Journal of Lipid Research Volume 56, 2015 1519have triggered intense interest in targeting HDL for therapeutic intervention. Several recent observations have cast doubt on the hypotheses that HDL-C levels relate to CAD risk in humans and that elevating HDL-C is therapeutic ( 3, 4 ). For example, genetic variations that alter levels of HDL-C do not always associate with CAD risk, and interventions that elevate HDL-C do not necessarily reduce cardiovascular events in humans with established CAD ( 5 ). Taken together, these observations indicate that HDL is complex and that simply quantifying HDL-C might be a poor way to assess HDL function ( 6, 7 ).The ability of HDL (or serum HDL, serum depleted of apoB-containing lipoproteins) to promote sterol effl ux from cultured macrophages incubated with radiolabeled cholesterol can vary markedly, despite similar levels of HDL-C and apoA-I ( 8 ). Therefore, HDL-C is not necessarily the major determinant of HDL's macrophage sterol effl ux capacity in this system. Importantly, the effl ux capacity of serum HDL is lower in individuals with prevalent CAD ( 9-11 ). A recent study of a large cohort, initially free of CAD, demonstrated that sterol effl ux associates strongly and negatively with the risk of future cardiac events ( 12 ). This association was strengthened by multivariate adjustment, suggesting that impaired HDL function affects incident cardiovascular risk by processes distinct from those involving HDL-C, LDL-cholesterol (LDL-C), and other traditional lipid risk factors. Together, these observations indicate that the sterol effl ux capacity of HDL might be a marker, and perhaps a mediator, of atherosclerotic Abstract Recent studies demonstrate that HDL's ability to promote cholesterol effl ux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol effl ux capacity during vascular disease are unclear. Infl ammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol effl ux capacity. We therefore tested the hypothesis that HDL's impaired effl ux capacity is mediated by specifi c changes of its protein cargo. Humans with acute infl ammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C effl ux capacity was signifi cantly impaired. Proteomic analyses demonstrated that HDL's cholesterol effl ux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute infl ammation caused a marked impairment of HDL-C effl ux capacity that correlated with a large increase in HDL SAA. In striking contrast, the effl ux capacity of mouse infl ammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the infl ammatory impairment of HDL-C effl ux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo. -Vaisar, T

Background It is critical to develop new metrics to determine whether high density lipoprotein (HDL) is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P) – the size and concentration of HDL in plasma or serum. However, the two methods currently used to determine HDL-P yield concentrations that differ more than 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. A calibration curve constructed with purified proteins was used 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) subjects, three subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4±2.4 µM (mean±SD). HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in subjects with cerebrovascular disease, and this difference remained significant after adjustment for HDL cholesterol levels. Conclusions Calibrated IMA accurately and reproducibly determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting the method could accurately quantify HDL particle concentration. Importantly, the estimated stoichiometry of apoA-I determined by calibrated IMA was 3–4 per HDL particle, in excellent agreement with current structural models. Furthermore, HDL-P associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.

Biomarkers are pressingly needed to assist with the clinical diagnosis of neurodegenerative diseases and/or the monitoring of disease progression. Glycoproteins are enriched in bodily fluids such as human cerebrospinal fluid (CSF), an ideal source for discovering biomarkers due to its proximity to the central nervous system (CNS), and consequently can serve as diagnostic and/or therapeutic markers for CNS diseases. We report here an in-depth identification of glycoproteins in human CSF using a complementary proteomic approach which integrated hydrazide chemistry and lectin affinity column for glycoprotein enrichment, followed by multidimensional chromatography separation and tandem mass spectrometric analysis. Using stringent criteria, a total of 216 glycoproteins, including many low-abundance proteins, was identified with high confidence. Approximately one-third of these proteins was already known to be relevant to the CNS structurally or functionally. This investigation, for the first time, not only categorized many glycoproteins in human CSF but also expanded the existing overall CSF protein database.