Component Of High-density Lipoprotein Particles Research Articles

A Streamlined Method for Quantification of Apolipoprotein A1 in Human Plasma by LC-MS/MS

To the Editor: Apolipoprotein A1 (apoA1) is the major protein component of high-density lipoprotein particles in blood, and its concentration in serum and plasma is a marker of atherosclerotic cardiovascular diseases. Clinical laboratories commonly use immunometric methods to measure apoA1; however, an alternative approach, LC-MS/MS, has been proposed (1–3). Uptake of these LC-MS/MS methods for routine testing in a clinical setting has been challenging owing to complex and time-intensive sample preparation work flows. Overnight digestion has been previously applied for quantification of apoA1 by use of proteolytic peptides VQPYLDDFQK, DYVSQFEGSALGK, and THLAPYSDELR (1, 2) and a 3-h digest using peptide VQPYLDDFQK (3). Toward the design of a streamlined work flow for implementation in a clinical laboratory, we simplified sample preparation by using only additive steps and eliminating the use of chemical denaturants, reduction, and alkylation. Herein we demonstrate that such a simple and rapid work flow, targeting a fast-forming proteolytic peptide, can be used to develop a quantitative apoA1 LC-MS/MS assay in agreement with an established immunonephelometric method. Our optimized protocol generated several proteotypic peptides suitable for apoA1 quantification by using a brief 20-min digestion without reduction or alkylation steps or the use of chemical denaturants. On the basis of several factors including the 24-h digestion profile (4), THLAPYSDELR was selected for further development. To generate the external 6-point calibration curve, the unlabeled THLAPYSDELR peptide was synthesized, with purity determined by high-performance LC and amino acid analysis (New England Peptide). The lyophilized peptide stocks were initially solubilized in a 5% acetonitrile and 0.1% formic acid solution and further diluted with …

Increased apolipoprotein M induced by lack of scavenger receptor BI is not activated via HDL-mediated cholesterol uptake in hepatocytes

BackgroundScavenger receptor BI (SR-BI) is a classic high-density lipoprotein (HDL) receptor, which mediates selective lipid uptake from HDL cholesterol esters (HDL-C). Apolipoprotein M (ApoM), as a component of HDL particles, could influence preβ-HDL formation and cholesterol efflux. The aim of this study was to determine whether SR-BI deficiency influenced the expression of ApoM.MethodsBlood samples and liver tissues were collected from SR-BI gene knockout mice, and serum lipid parameters, including total cholesterol (TC), triglyceride (TG), high and low-density lipoprotein cholesterol (HDL-C and LDL-C) and ApoM were measured. Hepatic ApoM and ApoAI mRNA levels were also determined. In addition, BLT-1, an inhibitor of SR-BI, was added to HepG2 cells cultured with cholesterol and HDL, under serum or serum-free conditions. The mRNA and protein expression levels of ApoM were detected by RT-PCR and western blot.ResultsWe found that increased serum ApoM protein levels corresponded with high hepatic ApoM mRNA levels in both male and female SR-BI−/− mice. Besides, serum TC and HDL-C were also significantly increased. Treatment of HepG2 hepatoma cells with SR-BI specific inhibitor, BLT-1, could up-regulate ApoM expression in serum-containing medium but not in serum-free medium, even in the presence of HDL-C and cholesterol.ConclusionsResults suggested that SR-BI deficiency promoted ApoM expression, but the increased ApoM might be independent from HDL-mediated cholesterol uptake in hepatocytes.

Associations between follicular fluid high density lipoprotein particle components and embryo quality among in vitro fertilization patients.

Follicular redox balance is likely to be important for embryo quality during in vitro fertilization (IVF), and the anti-oxidative high desity lipoprotein (HDL) particle is the sole lipoprotein measured in follicular fluid (FF). Therefore, we investigated FF HDL particle components as predictors of embryo quality during IVF. Two research follicles collected from each participant were individually tracked, and 103 women having at least one developed embryo were included in the analysis. Concentrations of 15 non-cholesterol HDL particle components and 26 HDL-cholesterol (HDL-C) particle size subfractions were determined. Embryo quality was assessed for embryo cell number, embryo fragmentation, and embryo symmetry. Multivariable Poisson regression with a sandwich variance estimator was used to evaluate associations between HDL particle components and embryo quality, adjusted for covariates. Higher γ-tocopherol concentration was associated with less embryo fragmentation (relative risk [RR] = 4.43; 95% confidence interval [CI] 1.78, 11.06), and higher apolipoprotein A-1 concentration was associated with full embryo symmetry (RR = 3.92; 95% CI 1.56, 9.90). Higher concentrations of HDL-C subfractions in the large and medium particle size ranges were associated with poorer embryo quality. FF HDL lipophilic micronutrients and protein components, as well as HDL-C particle size, may be important predictors of embryo quality during IVF.

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

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. 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. 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. 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.

ATP-Binding Cassette Transporter 1 Attenuates Ovalbumin-Induced Neutrophilic Airway Inflammation

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.

Sphingosylphosphorylcholine Stimulates CCL2 Production from Human Umbilical Vein Endothelial Cells

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.

Inverse relation of physical activity and apolipoprotein AI to blood pressure in elderly women

To study the relation of habitual physical activity, diet, and serum lipoproteins to blood pressure, a cross-sectional study was carried out in a cohort of 202 women, age 60-69 yr. Sitting, supine, and standing blood pressure was measured with a standard sphygmomanometer. Physical activity was assessed by questionnaire, diet by food records, serum lipoprotein cholesterol enzymatically, and apolipoprotein AI turbidimetrically. Among the women not taking antihypertensive medication (N = 127), the physically most active (physical activity 5 times per week or more) had sitting diastolic blood pressure of 86 mm Hg (adjusted for high-density-lipoprotein cholesterol, body mass index, and cardiovascular health status), which was 8 mm Hg lower (P = 0.007) than in the least active (physical activity twice per week or less) women. Subjects in the highest tertile of apolipoprotein AI (> 1.46 g.l-1) had a mean sitting systolic blood pressure of 147 mm Hg (adjusted for age, body mass index, and cardiovascular health status), which was 16 and 13 mm Hg lower (P = 0.001) than in women in the middle and lowest tertiles (< 1.32 g.l-1), respectively. The present data suggest that, in elderly women, regular physical activity is associated with a clinically significant lowering of diastolic blood pressure. Moreover, a higher level of serum apolipoprotein AI, the major protein component of high-density lipoprotein particles, is inversely associated with systolic blood pressure.