechanism. In the present study we have further investigated the regulation of E(2) esterification by LCAT and focused on the importance of HDL structure and composition in the esterification process. Isolated LDL, HDL(2), HDL(3), and reconstituted discoidal HDL (rHDL) were incubated with labeled E(2), with and without purified LCAT, at 37 degrees C for 24 h. After purification of the lipoprotein fractions, there was a significant peak of radioactivity representing esterified estradiol attached to HDL(3) and rHDL, but HDL(2) and LDL contained only trace amounts of labeled estradiol ester. TLC analysis confirmed that the radioactivity migrated in a position corresponding to that of 17beta-E(2) 17-monoester standard. The amount of radioactivity associated with HDL(3) and rHDL representing esterified E(2) was significantly increased by addition of purified LCAT. However, only limited increases of radioactivity were observed in HDL(2) and LDL. In conclusion, HDL subfractions differ in their potential to regulate estradiol esterification by LCAT.

A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass.

Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradients were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of LDL subclass pattern.

Catabolism of HDL1 cholesteryl ester in the rat. Effect of ethinyl estradiol treatment.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.