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Background Postprandial triglyceridemia may transitory affect the structure of HDL subclasses and probably their antiatherogenic properties but little is known in this field. We analyzed the HDL subclasses lipid content along postprandial period. Methods Fifteen metabolic syndrome (MS) patients and 15 healthy controls were enrolled. HDL were isolated from plasma samples obtained at fasting and every 2-h up to 8-h, after a 75-g fat meal. Cholesterol (C), triglycerides (TAG), and phospholipid (Ph) plasma concentrations of five HDL subclasses were determined by densitometry of electrophoresis gels enzymatically stained. Results The increase of postprandial triglyceridemia expressed as the incremental area under the curve (iAUC) was twice in MS patients than in controls. Only large HDL2b-TAG were higher in MS than controls at 4, 6 and 8 h after meal intake, whereas cholesterol of HDL2a, 3a and 3b were lower at 8 h. HDL size distribution shifted towards large HDL and HDL3a-, 3b- and 3c-subclasses had a lower content of cholesterol (estimated by the C-to-Ph ratio) in subjects whose iAUC > 289.5 mg h/dl ( n = 15) in comparison with those subjects with iAUC below this cutoff point ( n = 15), independently of the MS status and fasting TAG. Triglycerides content of HDL subclasses changed only discreetly along the postprandial period, whereas paraoxonase-1 remained unchanged. Conclusions A high postprandial triglyceridemia conditions the shift of HDL size distribution towards large particles and the decrease of cholesterol in HDL3 subclasses. These data demonstrate that postprandial hypertriglyceridemia contributes to a transitory hypoalphalipoproteinemia that may increase the risk of cardiovascular disease. [ABSTRACT FROM AUTHOR]

Abstract: High-density lipoproteins (HDL) are inversely related with coronary artery disease (CAD) and HDL-cholesterol is the only standardized and reproducible parameter available to estimate plasma concentration of these lipoproteins. However, pharmacological interventions intended to increase HDL-cholesterol have not been consistently associated to an effective CAD risk reduction. Among patients with a myocardial infarction, 43 and 44% of men and women, respectively, had normal plasma levels of HDL-cholesterol, whereas genetic studies have failed to show a causal association between HDL-cholesterol and CAD risk. Instead, HDL functionality seems to be the target to be evaluated, but the existing methods are still poorly reproducible and far to be adapted to the clinical laboratory. HDL subclasses rise as a potential alternative for the evaluation of CAD risk; HDL subclasses are a surrogate of intravascular metabolism of these lipoproteins and probably of their functionality. Low levels of large HDL and increased proportions of small particles are the most remarkable features associated to an increased risk of type 2 diabetes mellitus (T2DM) or CAD. However, inflammation and other environmental factors are related with abnormal HDL structure, and, as a consequence, more prospective studies are needed to better support the clinical usefulness of HDL subclasses. New insights from proteome and lipidome profiles of HDL will provide potential HDL-related biomarkers in the coming years. [Copyright &y& Elsevier]

Abstract: Background: Several studies have suggested that measurement of high-density lipoprotein (HDL) 2 and HDL3 subfractions might be more useful for evaluating coronary risk than total HDL-cholesterol (C). However, methods of measuring HDL2 and HDL3 are quite laborious for general clinical use. Development of a quick and easy method of measuring HDL subfractions has been long-awaited. Methods: Triglyceride (TG) rich lipoproteins (TRLs), low-density lipoprotein (LDL), HDL2, and HDL3 were used for screening of surfactants and enzymes to react selectively with HDL3-C and to decompose other lipoproteins. Results: In order to develop HDL3-C homogeneous assay, polyoxyethylene styrenated phenyl ether derivative, for which the hydrophilic lipophilic balance (HLB) value is 13.6, was adopted as the most effective and specific surfactant for selection of HDL3 from HDL. Sphingomyelinase (SMase) reacted with TRLs and LDL preferentially, and decomposed them. HDL2-C was estimated by subtracting measured HDL3-C from total HDL-C, directly measured by homogeneous method. The homogeneous assay exhibited excellent correlations with the results of HDL3-C and HDL2-C measured by standard ultracentrifugation (R2 =0.848 and 0.982, respectively). Conclusions: We established a rapid and effective, fully-automated assay for the measurement of HDL3-C. Furthermore, the subtraction of HDL3-C from total HDL-C allows concurrent determination of HDL2-C. [Copyright &y& Elsevier]

Abstract: Background: The antiatherogenic role of different HDL subclasses is still controversial. HDL particles of the same size can have different lipid contents in some physiopathological situations. However, little is known about the plasma lipid levels of HDL subclasses when they are separated by their hydrodynamic diameter. Methods: Triglycerides (Tg), phosphatidylcholine (Ph), and cholesterol (C) plasma concentrations of HDL subclasses, were determined by enzymatic staining on polyacrylamide gradient gel (PAGE) in 50 pediatric patients with metabolic syndrome (MS), and 50 control children paired by age and gender. Proteins of HDL subclasses were also stained for the assessment of the relative size distribution of HDL. Results: Relative HDL size distribution was shifted to small particles in MS pediatric patients when determined per protein. In contrast, cholesterol plasma concentrations corresponding to the HDL2b, 2a, 3a, and 3b subclasses were decreased; triglycerides of HDL3b and 3c, as well as plasma phospholipids from HDL3c, were elevated in MS patients as compared to controls. The C-to-Ph ratio, considered as indicative of HDL composition, was similar among the 5 HDL subclasses in control subjects, whereas this ratio gradually decreased from large HDL2b to small HDL3c in the MS group. Cholesterol plasma concentrations of HDL subclasses correlated with the components of the MS. Conclusions: Lipids of HDL subclasses provide more and accurate information than the relative HDL size distribution determined by protein staining, and may contribute to understand better HDL metabolism and the coronary risk associated to these lipoproteins. [Copyright &y& Elsevier]

Abstract: Background: The effect of ABCA1 genetic variation on HDL-C levels has been widely documented, although studies in children are scarce. We recently found a frequent non-synonymous ABCA1 variant (R230C) exclusive to populations with Native American ancestry, associated with low HDL-C levels and other metabolic traits in adults. Methods: We genotyped R230C variant in 1253 healthy unrelated Mexican school-aged children aged 6–15years (595 boys and 658 girls) to seek associations with HDL-C levels and other metabolic traits. HDL subclass distribution was analyzed in a subgroup of 81 age, gender and BMI-matched children. Results: Individuals carrying the C230 allele showed a significantly lower HDL-C levels (P =2.9×10−8), and higher TC/HDL-C ratio, BMI, BMI z-score and percent fat mass (P =0.001, 0.049, 0.032 and 0.039, respectively). HDL size was smaller in R230C heterozygotes as compared to R230R homozygotes (P <0.05). Moreover, the proportion of HDL2b was lower, while the proportion of HDL3a and HDL3b particles was higher in R230C heterozygous and/or C230C homozygous individuals as compared to R230R homozygotes (P <0.05). Conclusions: Our data suggest that the R230C ABCA1 gene variant plays an important role in HDL-C level regulation and HDL subclass distribution in healthy Mexican school-aged children. [Copyright &y& Elsevier]

Abstract: Background: Rosiglitazone is an agonist of the peroxisome proliferator-activated receptor (PPAR) gamma that may modify HDL metabolism in humans, but this effect has not been completely elucidated. Therefore, we determined the effect of rosiglitazone on apo AI turnover, HDL structure, and PON1 plasma activity. Methods: Kinetic studies of HDL-apo AI radiolabeled with 125I were performed in 7 chow-fed, male, New Zealand white rabbits after 6 weeks of 0.32 mg/kg/d rosiglitazone-treatment vs. vehicle-treated rabbits (n =11). HDL size distribution was determined by polyacrylamide gradient electrophoresis and paraoxonase-1 (PON1); plasma activity was assessed spectrophotometrically using phenylacetate as substrate. Results: Fractional catabolic rate (FCR) of HDL apo AI was higher in the rosiglitazone-treated group than in the control group (0.031±0.004 vs. 0.025±0.006 pools/h, respectively, p <0.05). The mean apo AI production rate (PR) was 62% higher in the rosiglitazone group as compared to controls (0.918±0.238 vs. 0.564±0.160 mg/kg/h, p <0.01). Accordingly, apo AI plasma levels in rosiglitazone-treated animals were about 37% higher than in the control group. Rosiglitazone-induced changes in apo AI turnover appeared concomitantly with a significant increase of phospholipids and a decrease in colesteryl esters content of the HDL. Compositional changes resulted in a relative increase of the HDL3b and HDL3c subfractions and a significant enhancement of the plasma PON1 activity (488.5±138.2 vs. 595.2±179.4 µmol/min/ml, p <0.05). Conclusions: Rosiglitazone increased apo AI plasma concentrations, resulting from an enhancement of apo AI synthesis, and induced the synthesis of smaller HDL particles with a concomitant increase of plasma PON1 activity. These modifications may contribute to the anti-atherogenic potential of rosiglitazone. [Copyright &y& Elsevier]

Abstract: Background: To elucidate the relationship between the apolipoproteins, especially apoA-I and the alteration of HDL subclasses in hyperlipidemic, HTC and HTG subjects. Methods: ApoA-I contents of plasma HDL subclasses were quantitated by two-dimensional gel electrophoresis coupled with immunodetection in 233 normolipidemic subjects and 312 hyperlipidemic subjects (132 HTC and 180 HTG subjects). Making use of the mean ±1 SD of apoA-I levels, we further subdivided normolipidemic, hyperlipidemic, HTC and HTG subjects into 3 subgroups, respectively. Results: Subjects in the middle and low apoA-I subgroups had decreased HDL-C and apoA-I while increased TG, apoB100, apoCII, apoCIII and apoE concentrations. With the reduction of apoA-I concentrations, the apoA-I contents of all HDL subclasses decreased successively and significantly. The relative percentage of small-sized HDL increased significantly while those of large-sized HDL2a, HDL2b decreased significantly in hyperlipidemic, especially in HTG group. Multiple liner regression result revealed that apoA-I was positively and significantly correlated with all HDL subclasses and apoA-I level influenced the distribution of HDL subclasses powerfully in hyperlipidemic subjects. Conclusions: Both the rate and efficiency of RCT might be weakened more seriously in hyperlipidemic, especially in HTG subjects with low apoA-I levels. ApoA-I level might be a powerful factor correlated with the distributions of HDL subclasses. [Copyright &y& Elsevier]

Abstract: High-density lipoprotein cholesterol (HDL-C) comprises a family of particles with differing physicochemical characteristics. Continuing progress in improving HDL-C analysis has originated from two separate fields—one clinical, reflecting increased attention to HDL-C in estimating risk for coronary heart disease (CHD), and the other analytical, reflecting increased emphasis on finding more reliable and cost-effective HDL-C assays. Epidemiologic and prospective studies established the inverse association of HDL-C with CHD risk, a relationship that is consistent with protective mechanisms demonstrated in basic research and animal studies. Atheroprotective and less atheroprotective HDL subpopulations have been described. Guidelines on primary and secondary CHD prevention, which increased the workload in clinical laboratories, have led to a revolution in HDL-C assay technology. Many analytical techniques including ultracentrifugation, electrophoresis, chromatography, and polyanion precipitation methods have been developed to separate and quantify HDL-C and HDL subclasses. More recently developed homogeneous assays enable direct measurement of HDL-C on an automated analyzer, without the need for manual pretreatment to separate non-HDL. Although homogeneous assays show improved accuracy and precision in normal serum, discrepant results exist in samples with atypical lipoprotein characteristics. Hypertriglyceridemia and monoclonal paraproteins are important interfering factors. A novel approach is nuclear magnetic resonance spectroscopy that allows rapid and reliable analysis of lipoprotein subclasses, which may improve the identification of individuals at increased CHD risk. Apolipoprotein A-I, the major protein of HDL, has been proposed as an alternative cardioprotective marker avoiding the analytical limitations of HDL-C. [Copyright &y& Elsevier]

Abstract: Background: Different high-density lipoprotein (HDL) subclasses have distinct but interrelated metabolic functions. HDL directly influences the atherogenic process, and changes in HDL subclasses distribution may be related to the incidence and prevalence of atherosclerosis. Lipoprotein lipase (LPL) is an important enzyme for hydrolysis of triglyceride-rich lipoproteins, and its activity is positively correlated with the plasma HDL cholesterol level. LPL gene HindIII polymorphism has been found associated with variations in lipid levels, but the impact on HDL subclasses distribution is less clearly established. Methods: The relative apolipoprotein (apo) A-I contents (% apoA-I) of plasma HDL subclasses were determined by two-dimensional gel electrophoresis coupled with immunodetection and LPL gene HindIII polymorphism was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 173 hyperlipidemic and 155 normolipidemic subjects. Results: The frequencies of 495TT genotype and allele T were the highest both in the hyperlipidemic and control groups. Compared with the control group, the frequency of 495TT genotype was higher, while the frequencies of 495TG and 495GG genotypes were significantly lower (P <0.05) in the hyperlipidemic group. Two-dimensional gel electrophoresis and immunodetection showed that HDL subclasses distribution was altered in hyperlipidemia, and had a general shift toward smaller size. Compared with the control group, the hyperlipidemic group had significantly higher relative apoA-I contents of preβ1-HDL, preβ2-HDL, HDL3b and HDL3a (P <0.05) and lower HDL2a and HDL2b levels (P <0.001). In the hyperlipidemic group, allele T carriers'' frequency was higher than that in the control group (P <0.05), and the genotype of 495TT showed higher levels of plasma TG, apoB100, TG/HDL-C ratio, relative apoA-I contents of preβ1-HDL, HDL3b and lower HDL2a, HDL2b compared with that of the 495GG genotype subgroup (P <0.05). In the control group, the genotype of 495TT had higher plasma TG, HDL3c and lower HDL2a compared with that of 495GG subgroup (P <0.05). Conclusions: The 495TT genotype of LPL gene HindIII polymorphism was associated with changes of HDL subclasses distribution in Chinese population with hyperlipidemia. The particle size of HDL shifted toward smaller, which, in turn, indicated that RCT might be weakened and HDL maturation might be abnormal in hyperlipidemic subjects with 495TT genotype. [Copyright &y& Elsevier]

Abstract: Background: It is generally accepted that apolipoprotein (apo) A-I is the dominant structural apolipoprotein of HDL particles and different HDL subclasses have distinct but interrelated metabolic functions. HDL is known to directly affect the atherogenic process hence changes in HDL subclasses distribution may be related to the incidence and prevalence of atherosclerosis. Methods: The ApoA-I contents (mg/l) of plasma HDL subclasses were determined by 2-dimensional gel electrophoresis coupled with immunodetection and apoA-I genotypes were assayed by PCR-RFLP in 307 Chinese subjects (169 males, 138 females). Results: The G/G and C/C genotypes were the most frequent at −78 bp and +83 bp of apoA-I gene, respectively. There were no significant differences in the frequencies of rare A allele at −78 bp and rare T allele at +83 bp between males and females. Compared with the G/G carriers, G/A and A/A carriers had significantly higher plasma concentrations of TG, apoC-II, apoC-III, apoA-I contents of preβ1-HDL, HDL3a and TG/HDL-C ratio. And in addition, A/A carriers had significantly lower apoA-I contents of HDL2a and HDL2b. Females had increased plasma concentrations of apoA-I, HDL-C, apoA-I contents of HDL2a and HDL2b while decreased apoA-I contents of preβ1-HDL, HDL3b and TG/HDL-C ratio as compared to males carrying the same genotype. No significant differences were demonstrated on the concentrations of plasma lipids, lipoproteins, apolipoproteins and apoA-I contents of plasma HDL subclasses between the C/C and C/T subjects. Conclusion: The G/A polymorphism at −78 bp of apoA-I gene was associated with changes of HDL subclasses distribution. There was a general shift towards smaller-sized HDL, which, in turn, indicated that reverse cholesterol transport (RCT) might be weakened and HDL maturation might be abnormal in the subjects with G/A mutation. [Copyright &y& Elsevier]

Background: It is generally accepted that different high-density lipoprotein (HDL) subclasses have distinct but interrelated metabolic functions. HDL is known to directly influence the atherogenic process and changes in HDL subclasses distribution may be related to the incidence and prevalence of atherosclerosis. Method: The relative apolipoprotein (apo)A-I contents (% apoA-I) of plasma HDL subclasses were determined by two-dimensional gel electrophoresis coupled with immunodetection for apoA-I, in 39 hypercholesterolemic (HTC) subjects, 97 hypertriglyceridemic (HTG) subjects and 32 mixed hyperlipidemic (MHL) subjects, and 124 normolipidemic subjects. Results: The relative apoA-I contents of preβ1-HDL, preβ2-HDL, HDL3c, HDL3b and HDL3a significantly increased while HDL2a and HDL2b significantly decreased in hyperlipidemic subjects. In HTC subjects of hyperlipidemia, the concentrations of preβ1-HDL were significantly lower and HDL2b concentrations were significantly higher than in HTG and MHL subjects. In HTG subjects, the concentrations of HDL3a were significantly higher and the concentrations of HDL2b were lower than in HTC and MHL subjects. In total hyperlipidemic subjects, plasma triglyceride (TG) concentrations showed positive correlation with preβ1-HDL, preβ2-HDL, HDL3b and HDL3a and negative correlation with HDL2a and HDL2b. The total cholesterol (TC) concentrations showed positive correlation with the relative apoA-I contents of preβ1-HDL and HDL3b, whereas the HDL-C concentrations showed negative correlation with the relative apoA-I contents of preβ1-HDL and HDL3a and positive correlation with those of HDL2a and HDL2b. The relative apoA-I contents of preβ1-HDL, preβ2-HDL, HDL3b, and HDL3a were positively correlated whereas those of HDL2a and HDL2b were negatively correlated with TG/HDL-C ratio. Conclusion: The particle size of HDL in hyperlipidemic subjects shifted towards smaller sizes, which, in turn, indicates that the maturation of HDL may be abnormal in hyperlipidemic subjects. [Copyright &y& Elsevier]

Background: Lipoprotein subclass determinations provide a more detailed reflection of lipoprotein metabolism and an accurate prediction for risk of cardiovascular disease. Gradient gel electrophoresis for lipoprotein separation on Pharmacia electrophoretic apparatus has been most commonly used for many years. Methods: In this paper, we describe a new method for separating LDL and HDL subclasses by nondenaturing polyacrylamide gradient (3–31%) gel electrophoresis, using BioRad Mini Protean II electrophoretic cells. Results: The mean particle diameters of cholesterol-stained LDL and HDL lipoproteins were estimated after calibrating the gels with size standards, using fractional absorbance profiles. For the first time in the Republic of Macedonia, lipoprotein distribution and size phenotyping were studied in 345 healthy individuals. Large LDL subclasses (phenotype A) were dominant in 88.5% of the population, whereas small LDL subclasses (phenotype B) were dominant in 11.5%. The mean dominant LDL size was 26.08±0.8 nm. Five HDL subclasses were separated on the same gels, and HDL2b and HDL2a (larger) were dominant in healthy Macedonians. Conclusion: Antiatherogenic, larger LDL and HDL particles are most commonly found in healthy populations in the Republic of Macedonia. [Copyright &y& Elsevier]